1

ABSTRACT
The microtubule-targeting antineoplastic agent, paclitaxel, obtained from Yew tree is highly efficacious against a wide spectrum of human cancers. However, dose-limiting toxicity and development of drug resistance limit its clinical application. Development of novel strategies that overcome chemoresistance and sensitize cancer cells to paclitaxel can enhance the therapeutic effect of this drug. The NF-B (nuclear factor kappaB) family of transcription factors and its specific intracellular inhibitor IB, participate are involved in a myriad of activities, including the regulation of immune responses, maturation of immune cells, development of secondary lymphoid organs, inflammation, cell proliferation and apoptotic cell death. Activation of transcription factor NF-B is frequently encountered in tumor cells and contributes to aggressive tumor growth and resistance to chemotherapy and ionizing radiation during cancer treatment. Accumulating evidence over the last few years indicate that most chemotherapeutic agents and radiation therapy activate NF-B in vitro and in vivo. Curcumin a natural polyphenol obtained from the rhizome of Curcuma longa has been shown to exhibit antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, and anticancer activities and thus has a potential against various malignant diseases, diabetes, allergies and several cancers. The major disadvantage of this compound is their low bioavailability. Several recent studies showed synergistic effect of curcumin in combination with other anticancer drugs. In this study, the role of NF-B in synergism of paclitaxel and curcumin in cervical cancer cell line HeLa was explored by MTT assay and immunoblot was performed to investigate NF-Bs role in paclitaxel induced upregulation cox-2 which involve in conversion of arachidonic acid to prostaglandins. Present study showed that curcumin inhibited paclitaxel-induced activation of NF-B and potentiated the growth inhibitory effect of paclitaxel in cervical cancer cell line HeLa. We observed that the combined treatment of curcumin and paclitaxel induced a synergistic reduction in cancer cell growth compared with the individual treatments of paclitaxel or curcumin. Curcumin suppresses the constitutively active NF-B activation which led to the down regulation of cyclooxygenase-2 (COX2.

1. INTRODUCTION
1.1. CANCER
Cancer is the abnormal growth of cells caused by multiple changes in gene expression leading to disregulated balance of the cell proliferation and the cell death and ultimately evolving into a population of cells that can invade tissues and metastasize to distinct sites causing significant mobility and if untreated death of the host. Cancer has been around since before the first humans walked the Earth. Fossilized dinosaur bones show evidence of tumors, and archaeologists have discovered a 2,700-year-old human skeleton with evidence of prostate cancer that had spread through its bones. The Greek physician Hippocrates named the disease after the Greek word for crab, perhaps because the tumor and its branching network of blood vessels reminded him of the multi legged creature. Cancer is a major public health problem in the all parts of the world. Currently, one in 4 deaths is happened due to cancer. It is considered as the most dominant disease of this century. It is estimated that around a total of 1,596,670 new cancer cases and 571,950 deaths from cancer are projected to occur in the United States in 2011. Overall cancer incidence rates were stable in men in the most recent time period after decreasing by 1.9% per year from 2001 to 2005; in women, incidence rates have been declining by 0.6%annually since 1998. Overall cancer death rates decreased in all racial/ethnic groups in both men and women from 1998 through 2007, with the exception of American Indian/Alaska Native women, in whom rates were stable. African American and Hispanic men showed the largest annual decreases in cancer death rates during this time period (2.6% and 2.5%, respectively). Lung cancer death rates showed a significant decline in women after continuously increasing since the 1930s. The reduction in the overall cancer death rates since 1990 in men and 1991 in women translates to the avoidance of about 898,000 deaths from cancer. Cancer prevalence in India is estimated to be around 2.5 million, with over 8,00,000 new cases and 5,50,000 deaths occurring each year due to this disease.4 More than 70% of the cases report for diagnostic and treatment services in the advanced stages of the disease, which has lead to a poor survival and high mortality rate.

Courtesy: (Siegel et al. 2011, Cancer Statistics, A Cancer Journal for Clinicians.)

1.1.1. TYPES
1.1.1.1. Carcinoma A malignant new growth that arises from epithelium, found in skin or, more commonly, the lining of body organs, for example: breast, prostate, lung, stomach or bowel. Carcinomas tend to infiltrate into adjacent tissue and spread (metastasize) to distant organs, for example: to bone, liver, lung or the brain. 1.1.1.2. Sarcoma One of a group of tumor usually arising from connective tissue. Most sarcomas are malignant. Many types are named after the type of cell, tissue, or structure involved, as in angiosarcoma, chondrosarcoma, fibrosarcoma, liposarcoma,and osteosarcoma. 1.1.1.3. Leukemias Malignant neoplasm of blood-forming tissues; characterized by abnormal proliferation of leukocytes. First recognized by the German pathologist Rudolf Virchow in 1847. 1.1.1.4. Lymphomas Lymphomas affect the lymphatic system. There are over 20 types of lymphoma Hodgkin`s disease is one type of lymphoma. All other lymphomas are grouped together and are called non-Hodgkin`s lymphoma. Non-Hodgkin`s lymphoma may occur in a single lymph node, a group of lymph nodes, or in another organ. This type of cancer can spread to almost any part of the body, including the liver, bone marrow, and spleen.

1.1.2. GENETICS OF CANCER

Cancer (neoplasm) is a family of diseases that involve uncontrolled cell division and tissue invasiveness (metastasis). During metastasis, malignant cells travel among tissues via the circulatory and/or lymphatic system. Unregulated cell growth and metastasis are caused by mutations in the genes (DNA) of proteins involved in the regulation of the cell cycle. Agents that cause DNA damage leading to the transformation of a cell are called carcinogens. Cancers result from a series of gene mutations that typically involve two categories of function: promotion of cell division and inactivation of cell cycle suppression. Proto-

oncogenes are normal genes that promote cell growth and mitosis, whereas tumor suppressor genes discourage cell growth. Proto-oncogenes can be mutated by carcinogenic agents to become oncogenes. Oncogenes produce excessive levels of growth promoting proteins. Tumor suppressor gene products typified by p53 are frequently transcription factors that suppress mitosis and cell growth to allow for DNA repair. Nearly half of all cancers involve altered p53 genes. Other suppressor genes include Rb (retinoblastoma family), APC (adenomatous polyposis coli), SMAD4, TP53, p16/CDKN2A and BRCA (breast cancer susceptibility protein) types 1 and 2. Cancer results from cumulative mutations of protooncogenes and suppressor genes which together allow the unregulated growth of cells. Oncogenes are typically dominant because they provide gain-of-function, whereas suppressor genes are recessive. They contain loss-of function mutations. Both copies of a suppressor gene need to mutate to cause loss-of-suppressor function. Only one copy of a proto-oncogene needs to mutate for gain-of-function. Mutations of tumor suppressor genes can be inherited.

1.1.3. CERVICAL CANCER

Cervical cancer is the second most common type of cancer in women worldwide, after breast cancer. A preponderance of evidence supports a causal link between human papillomavirus infection and cervical neoplasia. The presence of high-risk human papillomavirus genital subtypes increases the risk of malignant transformation. Widespread use of the Papanicolaou smear has dramatically reduced the incidence of cervical cancer in developed countries. Accurate and early recognition of abnormal cytological changes prevents progression of the disease from pre invasive to invasive. Research is under way to determine if efforts to reduce the false-negative rate of the Papanicolaou smear should include rescreening programs and fluid-based technology. Once cervical cancer is diagnosed, clinical staging takes place. Early-stage tumors can be managed with cone biopsy or simple hysterectomy. Higher stage tumors can be treated surgically or with radiotherapy. Advanced metastatic disease may respond to radiation therapy and concurrent chemotherapy. Protein markers for detection of recurrence and vaccines for prevention of cervical cancer are under investigation.

Fig 1: Cervical cancer [Courtesy: http://ziviso.wordpress.com/2011/10/26/cervical-cancer-kills-women-in-developingcountries/

1.1.4. TYPES OF CANCER OF THE CERVIX There are three main types of cervical cancer: Squamous cell carcinoma (SCC) is the most common type of cervical cancer, accounting for 85% to 90% of all cases. It develops from the cells that line the inner part of the cervix, called the squamous cells. It usually begins where the part of the cervix that connects with the vagina (called the ectocervix) meets the part of the cervix that opens into the uterus (called the endocervix). Adenocarcinoma develops from the column-shaped cells that line the mucousproducing glands of the cervix. In rare instances, adenocarcinoma originates in the supportive tissue around the cervix. Adenocarcinoma accounts for about 10% of all cervical cancers. Mixed carcinomas (for example, adenosquamous carcinomas) combine features of both squamous cell carcinoma and adenocarcinoma. 1.1.5. RISK FACTORS A risk factor is anything that changes your chance of getting a disease such as cancer. Different cancers have different risk factors. For example, exposing skin to strong sunlight is a risk factor for skin cancer. Smoking is a risk factor for many cancers. But having a risk factor, or even several, does not mean that you will get the disease. Several risk factors increase your chance of developing cervical cancer. Women without any of these risk factors rarely develop cervical cancer. Although these risk factors increase the odds of developing cervical cancer, many women with these risks do not develop this disease. When a woman develops cervical cancer or pre-cancerous changes, it may not be possible to say with certainty that a particular risk factor was the cause. Risk factors include 1.1.5.1. Human papillomavirus infection Of the many types of human papillomavirus (HPV), more than 30 infect the genital tract.. Although HPV is essential to the transformation of cervical epithelial cells, it is not sufficient, and a variety of cofactors and molecular events influence whether cervical cancer will develop. Persistent infection with one of about 15 genotypes of carcinogenic human papillomavirus (HPV) causes almost all cases. There are four major steps in cervical cancer

development: infection of metaplastic epithelium at the cervical transformation zone, viral persistence, progression of persistently infected epithelium to cervical precancer, and invasion through the basement membrane of the epithelium. 1.1.5.2. Smoking Women who smoke are about twice as likely as non-smokers to get cervical cancer. Smoking exposes the body to many cancer-causing chemicals that affect organs other than the lungs. These harmful substances are absorbed through the lungs and carried in the bloodstream throughout the body. Tobacco by-products have been found in the cervical mucus of women who smoke. Researchers believe that these substances damage the DNA of cervix cells and may contribute to the development of cervical cancer. Smoking also makes the immune system less effective in fighting HPV infections. 1.1.5.3. Immunosuppression Human immunodeficiency virus (HIV), the virus that causes AIDS, damages the body's immune system and places women at higher risk for HPV infections. This may explain the increased risk of cervical cancer for women with AIDS. Scientists believe that the immune system is important in destroying cancer cells and slowing their growth and spread. In women with HIV, a cervical pre-cancer might develop into an invasive cancer faster than it normally would. Another group of women at risk of cervical cancer are women receiving drugs to suppress their immune response, such as those being treated for an autoimmune disease (in which the immune system sees the body's own tissues as foreign and attacks them, as it would a germ) or those who have had an organ transplant. 1.1.5.4. Chlamydia infection Chlamydia is a relatively common kind of bacteria that can infect the reproductive system. It is spread by sexual contact. Chlamydia infection can cause pelvic inflammation, leading to infertility. Some studies have seen a higher risk of cervical cancer in women whose blood test results show evidence of past or current chlamydia infection (compared with women who have normal test results). Infection with chlamydia often causes no symptoms in women. A woman may not know that she is infected at all unless she is tested for chlamydia when she gets her pelvic exam.

1.1.5.5. Diet Women with diets low in fruits and vegetables may be at increased risk for cervical cancer. Also overweight women are more likely to develop adenocarcinoma of the cervix. 1.1.5.6. Oral contraceptives (OCS) There is evidence that taking oral contraceptives (OCs) for a long time increases the risk of cancer of the cervix. Research suggests that the risk of cervical cancer goes up the longer a woman takes OCs, but the risk goes back down again after the OCs are stopped. In one study, the risk of cervical cancer was doubled in women who took birth control pills longer than 5 years, but the risk returned to normal 10 years after they were stopped. 1.1.5.7. Use of intrauterine device (IUD) A recent study found that women who had ever used an intrauterine device (IUD) had a lower risk of cervical cancer. The effect on risk was seen even in women who had an IUD for less than a year, and the protective effect remained after the IUDs were removed. Using an IUD may also lower the risk of endometrial (uterine) cancer. However, IUDs do have some risks. A woman interested in using an IUD should first discuss the potential risks and benefits with her doctor. Also, a woman with multiple sexual partners should use condoms to lower her risk of sexually transmitted illnesses no matter what other form of contraception she uses. 1.1.5.8. Multiple full term pregnancies Women who have had 3 or more full-term pregnancies have an increased risk of developing cervical cancer. No one really knows why this is true. One theory is that these women had to have had unprotected intercourse to get pregnant, so they may have had more exposure to HPV. Also, studies have pointed to hormonal changes during pregnancy as possibly making women more susceptible to HPV infection or cancer growth. Another thought is that the immune system of pregnant women might be weaker, allowing for HPV infection and cancer growth.

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1.1.6. TREATMENTS Different types of treatment are available for patients with cervical cancer. Some treatments are standard (the currently used treatment), and some are being tested in clinical trials. A treatment clinical trial is a research study meant to help improve current treatments or obtain information on new treatments for patients with cancer. 1.1.6.1. Surgery Surgery (removing the cancer in an operation) is sometimes used to treat cervical cancer. The following surgical procedures may be used:

Conization: A procedure to remove a cone-shaped piece of tissue from the cervix and cervical canal.

Total hysterectomy: Surgery to remove the uterus, including the cervix. If the uterus and cervix are taken out through the vagina, the operation is called a vaginal hysterectomy. Total laparoscopic hysterectomy.

1.1.6.2. Radiation therapy Radiation therapy is a cancer treatment that uses high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing. There are two types of radiation therapy. External radiation therapy uses a machine outside the body to send radiation toward the cancer. Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer. The way the radiation therapy is given depends on the type and stage of the cancer being treated. 1.1.6.3. Chemotherapy Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. When chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the cerebrospinal fluid, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.

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1.2. HeLa CELLS

HeLa cells are a human epithelial cervical cancer (cervical cancer) and the first human cells, from which a permanent cell line was established. On 9 February 1951.surgeon Lawrence Wharton Jr. removed the tissues from the patient Henrietta lacks, 31 year old African American women from Baltimore, in the womans clinic of the john Hopkins hospital. The cells were from the carcinoma of the cervix where expected to examine for the malignancy patient died 8 months later from the disease. A portion of the cell from the biopsy where sent to the George Gey then head of the cell culture laboratory at johns Hopkins hospital. The cells where cultivated and propagated in cell culture so well that since they are widely used in research. HeLa cells where used in the establishment of the first polio vaccine by Jonas Salk HeLa cells are now available in many laboratories of the world wide sale of HeLa cell suggest that Ms. Lack is probably the most valuable human individual had previously lived.

1.4. PACLITAXEL (TAXOL)

Paclitaxel is a mitotic inhibitor used in cancer chemotherapy. Paclitaxel is a novel antimicrotubule agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stability inhibits the normal dynamic reorganization of the microtubule network that is essential for vital interphase and mitotic cellular functions. In addition, paclitaxel induces abnormal arrays or bundles of microtubules throughout the cell cycle and multiple asters of microtubules during mitosis, further disrupting cell function. It was discovered in a National Cancer Institute program at the Research Triangle Institute in 1967 when Monroe E. Wall and Mansukh C. Wani isolated it from the bark of the Pacific yew tree, Taxus brevifolia and named it 'taxol'. When it was developed commercially by Bristol-Myers Squibb (BMS) the generic name was changed to 'paclitaxel' and the BMS compound is sold under the trademark 'Taxol'. In this formulation paclitaxel is dissolved in Cremophor EL, a polyoxyethylated castor oil, as a delivery agent since paclitaxel is not soluble in water. A newer formulation, in which paclitaxel is bound to albumin as the delivery agent (Protein-bound paclitaxel), is sold commercially by Abraxis BioScience under the trademark Abraxane

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Fig 4 Pacafic Yew Tree (Taxus brevifolia) [Courtesy: http://www.wildflowerfinder.org.uk/Flowers/Y/Yew%5BTree%5D/Yew%5BTree%5D.htm]

Fig 5 Paclitaxel Molecular Structure [Courtesy: http://www.wildflowerfinder.org.uk/Flowers/Y/Yew%5BTree%5D/Yew%5BTree%5D.htm]

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Paclitaxel is now used to treat patients with lung, ovarian, breast cancer, head and neck cancer, and advanced forms of Kaposi's sarcoma. Paclitaxel is also used for the prevention of restenosis.

1.5. CURCUMIN
Imagine if the key to disease prevention was as close as your kitchen shelf. Its not the product of someones imagination, but the product of years of medical research. Scientists are beginning to take notice of a well-known spice as a potent new preventive therapy against disease, especially cancerJohn C. Martin, LE Magazine, September 2001 More than one billion people consume curcumin regularly in their diets. Curcumin has long been used in Eastern medicine and is gaining attention in Western medicine, not only as a nonsteroidal anti-inflammatory drug (NSAID) but also for its chemopreventive properties. Essentially, curcumin is believed to possess generalized protective properties. Curcumin, a yellow pigment from Curcuma longa, is a major component of turmeric and is commonly used as a spice and food-coloring material. It exhibits anti-inflammatory, antitumor, and antioxidant properties. Curcumin is a low molecular-weight polyphenol, first chemically characterized in 1910, with the molecular formula of C21H20O6. It is generally regarded as the most active constituent of and comprises 28% of most turmeric preparations. It has long been used as the yellow spice in Indian food and as a naturally occurring medicine for the treatment of inflammatory diseases. The desirable preventive or putative therapeutic properties of curcumin have also been considered to be associated with its antioxidant property. Because free radical-mediated peroxidation of membrane lipids and oxidative damage of DNA and proteins are believed to be associated with a variety of chronic pathological complications such as cancer, atherosclerosis, neurodegenerative diseases, and aging, curcumin is thought to play a vital role against oxidative-stress-mediated pathological conditions. Hence, the past few decades have witnessed intense research devoted to the antioxidant activity of curcumin. Before pointing out the potential antioxidant property of curcumin, it is worthwhile to outline the role of free radicals and antioxidants in health and disease.

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Fig 1 Turmeric plant (Curcuma longa Linn)

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Fig2 Curcumin

Fig 3 Curcumin Molecular Structure (Keto form) [Courtesy: http://www.avaplant.com/products/herbal-extracts/water-soluble-curcumin/]

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1.6. NF-B
NF-B refers to a family of transcription factors that has been highly conserved through evolution and is present in the cytoplasm of all cells. NF-B has been called a stress sensor because its activity is induced by a wide variety of stimuli, 7 including tumor necrosis factor (TNF-), PMA and other tumor promoters, cigarette smoke extract (CSE), lipopolysaccharide (LPS), oxidants, and pathogenic bacteria. The NF-B family comprises five members: p50 (NF-B1), p52 (NF-B2), RelA (p65), RelB, and c-Rel. p50 and p52 are cleaved from inactive precursor proteins, p105 and p100, respectively, prior to translocation to the nucleus. NF-B family members are characterized by having:

A Rel homology domain that binds to DNA A dimerization domain The ability to bind to the intracellular inhibitor complex, IB The most widely studied NF-B heterodimers are p50/p65 and p50/c-Rel (both

associated with the classical or canonical pathway) and p52/RelB (alternative pathway). The classical pathway is activated by inflammatory cytokines, bacterial and viral infections, and oxidative stimuli and induces gene expression responsible for the antiapoptotic actions of NFB. The alternative pathway is primarily involved in B cell survival. Cytoplasmic NF-B is sequestered as an inactive complex with its regulatory subunit, IB. The most abundant member of the IB family of proteins is IB. Phosphorylation of two conserved serine residues in the N-terminal domain of the NF-B/IB complex induces the rapid dissociation and polyubiquitination of IB followed by its degradation by the 26S proteasome. Activated NF-B translocates to the nucleus where specific subunit lysines are acetylated by SRC-1 and p300 histone acetyl transferases. Acetylation promotes DNA binding and NF-B-induced gene transcription.10. Many of the genes regulated by NF-B code for inflammatory cytokines and proteins that mediate cell survival, cellular adhesion, cell cycle activation, cell proliferation, angiogenesis, and oncogenesis. However, not all the actions of NF-B promote cell survival. Activation of NF-B also appears to be essential for p53-induced apoptosis in response to oxidative stress or to the anticancer agents, doxorubicin and etoposide.

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1.7. COX-2
Cyclooxygenase-2, an enzyme that acts to speed up the production of certain chemical messengers, called prostaglandins that play a key role in in promoting inflammation. When cox-2 activity is blocked, inflammation is reduced. Unlike cox-1, cox-2 is active only at the site of inflammation, not in the stomach. COX-2 was discovered in 1991 by the Daniel Simmons laboratory at Brigham Young University. COX-2 exists as a homodimer, each monomer with a molecular mass of about 70 kDa. The tertiary and quaternary structures of COX-1 and COX-2 enzymes are almost identical. Each subunit has three different structural domains: a short N-terminal epidermal growth factor (EGF) domain; an -helical membrane-binding moiety; and a C-terminal catalytic domain. COX enzymes are monotopic membrane proteins; the membrane-binding domain consists of a series of amphipathic helices with several hydrophobic amino acids exposed to a membrane monolayer. Cyclooxygenase 2 (COX-2) catalyzes the conversion of arachidonic acid to prostaglandin H2 in the first step in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins. COX-2 inhibition by nonsteroidal anti-inflammatory agents has been shown to decrease angiogenesis and tumor growth, and promote apoptosis. COX-2 over expression has been associated with increased microvascular density. COX-2 overexpression has also been suggested as a poor prognostic indicator in carcinomas of the colon, breast, pancreas, and adenocarcinoma of the lung.

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Schematic representation of the human COX-2 primary protein structure [Courtesy: Maria Teresa Rizzo, Cyclooxygenase-2 in oncogenesis. Clinica Chimica Acta 412 671687]

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2. OBJECTIVES
The objectives of the present study are, To study the role of Nuclear factor Kappa B on the synergistic effect of paclitaxel and curcumin in the cervical cell line, HeLa. To study the effect of paclitaxel on the expression COX 2 in HeLa cell. To study the regulatory role of Nuclear Factor kappa B in the expression level of COX 2 induced by paclitaxel.

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3. REVIEW OF LITERATURE
Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. If the spread is not controlled, it can result in death. Cancer is caused by both external factors (tobacco, infectious organisms, chemicals, and radiation) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). These causal factors may act together or in sequence to initiate or promote carcinogenesis. Ten or more years often pass between exposure to external factors and detectable cancer. Cancer is treated with surgery, radiation, chemotherapy, hormone therapy, biological therapy, and targeted therapy (Anonymous, 2011) The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. (Hanahan and Weinberg, 2011) Cancer cells can acquire the capability to sustain proliferative signaling in a number of alternative ways: They may produce growth factor ligands themselves, to which they can respond via the expression of cognate receptors, resulting in autocrine proliferative stimulation. Alternatively, cancer cells may send signals to stimulate normal cells within the supporting tumor-associated stroma, which reciprocate by supplying the cancer cells with various growth factors (Cheng et al., 2008). Four decades of research have demonstrated that the cell-to cell contacts formed by dense populations of normal cells propagated in two-dimensional culture operate to suppress further cell proliferation, yielding confluent cell monolayers. Importantly, such contact inhibition is abolished in various types of cancer cells in culture, suggesting that contact inhibition is an in vitro surrogate of a mechanism that operates in vivo to ensure normal tissue homeostasis, one that is abrogated during the course of tumor genesis. Until recently, the

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mechanistic Basis for this mode of growth control remained obscure. Now, however, mechanisms of contact inhibition are beginning to emerge (Hanahan and Weinberg, 2011). The mechanism involves the product of the NF2 gene, long implicated as a tumor suppressor because its loss triggers a form of human neurofibromatosis. Merlin, the cytoplasmicNF2 gene product, orchestrates contact inhibition via coupling cell-surface adhesion molecules (e.g., E-cadherin) to transmembrane receptor tyrosine kinases (e.g., the EGF receptor). In so doing, Merlin strengthens the adhesivity of cadherin-mediated cell-tocell attachments. Additionally, by sequestering growth factor receptors, Merlin limits their ability to efficiently emit mitogenic signals (Curto et al., 2007; Okada et al., 2005). The concept that programmed cell death by apoptosis serves as a natural Barrier to cancer development has been established by compelling functional studies conducted over the last two decades. Although the cellular conditions that trigger apoptosis remain to be fully enumerated, several abnormality sensors that play key roles in tumor development have been identified (Adams and Cory, 2007; Lowe et al., 2004). Most notable is a DNA damage sensor that functions via the TP53 tumor suppressor TP53 induces apoptosis by up regulating expression of the Noxa and Puma BH3-only proteins, doing so in response to substantial levels of DNA breaks and other chromosomal abnormalities (Junttila and Evan, 2009). Insufficient survival factor signaling (for instance inadequate levels of interleukin-3 in lymphocytes or of insulin-like growth factor 1/2 [Igf1/2] in epithelial cells) can elicit apoptosis through a BH3-only protein called Bim. Yet another condition leading to cell death involves hyperactive signaling by certain oncoproteins, such as Myc, which triggers apoptosis (in part via Bim and other BH3-only proteins) unless counter Balanced by antiapoptotic factors (Junttila and Evan, 2009; Lowe et al., 2004). Cervical cancer continues to remain a major public health problem, as it remains a major cause of cancer-related deaths among women worldwide. Infection with oncogenic human papillomavirus (HPV) is the primary etiological factor for cervical cancer and its precursor lesions. HPV infections are often transient; however, persistent infections, especially with oncogenic or high-risk HPV types (e.g. HPV-16 and -18), further increase the likelihood of

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developing cervical cancer. Persistent HPV infections cause virtually all of the more than 500 000 cases of invasive cervical cancer per year worldwide (Bosch et al., 2010). HPV-induced oncogenesis in cervical carcinoma is largely attributable to expression of the viral oncoproteins E6 and E7, but HPV infection alone is insufficient to induce malignant transformation of cervical epithelium. Other significant cofactors, such as individual genetic variation and environmental exposures, contribute to the multistep process of tumor formation (Delmas et al., 2011). Strong evidence now supports the adoption of cervical cancer prevention strategies that explicitly focus on persistent infection with the causal agent, human papillomavirus (HPV). New HPV infections acquired at any age are virtually always benign, but persistent infections with one of approximately 12 carcinogenic HPV types explain virtually all cases of cervical cancer. In the absence of an overtly persistent HPV infection, the risk of cervical cancer is extremely low. Thus, HPV test results predict the risk of cervical cancer and its precursors (cervical intraepithelial neoplasia grade 3) better and longer than cytological or colposcopy abnormalities, which are signs of HPV infection. The logical and inevitable move to HPVBased cervical cancer prevention strategies will require longer screening intervals that will disrupt current gynecologic and cytology laboratory practices built on frequent screening (Schiffman et al., 2010). Cervical cancer cells are addicted to the expression of Human Papillomavirus (HPV) oncoproteins E6 and E7. The oncogencity of E6 is mediated in part by targeting p53 and PDZ-family tumor suppressor proteins for rapid proteasomal degradation, whereas E7

oncoprotein acts in part by co-opting histone deacetylases (HDAC)1/2 (Lin et al., 2009). Cervical cancers occur primarily at the cervical transformation zone. The transformation zone is a ring of tissue located where the squamous epithelium of the vagina meets, undermines, and replaces the glandular epithelium of the endocervical canal (Schiffman et al., 2010). Two prophylactic vaccines (Cervarix and Gardasil) targeting the 2 most prevalent high-risk HPV types (HPV types 16 and 18) found in cervical cancer are available. These

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vaccines are expected to prevent 70% of cervical cancers worldwide. There remain at least 13 high-risk HPV types not targeted by the current vaccines that account for the remaining cervical cancers (Chan et al., 2011). Cervical cancer can be prevented in two ways. Primary prevention, aimed at preventing or neutralizing the infection with human papillomavirus (HPV), as for example through prophylactic HPV vaccination, and secondary prevention, aimed at preventing precancerous lesions from progressing to invasive cancer through screening, early detection, diagnosis, and treatment, as clinically indicated (Ngan et al., 2011). Discovered 25 years ago, the transcription factor nuclear factor-kappa B, also known as NF-B is still one of the most studied proteins due to its pleiotropic functions and its involvement in inflammatory diseases and cancer (Ghosh and Hayden, 2008) NF-B directly regulates a wide range of genes that control the inflammatory and immune responses, programmed cell death, cell proliferation and differentiation (Chen and Greene, 2004). The important role played by NF-B proteins is illustrated not only by the diversity of genes that they regulate, but also by the large variety of stimuli that lead to their activation (Wan and Lenardo, 2010). In mammalian cells, five NF-kappaB family members are found: p65 (RelA), RelB, cRel, p50/p105 (NF-kappaB1) and p52/p100 (NF-kappaB2). These proteins share a unique N terminalRel homology domain (RHD) for forming hetero- or homodimer dimmers and binding DNA. Having a C-terminal transactivation domain (TAD) p65, RelB, and c-Rel function as transactivators when associated with p50 or p52, while p50 and p52 lack TADs, and their homodimers serve as transcription repressors that provide a threshold for NFkappaB activation (Hayden and Ghosh, 2004). NF-B transcription factors are assembled through dimerization of five subunits: RelA(p65), p50(NF-B1), p52(NF-B2), c-Rel and RelB. Each dimer is bound in the cytoplasm by an inhibitor-B (IB), which prevents its nuclear translocation (Perkins, 2007).

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NF-kappaB is a transcription factor that induces expression of more than 200 genes involved in diverse process such as cell survival, cell adhesion, inflammation, differentiation and growth ((Hayden and Ghosh, 2004). NF-kappaB is a positive mediator of cell growth and proliferation. NF-kappaB increases the expression of several factors involved cell cycle progression such as cyclins D and E. Upregulation of cyclin D1 expression by NF-kappaB is associated with enhanced transition from G1 to S phase. Furthermore, NF-kappaB negatively regulates expression of growth arrest and DNA damage-inducible protein 45 (GADD45), a cell cycle checkpoint protein that keeps cell at the G2/M phase transition (Chen et al., 2001). NF-kappaB plays a critical role in blocking apoptosis through various mechanisms, of which induction of antiapoptotic protein expression is regarded as the major one. The NF kappaB responsive anti-apoptotic genes include Bcl-XL, cIAP1, cIAP2, XIAP, A20, TRAF-2 and c- FLIP, which promote cell survival by desensitizing the cells to apoptosis induced by a variety of stimuli such as cytokines and chemotherapeutics. Second, NF-kappaB can suppress cellular stress-mediated apoptosis through removal of reactive oxygen species (ROS) via increasing expression of manganese superoxide dismutase (MnSOD) (Chen et al., 2011). The nuclear factor-B (NF-B) signaling pathway plays a major role in the development, maintenance, and progression of most chronic diseases. NF-B controls the expression of genes involved in a number of physiological responses, including immune inflammatory responses, acute-phase inflammatory responses, oxidative stress responses, cell adhesion, differentiation, and apoptosis. Recent studies have suggested that NF-B dis regulation is associated with many diseases including AIDS, atherosclerosis, asthma, arthritis, diabetes, inflammatory bowel disease, stroke, muscle wasting and viral infections (Gupta et al., 2010). Numerous lines of evidence from preclinical and clinical studies have shown that NFB and NF-B-mediated pro inflammatory cytokines play a central role in the progression of cancer related symptoms.1, 11, 48 50 For instance, administration of selective inhibitors of the NF-B pathway was shown to abrogate the expression of cytokines such as IL-1 and TNF- in animal models of chronic inflammation (Gupta et al., 2011).

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Nuclear factor (NF)-B, activated by IB kinase (IKK), is a key regulator of inflammation, innate immunity, and tissue integrity. NF-B and one of its main activators and transcriptional targets, tumor necrosis factor (TNF), are up-regulated in many inflammatory diseases that are accompanied by tissue destruction. NF-B activity is of particular importance for maintenance of epithelial Barriers, but it was also proposed that NF-B activation in epithelial cells can lead to production of inflammatory chemokines that recruit immune cells to the tissue, thereby initiating an inflammatory amplification cascade. The etiology of many inflammatory diseases is poorly understood, but often depends on genetic factors and environmental triggers that affect NF-B and related pathways (Guma et al., 2011). NF-B transcription factors activate expression of genes encoding cytokines, chemokines, adhesion molecules, matrix metalloproteinases, cyclooxygenase 2, inducible nitric oxide synthase, and enzymes and molecules with microbicidal activity (Li and Verma, 2002; Lawrence, 2009; Pasparakis, 2009). NF-B activation is tightly regulated mainly through its localization. In resting cells, NF-B proteins are kept in the cytoplasm in association with inhibitory IB proteins including IBa, IB, and IB (Ghosh and Karin, 2002) among which IBa is the most abundant. NFB signaling occurs through the canonical (classical) pathway initiated by NF-B1 (p50/p105) and a non-canonical (alternative) pathway initiated by NF-B2 (p52/p100). Before the active NF-B is translocated into the nucleus, NF-B1 and NF-B2 are cleaved to the active p50 and p52 subunits, respectively. While the classical pathway depends on IKK complex consisting of IKK, IKK, IKK and the inhibitory subunit IB, the alternative pathway depends on IKK homodimers and NF-B inducing kinase (NIK) (Dejardin, 2006). NF-B activation requires the phosphorylation, polyubiquitination, and subsequent degradation of its inhibitory subunit, IBa. Hence, inhibiting IBa phosphorylation ultimately inhibits NF-Bs transcriptional activity (Karin, 1999; Liu et al., 2000).

26

Fig: Classical and alternative NF-B activation pathways [Courtsey: Nishikori, 2005]

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One approach for inhibiting NF-B activation is to use small peptides that cross the cell membrane and block the nuclear translocation of the NF-B complex (Letoha et al., 2005). For example, SN50, a forty-one-residue synthetic peptide that contains a hydrophobic membrane-translocating region and the nuclear localization sequence of NF-B p50, can enter cells and compete with NF-B complexes for the machinery responsible for the nuclear translocation of NF-B. SN50 effectively inhibits the LPS- and TNF--induced nuclear translocation of NF-B in different cell lines (Koulich et al., 2001; Lin et al., 1995) and mitigates inflammatory responses in vivo (D'Acquisto et al., 2001). NF-B is aberrantly activated in tumor cells, contributing to the cells' advantage in survival and proliferation. The mechanism of NF-B activation in tumor cells is not well elucidated, but it is apparently complex and varies in different tumor types. Undoubtedly, understanding the mechanism of NF-B activation in tumor cells will facilitate development of means for cancer prevention and therapy (Lin et al., 2010). No bioactive compound discovered over the last 30 years has attracted more public attention than paclitaxel (Pujol et al., 2007). Paclitaxel is a complex taxane diterpene isolated from the Bark of Taxus brevifolia (Kovacs et al., 2007). The cytotoxic activity of the Bark extract was first reported in 1963, utilizing B cytotoxicity assay. Subsequently, Paclitaxels in vivo activity against mouse leukemia was discovered in1966 (Kingston, 2007), and its structure was described in 1971 (Wani et al., 1971). Paclitaxel (Taxol) is an anti-tumor agent that has proved to be effective against a number of cancers. Its primary mechanism of action in cells is to cause a mitotic block by stabilizing microtubules, thereby decreasing the dynamic nature of these cytoskeletal structures (Jordan, 2002). Prolonged exposure of mammalian cells to paclitaxel leads to apoptotic cell death (Woods et al., 1995). Microtubules are composed of the protein tubulin, a heterodimer consisting of - and -subunits. Paclitaxel binds to the -subunit of tubulin in a 1: 1 stoichiometry. It is essential that the binding interactions between the drug and tubulin be defined in detail so that rational drug design can be used to create more efficient paclitaxel derivatives (Foland et al., 2005).

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Paclitaxel resistance has been attributed to a variety of mechanisms, including upregulation of antiapoptotic Bcl-2 family members such as Bcl-2 and Bcl-XL (Blagosklonny et al., 1997); upregulation of membrane transporters such as mdr1, which increases drug efflux (Yu et al., 1998); point mutations in -tubulin residues or altered expression of tubulin isotypes, which impair drugtubulin binding (Sve et al., 2005); upregulation of ErbB2 (HER-2) by inhibition of cyclin-dependent kinase (CDK) 1, which causes delayed mitosis (Yu, 2001); and high expression of microtubule-associated protein tau mRNA, which decreases paclitaxel binding to microtubules and microtubule polymerization (Andre, 2007). Microtubule-targeting drugs inhibit the metaphase anaphase transition through suppressing spindle microtubule dynamics, which block mitosis and induce apoptosis (Jordan and Wilson, 2004). Microtubule stabilizing agents (MSA) is a class of these drugs that includes taxanes (paclitaxel and docetaxel), epothilones A and B, discodermolide, eleutherobin (Jordan, Wilson), and monastrol (Jiang et al., 2006; Cochran et al, 2005). These agents stabilize microtubules by binding to polymeric tubulin, thus preventing disassembly (Zhou et al., 2005; McGrogan et al., 2008). Paclitaxel causes polymerization and stabilization of microtubules in tumor cells, thereby inhibiting cell replication through disruption of normal mitotic spindle formation (Amos and Lowe, 1999). Therefore, cells treated with paclitaxel are unable to proceed normally through the cell cycle and arrest in G2/M phase (Mullan et al., 2001). This halt of the cell cycle at mitosis has been considered the cause of paclitaxel-induced cytotoxicity (Pieiro et al., 2007). Paclitaxel is a microtubule-stabilizing drug linked to the activation of numerous caspases, including caspase 3, 8 and 10 (Park et al., 2004; von Haefen et al., 2003). In these cases, apoptosis is blocked by broad-spectrum caspase inhibitors, and occurs in a 624 hour time period. In contrast the induction of apoptosis following cell cycle crisis is observed between 24 and 72 hours (Mielgo et al., 2009). Serious draw Backs hamper PTXs clinical usefulness. For instance, paclitaxel lacks selective cytotoxicity between cancer cells and normal cells, which frequently leads to serious

29

unwanted side effects. The poor water solubility of paclitaxel is another problem that significantly reduces its wider clinical application (Ndungu et al., 2010) Different strategies have been explored to circumvent these side effects. One of the most attractive tactics is the selective targeting of tumor cells over normal cells exemplified by employing suitable monoclonal antibodies (Ojima et al., 2002). There are a variety of mechanisms responsible for the development of chemoresistence in cancer cells; reduced drug uptake or increased drug efflux, increased repair of drug targets, alteration of drug targets that prevent drug binding or action and blocks in biochemical pathways that mediate drug-induced cytotoxicity (Kutuk and Letai, 2008). Paclitaxel is one of the most successful drugs for the treatment of cancer, but many patients show resistance to paclitaxel. Previous studies have identified several different mechanisms involved in paclitaxel resistance. Because the primary target of paclitaxel is microtubules, it is natural that molecules related to paclitaxel resistance are involved in microtubule-related mechanisms (Xu et al., 2012). Development of acquired paclitaxel-resistance has been attributed to such mechanisms, including overexpression of P-glycoprotein, alterations in tubulin composition and mutations in -tubulin (Sangrajrang and Fellous, 2000). Many chemotherapeutics, including paclitaxel, have been shown to exert their effect through engagement and activation of the intrinsic apoptotic pathway (Pommier et al., 2004; Makin and Dive, 2001). However, most studies of paclitaxel resistance have focused on events upstream of engagement of the intrinsic apoptotic pathway, either on drug interacting with target or entry of drug into cell. Paclitaxel binds to the N-terminal domain of -tubulin to initiate microtubule polymerization. Resistance to paclitaxel treatment was shown to be mediated by alterations in tubulin composition or mutations in the paclitaxel-binding region of -tubulin (Hari et al., 2006). Today, Paclitaxel is the No. 1 Anti-cancer Drug in the Battle of we humans against cancer. It constitutes about 22% of all major cancer chemotherapy drugs on the world market. In 2000. BMS reported its annual sales of Taxol (Paclitaxel Injection) was $1.592

30

billion - equal to excess $4.3 million per day. In January 2001. Ivax announced that $30 million of its generic Paclitaxel Injection (Onxol) has been sold in a 10-week period - in USA alone. And in the second quarter of 2001, Paclitaxel sales accounted for in excess of 25% of Ivax's total revenue (http://www.21cecpharm.com/px/). Curcumin (diferuloylmethane) is the chief component of the spice turmeric and is derived from the rhizome of the East Indian plant Curcuma longa. Curcuma longa is a member of the Zingiberacae (ginger) family of botanicals and is a perennial plant that is native to Southeast Asia (Chattopadhyay et al., 2004). Curcumin has been used extensively in Ayurvedic medicine for centuries, as it is nontoxic and has a variety of therapeutic properties including anti-oxidant, analgesic, antiinflammatory and antiseptic activity. More recently curcumin has been found to possess anticancer activities via its effect on a variety of biological pathways involved in mutagenesis, oncogene expression, cell cycle regulation, apoptosis, tumorigenesis and metastasis (Wilken et al., 2011) Turmeric contains a class of compounds known as the curcuminoids, comprised of curcumin, demethoxycurcumin and bisdemethoxycurcumin (Jurenka, 2009). Curcumin is the principal curcuminoid and comprises approximately 2-5% of turmeric; it is responsible for the yellow color of the spice as well as the majority of turmerics therapeutic effects (Chattopadhyay et al., 2004) Curcumin is a lipophilic polyphenol and thus is insoluble in water, but is readily soluble in organic solvents such as dimethylsulfoxide, acetone and ethanol (Aggarwal, 2007). The antioxidant activity of the curcuminoids comes by virtue of their chemical structure. The curcuminoids consist of two methoxylated phenols connected by two a, B unsaturated carbonyl groups that exist in a stable enol form (SreejayanRao, 1994). Curcumin has been shown to suppress the activation of NF-B, an inducible transcription factor that regulates the expression of a host of genes involved in inflammation, cellular proliferation and cell survival (Wilken et al., 2011).

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Curcumins inhibitory effect on the NF-B pathway is central to providing the compound with its anti-inflammatory properties. Curcumin blocks the IkK-mediated phosphorylation and degradation of IBa, thus NF-B remains bound to IBa in the cytoplasm and is not able to enter the nucleus to activate transcription (Wang et al., 2008). Curcumin has been shown to decrease the metabolism of arachadonic acid by down regulating the activity of LOX and COX-2, both at the transcriptional level as well as via post-translational enzyme inhibition (Rao, 2007). Curcumin has been shown to selectively induce apoptosis in tumor cells at the G2 phase via upregulation of p53 expression and initiation of the mitochondrial apoptotic pathway via increased Bax expression and cytochrome c release (Choudhari et al., 2005; Weir et al., 2007). Curcumin also has a stimulatory effect on the extrinsic apoptotic pathway, which is triggered by the binding of death activators such as TNF-a, and Fas Ligand to their corresponding cell surface receptors. Activation of these receptors results in activation of caspase-8 via the receptor-attached FADD adapter molecule and initiation of the caspase cascade (Lu et al., 2009). Curcumin has demonstrated an anti-angiogenic effect in vivo in xenograft models of various tumors including glioblastoma, hepatocelluar carcinoma, prostate and ovarian carcinomas (Yoysungnoen et al., 2008; Perry et al., 2010). Curcumin has been shown to regulate a variety of pro-angiogenic growth factors, enzymes and transcription factors including bFGF, VEGF, angiopoetin-1 and 2, COX-2, matrix metalloproteinase-9 (MMP-9), AP-1 and NF-B (Gururaj, 2002). Extensive research conducted within the past two decades has revealed that cancer is a result of the disregulation of multiple cell signaling pathways. Curcumin is a highly pleiotropic molecule that modulates numerous targets , including the activation of transcription factors (e.g., NF-jB, STAT3, AP-1, NRF-2, PPAR-c, and HIF-1), receptors (e.g., HER-2, IL-8, and CXCR-4), kinases (e.g., EGFR, ERK, JAK, and AAPK), cytokines (e.g., TNF, IL, MIP, and MCP), enzymes (e.g., MMP, iNOS, GST, and ATPase), and growth factors (e.g., EGF,NGF, HGF, and PDGF) (Anand et al, 2008).

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Curcumin was found to induce G0/G1 and/or G2/M phase cell cycle arrest, upregulate CDKIs, p21WAF1/CIP1, p27KIP1, and p53, and slightly down-regulate cyclin B1 and cdc2 in ECV304 cells (Park et al., 2002). The cyclin D1 proto-oncogene is an important regulator of G1 to S phase transition in numerous cell types from diverse tissues. A study has demonstrated that curcumin induces apoptosis in the G2 phase of the cell cycle in deregulated cyclin D1-expressing mammary epithelial carcinoma cells, leaving its normal cells unaffected (Chodhuri et al., 2005). Curcumin induces the degradation of cyclin E expression through the ubiquitindependent pathway and up-regulates cyclin-dependent kinase inhibitors p21 and p27 in multiple human tumor cell lines (Aggarwal et al., 2007). In human mantle cell lymphoma, curcumin causes cell cycle arrest at the G1/S phase of the cell cycle and induces apoptosis (Shishodia et al., 2005). Curcumin enhances the expression of tumor cyclin-dependent kinase (CDK) inhibitors p21 andp27 as well as tumor suppressor protein p53 but suppressed the expression of retinoblastoma protein and also induced the accumulation of cells in G1 phase of the cell cycle in multiple human tumor cell lines (Aggarwal et al., 2007). Paclitaxel activated NF-B in breast cancer cells and curcumin inhibited this activation through inhibition of IKK activation, IB phosphorylation, and IB degradation. Curcumin also suppressed paclitaxel-induced expression of antiapoptotic, proliferative, and metastatic proteins and enhanced apoptosis (Aggarwal et al., 2005). Paclitaxel activates NF-B in human breast cancer cells through a classic NF-B activation pathway consisting of IKK activation, IBa phosphorylation and degradation, and NF-Bregulated gene expression, including cyclin D1, MMP-9, and COX-2. Treatment of breast cancer cells with curcumin completely suppressed the paclitaxel inducedIKK activation, leading to suppression of NF-B activation. Curcumin also suppressed paclitaxelinduced cyclin D1, MMP-9, and COX-2 in breast cancer cells (Aggarwal et al., 2005).

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Cyclooxygenase (COX) or prostaglandin endoperoxide synthase (PGHS) is the key and rate-limiting enzyme that catalyses the initial step of arachidonic acid (AA) metabolic transformation into prostanoids (prostacycline, thromboxane, prostaglandins E2, D2, F2). Sir John Vane was the first to demonstrate in 1971 that non-steroid anti-inflammatory drugs (NSAIDs) block cyclooxygenase, then five years later; COX was isolated, and cloned in 1988 by three separate groups (Vane, 1971; Kajubu et al., 1991). The COX-2 gene for human is approximately 8.3 kilo Base long, consist of 10 exons and 9 introns and is located on chromosome 1 (Appleby et al, 1994). The COX-2 gene encodes for a homodimeric protein of 604 amino acids in size, which is highly similar in structure and enzymatic activity to COX-1 (Garavito et al, 2002). In contrast to COX-1, the COX-2 promoter is not basally active in most cell types, but can be strongly and rapidly induced by growth factors and pro-inflammatory mediators. COX-2 is undetectable in most normal tissues (except for the central nervous system, kidneys, and seminal vesicles) but is induced by various inflammatory and mitogenic stimuli. Growth factors (epidermal growth factor, platelet derived growth factor, pro-inflammatory cytokines ((IL) 1, IL2) and tumor necrosis factor, tumor promoters, bile acids and ultraviolet B irradiation are all stimulators of COX-2 expression(Chen et al, 2001;Dempke et al, 2001). The activation of carcinogens due to COX-2 activity is a first step before tumor initiation. The metabolism of arachidonic acid produces mutagens (e.g. a by-product of the oxidation of AA, malondialdehyde, is highly chemically reactive and form adducts with DNA) (Wiese et al, 2001). Tumor initiation induced by COX-2 over-expression was first demonstrated by the studies of Liu et al (Liu et al, 2001). Deregulation of COX-2 expression leads to an increased abundance of eicosanoids that affect the hallmarks of cancer. For examples, COX2/prostaglandin E2 signal is thought to protect tumor cells or tumor initiating cells from apoptosis induction by regulating pro- and/or anti-apoptotic molecules (Shimada et al, 2011). COX-2 promoter region contains multiple regulatory elements, such as nuclear factorB (NFB) binding site, nuclear factor interleukin-6(NF-IL6)/CCAAT/enhancer-binding

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protein(C/EBP) binding site, cyclic AMP-response element (CRE) and activation protein 1(AP-1). The regulation of COX-2 gene expression could involve complex interaction among them (Wang et al., 2006).

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4. MATERIALS AND METHODS

4.1. MATERIALS
HeLa cells are one of the oldest and most commonly used human cell lines. The cell line was derived from cervical cancer cells taken from Henrietta Lacks. Horizontal gene transfer from HPV 18 to human cervical cells created the HeLa cells genome which is different from either parent genome in various ways including its number of chromosomes. HeLa cells have a model chromosome number of 82 with 4 copies of chromosome 12 and 3 copies of chromosome 6,8,17. HeLa cells have been reported to contain HPV 18 sequences and P53 expression was reported to be low, and normal levels of pRB (Retinoblastoma suppresser) were found. HeLa cells were purchased from national center for cell science (NCCS), Pune. Curcumin, fetal bovine serum, mouse monoclonal antibody to actin, IB, and antimouse HRP were purchased from sigma. Dulbeccos Modified Eagles Medium (DMEM) was purchased from invitrogen. MTT was obtained from Amarsham (now GE healthcare). ECL pulse reagent was obtained from Millipore. Antibodies against XIAP, cIAP1, surviving were purchased from Santa Cruz Biotechnology. Prestained protein marker was purchased from New England Biolabs. Paclitaxel was purchased from Calbiochem. All other chemicals from sigma unless mentioned above.

4.2. EQUIPMENTS
4.2.1. Cell culturing T25 and T75 Culture Flasks (Nunc) Micropipettes (Eppendorf) Centrifuge tubes (Nunc) Cryovial (Tarsons) Centrifuge (Eppendorf) CO2incubator (Sanyo) Inverted microscope (Nicon) -20 Deep freezer (Evolution)

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-80 Deep freezer (Thermo) Liquid Nitrogen Container (ARPEGE 170) 4.2.2. Mtt assay 96 well Microtitre Plate (BD-Falcon) 60 mm Culture plates (BD-Falcon) ELISA Plate Reader (BIORAD) 4.2.3. Western blotting Western blotting apparatus (Bio-Rad mini PROTEIN III)

4.3. METHODS
4.3.1. Maintenance of the cells HeLa cells were cultured in Dulbeccos Modified Eagles Medium (Invitrogen) supplemented with 10 % fetal bovine serum (sigma) alone with 100 units/ML of penicillin, 50 g /mL of streptomycin. The cells were maintained at 370 C in humidified atmosphere of 3% CO2 and 95% air. These cells were observed every day and allowed to become 80% confluent. 4.3.2. Sub culturing of the Cells When the cells were grown to 80 % confluency, the medium was removed and the monolayer was washed once or twice with room temperature 1X PBS-EDTA. 0.25 % Trypsin-EDTA solution was added and incubated to 3-5 minutes at 370C for trypsinisation. Fresh medium (with serum) as added and cells were dispensed gently and transferred to small vials for centrifugation. After centrifugation the supernatant was removed, fresh medium was added and the number of cells /mL were counted using haemocytometer. A known number of cells were seeded to microtitre plates or petridishes for further experiments. 4.3.3. Drug treatment for cells Stock solutions of curcumin and paclitaxel were made in Dimethyl sulfoxide (DMSO), the concentration of which in media should not exceed 0.2 %. After 24 hours of seeding; the

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cells were treated with curcumin (5M) and/or paclitaxel (5nM). In combination treatments, curcumin (5M) was added 2h before adding paclitaxel (5nM). 4.3.4. Storage of cells For future use of cells they can be stored by freezing. For this some amount of cells were transferred along with the medium to freezing vials. After this the cells were pelleted by spinning the vials at 3000 rpm for 5-10 minutes. To the pelletized cells freezing mixture (10% DMSO and 90% serum) is added and mixed well. The vials are then transferred to -200C for 1-2 hours and then to 800C. It is stored for overnight and on the next day vials transferred to 1960C in liquid nitrogen for long term storage. 4.3.5. MTT Assay MTT Assay is a colorimetric assay for measuring cell viability, cellular proliferation and activation. It is also used to determine the cytotoxicity of potential medical agents and other toxic materials. MTT Assay was first described by Mosman in 1983. Yellow MTT 3-(4, 5 Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, is reduced to purple formazan in mitochondria of living cells. A solubilization solution is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at certain wave length (usually between 500 to 600 nm) by a spectrophotometer. The reduction takes place only when mitochondrial reductase enzyme is active and therefore conversion is directly related to number of viable cells. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, effectiveness of the agent is causing death of the cell can be reduced. 4.3.5.1. Procedure For the MTT Assay, 3000 cells were seeded per well in a 96 well plate and incubated for 24 hours at 370C. The cells were treated with 5 nM of paclitaxel 5 M of curcumin, and in a combination of both. Six wells were kept as control. i.e., cells in drug free medium to determine cells survival and the percentage of live cell after culture. The cells were then

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incubated for 24 hours at 370C. Then the medium was removed and after a PBS wash, 100 L of fresh media containing 20% MTT (95 mg /mL) was added to each well. The cells were incubated for 2 hours. The yellowish colored MTT was reduced to dark blue coloured formazan by the viable cells only. The cells were solubilized with 0.1 ml of lysis buffer (20% SDS in 50% Dimethyl formamide). The plates were then kept for one hour incubation at 370 C. The optical densities at 570 nm are measured using ELISA plate reader (Bio-Rad). To analyze the effect of curcumin in paclitaxel treated cells the cells were pretreated with curcumin (5M) for 2 hours and after 2 hours the medium was changed and treated with paclitaxel (5nM) in combination with curcumin 5mm for 24 h. after removing medium 100 ml of 20% MTT 5mg/ml was added to each well and incubated for 2 h. the cells are solubilize with .1 ml of lysis buffer 20 % SDS in 50 % dimethyl formamide. The plates were then kept for 1 hour incubation at 370 C. The optical densities were 570 nm was measured using ELISA plate reader. The relative cell viability in percentage was calculated by comparing the viability of the treated cells with that of the control and the cell survival was expressed as: Cell survival (%) = (Mean OD (Drug exposed cells) / Mean O.D (Control) 100 Graph was platted by taking percentage viability own Y axis and the concentration of drug on the X-axis. 4.3.6. Western blotting (Immunoblotting) The term refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Western blotting (also called immunoblotting because an antibody is used to detect its antigen) was introduced by Towbin et al in 1979 and is now a routine technique for protein analysis. The specificity of the antibody antigen interaction enables a single protein to be identified in the midst of a complex protein mixture. Western blotting is commonly used to positively identify a specific protein in a complex mixture and to obtain qualitative and semi quantitative data about the protein. The process of Western blotting was following steps:

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4.3.6.1. Cell / Tissue lysate preparation Samples may be taken from whole tissue or cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (small volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be a source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed by to encourage lysis of cells and to solubilize proteins. Protease and phosphatases inhibitors are often added to prevent the digestion of the samples by its own enzyme. 4.3.6.2. Gel electrophoresis The proteins of the samples are separated using the gel electrophoresis. Separations of protein are made by isoelectric point (pI), molecular weight, electric charge or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl Sulphate (SDS). SDS-PAGE maintains polypeptide in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure and thus allows separation of proteins by their molecular weight. Samples are loaded into wells in the gel. One lane is usually reserved for a marker or a ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible colored bands. An example of a ladder is the GE Full Range Molecular weight ladder. When voltage is applied along the gel, proteins migrate into at different speeds. These different rates of advancement in separate into bands within each lane. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension and according to their molecular weight in the second dimension.

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4.3.6.3. Protein transfer to a membrane Following electrophoresis, the protein must be transferred from the electrophoresis gel to a membrane. There are a variety of methods that have been used for this process, including the diffusion transfer, capillary transfer; heat accelerated conventional transfer, vacuum blotting transfer and electro elution. The transfer method that is used most commonly for proteins is electro elution or electrophoretic transfer, because of its speed and transfer efficiency. This method uses the electrophoretic mobility of protein to transfer them from the gel to the matrix. Electrophoretic transfer of proteins involves placing a protein containing polyacrylamide gel in direct contract with a piece of nitrocellulose or other suitable, protein binding support and sandwiching this between two electrodes submerged in a conducting solution. When an electric field is applied, the proteins move out of the polyacrylamide gel and onto the surface of the membrane, where the proteins become tightly attached. The resulting membrane is a copy of the protein pattern that was found in the polyacrylamide gel itself. The transfer efficiency can vary dramatically among proteins, based upon the ability of a protein to migrate out of the gel and its propensity to bind to the membrane under a particular set of conditions. The efficiency depends on factors such as the composition of the gel, complete contact of the gel with the membrane, the position of the electrodes, the transfer time, size and the composition of proteins, field strength and the presence of the detergents. Optimal transfer of proteins is generally obtained in low ionic strength buffers and with low electrical current. After transfer and before proceeding with the western blot, it is often desirable to stain all proteins on the membrane with a reversible stain to check the transfer efficiency. Ponceau S stain is the most widely used reagent for staining proteins on a membrane. However, it has limited sensitivity, does not photograph well, and fades with time. 4.3.6.4. Blocking nonspecific binding sites In a western blot, it is important to block the unreacted sites on the membrane to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins

41

have been used to block unreacted sites on the membrane. Individual blocking buffers are not compatible with every system. For this reason, a variety of blockers in both Tris buffered saline (TBS) and phosphate buffered saline (PBS) are available. The proper choice of blocker for a given blot depends on the antigen itself and on the type of the enzyme conjugate to be used. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding. 4.3.6.5. Primary and secondary antibodies The choice of primary antibody for western blot will depend on the antigen to be detected and what antibodies are available to that antigen. Alternatively a primary antibody may be made to recognize the antigen of interest. Both polyclonal and monoclonal antibodies work well for western blotting. Polyclonal antibodies are less expensive and less time consuming to produce and they often have a high affinity for the antigen. Monoclonal antibodies are valued for the specificity, purity and consistency that result in lower background. To obtain antibodies with the greatest specificity, they can be affinity purified using the immobilized antigen. A wide variety of labeled, secondary antibodies can be used for Western blot detection. The choice of secondary antibody depends upon the species of animal in which the primary antibody was raised (the host species). For example, if the primary antibody is a mouse monoclonal antibody then the secondary antibody must be an anti-mouse antibody obtained from a host other than mouse. The host species of the secondary antibody often will not affect the experiment. However, secondary antibody causes high background in a particular assay, another host species may be chosen. Another option to reduce background is to use a secondary antibody that has been pre-absorbed o serum proteins from other species. This pre adsorption process removes antibodies that have the potential to cross-react with serum proteins, including antibodies, from those species. Antibodies for western blotting are typically use as dilute solutions, ranging from 1/1001/500,000 dilutions beginning from a 1 mg/mL stock solution. The optimal dilution of a given antibody with a particular detection must be determined experimentally. More sensitive detection systems requires that less antibody be used, which can result in substantial saving on antibody costs and allow a limited

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supply of antibody to be stretched out over more experiments. It also produces a side benefit of reduced background because the limited amount of antibody shows increased specificity for the target with the highest affinity. Antibody dilutions are typically made in the wash buffer containing a blocking agent. The presence of small amount of blocking agent and detergent in the antibody diluents often helps to minimize background. 4.3.6.6. Washing Like other immunoassay procedures, Western blotting consists of series of incubations with different immunochemical reagents separated by wash steps. Washing steps are necessary to remove unbound reagents and reduce background, thereby increasing the signal: noise ratio. Insufficient washing will allow high background, while excessive washing may resulted in decreased sensitivity cause by elution of the antibody/ or antigen from the blot. As with other steps in performing a western blot, a variety of buffers may be used. Occasionally, washing is performed in a physiological buffer such as TBS or PBS without any additives. More commonly, a detergent such as 0.05 % Tween-20 is added to the buffer to help remove nonspecifically bound material. 4.3.6.7. Detection Detailed procedures for detection of a western blot vary widely. One common variation involves direct vs. indirect detection. With the direct detection method, the primary antibody that is used to detect an antigen on the blot is also labeled with an enzyme or fluorescent dye. This method is not widely used as most researchers prefer the indirect detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons. In the indirect detection method, a primary antibody is added first to bind to the antigen. This is followed by a labeled secondary antibody that is directed against the primary antibody. Labels include biotin, fluorescent probes such as fluorescein or rhodamine, and enzyme conjugates such as horseradish peroxidase or alkaline phosphatases. The indirect method offers many advantages over the direct method. The advantages of indirect methods are:

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Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. A wide variety of labeled secondary antibodies are available commercially. Since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection, it is versatile. Immunoreactivity of the primary antibody is not affected by labeling. Different visualization markers can be used with the same primary antibody. One common method of indirect method is Enhanced Chemiluminescence. Enhanced Chemiluminescence (ECL) is a common technique for a variety of detection

assays in biology. A horseradish peroxidase enzyme (HRP) is tethered to the molecule of interest (usually through labeling an immunoglobulin that specifically recognizes that molecule). This enzyme complexes then catalyzes the conversion of the ECL substrate into a sensitized reagent in the vicinity of the molecule of interest, which on further oxidation by hydrogen peroxide, produces a triplet (excited) carbonyl which emits light when it decays to the singlet carbonyl. Enhanced Chemiluminescence allows detection of minute quantities of a biomolecules. Protein can be detected down to femtomole quantities well below the detection limit for most assay systems. ECL western blotting is based on the oxidation of the cyclic Diacylhydrazide, luminal. ECL plus utilizes new technology developed by Lumigen Inc. based on the enzymatic generation of an acridium ester, which produces a more intense light emission of longer duration. Combined HRP and peroxide catalyzed oxidation of the Lumigen PS-3 Acridan substrate generate thousands of acridinium intermediates per minute. These intermediates react with peroxide under slight alkaline conditions to produce a sustained, high intensity chemiluminescence with maximum emission at a wavelength of 430 nm. The resulting light is detected on autoradiography film or CCD cameras.

4.4. STEPS FOLLOWED

4.4.1. Cell lysate preparation 1.6 106cells were seeded in 60 mm plates and incubated. After treatment with paclitaxel and/or curcumin as needed for each experiment, the medium was removed and scraped using 1X PBS. Centrifuged for 2 minutes at 13,000 rpm at 4C and to the obtained

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pellet added 150 L of WCL solution. Protease inhibitors are often added in WCL to prevent digestion of the sample proteins by its own enzymes. Mix well and keep in ice for 30 minutes with intermittent quick vortexing after every 5 minutes. Spin down at 13,000 rpm for 10 minutes and obtain the supernatant. Transfer the supernatant to a fresh tube. To this 5X loading dye and boil for 5 minutes and store at -70 C. The tube with the pellet should be kept for protein estimation. 4.4.2. Protein estimation The amount of protein was estimated by Bradford method. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. The degree of binding to different proteins varies with the number of basic amino acids in the protein. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-protein complex solution is constant over a 10-fold concentration range. Within the linear range of the assay (~5-25 g/mL), the more protein present, the more Coomassie binds. The protein concentration of a test sample is determined by comparison to that of a series of protein standards known to reproducibly exhibit a linear absorbance profile in this assay. Protein estimation was done to provide equal loading of the sample protein for gel electrophoresis. 2.5l of the protein sample was mixed with 122.5l of distilled water and 50l from this solution was transferred to corresponding two wells of the microtitre plates. Then the 200l of Bradfords reagent was added the reading was taken at 570nm. The concentration of protein (g) per l X can be calculated as: X= (Protein average Blank average) x 6 80g/well of protein sample was loaded on a gel 10% gel. 4.4.3. Gel electrophoresis The proteins of the samples were separated using SDS-PAGE. Polyacrylamide gel electrophoresis (PAGE) is probably the most common analytical technique used to separate and characterize proteins. A solution of acrylamide and bisacrylamide is polymerized. Acrylamide alone forms linear polymers. The bisacrylamide introduces

45

crosslinks between polyacrylamide chains. The 'pore size' is determined by the ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. A high ratio of bisacrylamide to acrylamide and a high acrylamide concentration cause low electrophoretic mobility. Polymerization of acrylamide and bisacrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form free radicals. TEMED, a free radical stabilizer, is generally included to promote polymerization. Comparative analysis of multiple samples is accomplished using one-dimensional (1D) electrophoresis. Gel sizes range from 2 x 3 cm (tiny) to 15 x 18 cm (large format). The most popular size (8 x 10 cm) is usually referred to as a "mini-gel". Small gels require less time and reagents than their larger counterparts and are suited for rapid screening. However, larger gels provide better resolution and are needed for separating similar proteins or a large number of proteins. Samples are added to sample wells at the top of the gel. When the electrical current is applied, the proteins move down through the gel matrix, creating what are called "lanes" of protein "bands". Samples that are loaded in adjacent wells and electrophoresed together are easily compared to each other after staining or other detection step. The intensity of staining and "thickness" of protein bands are indicative of their relative abundance. The position (height) of bands within their respective lanes indicates their relative sizes (and/or other factors affecting their rate of migration through the gel). Protein samples prepared for SDS-PAGE analysis are denatured by heating in the presence of a sample buffer containing 1% SDS with or without a reducing agent such as 20 mM DTT, 2-mercaptoethanol (BME) or TCEP. The protein sample is mixed with the sample buffer and boiled for 3-5 minutes, then cooled to room temperature before it is pipetted into the sample well of a gel. Loading buffers also contain glycerol so that they are heavier than water and sink neatly to the bottom of the buffer-submerged well when added to a gel. If a suitable, negatively charged, low-molecular weight dye is also included in the sample buffer, it will migrate at the buffer-front, enabling one to monitor the progress of electrophoresis. The most common tracking dye for sample loading buffers is bromophenol blue. To assess the relative molecular weight of a protein on a gel, protein molecular weight markers are run in the outer lanes of the gel for comparison. A standard curve can be constructed from the distances migrated by each marker protein. The distance migrated by the

46

unknown protein is then plotted, and the molecular weight is extrapolated from the standard curve. SDS PAGE separates proteins based on their molecular weight. Sample proteins become covered in the negatively charged SDS and move towards tha anode through the acrylamide mesh of the gel. Small proteins migrate faster through this mesh and proteins are thus separated according to their size (kDa). From the lysate obtained 80g/well of protein sample was loaded on to a 10% SDS PAGE gel. When the voltage of 40 mA was applied along with them, proteins migrated at different speed. The different rates of different electrical mobilities separated proteins into band within each lane formed under the wells. One lane reserved for the protein marker which is a mixture of proteins having defined molecular weight, typically stained so as to form visible colored bands. 4.4.4. Transfer The resolved bands in the SDS PAGE were electro blotted on to a PVDF membrane as described by Towbin. In order to make the proteins accessible to antibody detection they were moved from gel on to a membrane made of nitrocellulose or PVDF. PVDF membranes need to be pretreated as it is hydrophobic in nature. PVDF membrane was pre-treated using methanol for 10 seconds followed by 5 minutes treatment with distilled water, then followed by Towins buffer. After electrophoresis, both the geland PVDF membrane of s ame dimension was equilibrated using Towbins buffer. The proteins bands were transferred to on to the PVDF membrane (Hybond-P, Amersham) using a Bio-Rad mini PROTEAN III wet blot apparatus at 40 V overnight in cold room.

47

The method for transferring the protein is called electro blotting. Electrophoretic transfer of proteins involves placing a protein containing polyacrylamide gel in direct contact with a piece of PVDF or other suitable, protein binding support and sandwiching this between two electrodes submerged in a conducting solution. When an electric field is applied, the protein moves out of the polyacrylamide gel and on to the surface of the membrane were the proteins become tightly attached. Protein binding was based upon the hydrophobic interactions as well as charged interaction between membrane and proteins. After the transfer process, the membrane was stained with Ponceau stain to find if the transfer has taken place completely. The stain was later washed off completely using TBS-T. 4.4.5. Ponceau staining The uniformity and all over effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie or Ponceau S dyes. Ponceau S is the more common of the two; due to ponceau Ss higher sensit ivity and its water solubility make it easier to subsequently destain. It can be easily reversed with water washes, facilitating subsequent immunological detection. After transferring the proteins, place the membrane in an incubation tray (proteins staining up). Enough amount of Ponceau Staining was added to cover the membrane and incubated at least 30 seconds with gentle agitation. Then the membrane was rinsed with running distilled water until the background is clear. The membrane was distained with running distilled water for 2-3 minutes. The membrane was then placed in blocking solution. 4.4.6. Storage Membrane blots can be dried and stored at 40C for use at a later date. Rewet the membrane by replacing it in small volume of 100% methanol for 1-2 seconds and it was placed in a large volume of deionized water to remove the methanol. If exposure to methanol is to be avoided, the blot can be taken directly to protein blocking and then air dried. This will coat the membrane with hydrophilic protein and allows for rewetting in antibody incubation solution. When stored dry, blots will remain stable up to one year.

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4.4.7. Blocking Since the membrane has high affinity for protein, both the antibodies are the target are proteins, steps had to be taken to prevent interactions between the antibodies used in for the detection and the membrane. Blocking of nonspecific binding was achieved by placing the membrane in a dilute solution of protein typically BSA or nonfat dry milk (5%) for 1 hour with a minute percentage of detergent such as Tween 20. The proteins in the dilute solution attach to the membrane, where the sample protein had not attached during the process of transfer. Thus when the antibody was added, no place will be available on the membrane for the antibody to attach other than on the binding sites of the specific target proteins. This reduced noise in the final product of the western blot, leading to the clearer results, and eliminated false positives. 4.4.8. Washing The blot that is blocked is washed with large volume of TBS-T buffer in order to remove excess milk contents and unbound proteins. 4.4.9. Addition of primary antibody After blocking, a dilute solution of primary antibody against IB, -actin and cox-2 (generally between 0.5 and 5mg/ml; the concentration varies from 1:1000 and 1:5000) were added and incubated with the membrane under gentle agitation. Typically the solution was comprised of Tris Buffered Saline (TBS) solution with a small percentage of detergent like Tween 20 and sometimes powdered milk or BSA. The antibody solution and blot could be sealed and incubated together for overnight in cold room in order to protect antibodies. When antibody is prepared in BSA the life time of the antibody will be increased and hence primary antibody is usually prepared in BSA. Usually the membrane is incubated overnight. 4.4.10. Washing The primary antibody solution was poured off and then washed quickly with large volumes of TBS-T buffer.

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4.4.11. Addition of secondary antibody After removing the primary antibody the blot was washed thrice with TBS-T to remove unbound primary antibody and then was exposed to secondary antibody (anti-mouse HRP; dilute 1:3000), directed at the species specific portion of the primary antibody. This is known as secondary antibody. The secondary antibody is usually linked to biotin or to a reporter enzyme such as Alkaline Phosphatases or Horse Radish Peroxidase. It is usually made in 5% fat free milk solution. The membrane is incubated with the secondary antibody for three hours with gentle agitation in cold room or incubated in room temperature for 1 hour. 4.4.12. Washing The secondary antibody solution was poured off and then the membrane was washed three times each with TBS-T and once for 5 minutes with larger volume of TBS-T. It is a critical step. All wash steps are critical for reducing general background signal and nonspecific binding to discrete bands. If high back ground is a problem, the number, length and composition of the wash can be increased.

4.4.13. Detection by chemiluminescence The unbound secondary antibody was washed off with TBS-T and detected by Enhanced Chemiluminescence (ECL) method (Amersham ECL Plus kit). Chemiluminescent detection methods depend on the incubation of the western blot with a substrate that will luminesce when exposed to the reporter enzyme on the secondary antibody. First the ECL solutions A and B are mixed in a ratio of 1:1. After mixing, the solution was poured over the drained blot. Incubate it for 5 minutes. After this blot is transformed to a SaranwrapTM and the blot was wrapped within this. Then the wrapped blot was placed in an X-ray film cassette with the protein side facing up. An X-ray film is placed over the blot and the cassette is closed so that the film doesnt get exposed to light. The luminescence observed was transferred to X ray film in the dark and the film was developed and then visualized. ECL detection was considered to be the most sensitive detection methods for blotting analysis.

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5. RESULT
The objective of this study was to investigate the role of NF-B in the synergistic effect of paclitaxel and curcumin and also its role in paclitaxel induced up-regulation of cox-2 in cervical cancer cell line HeLa.

5.1. CLONE SELECTION

To investigate the effect of inactivation of NF-B in the synergism of paclitaxel and curcumin, stable clones of HeLa-Neo and HeLa IB-DM, which were developed earlier in the lab by G418 selection, were used. Expression levels of IB was analyzed and detected by Western blot. The whole cell lysate of clones transfected with pcDNA3 -IB-DM vector and that of pc DNA3 empty vectors were prepared. After western blotting, the membrane was probed with the antibody against IB. Results obtained from the blot illustrate that clone 2 has maximum expression. So clone 2 was selected for further studies. 5.2. CELL VIABILITY ASSAY To study the role of NF-B in the synergism of paclitaxel and curcumin in the cervical cancer cell line HeLa both HeLa Neo and HeLa-IB DM cells were treated with paclitaxel (5nm), curcumin (5M) either alone or in combination. The relative cell viability was determined after 72 hours of treatment by MTT assay, which is a very convenient method for assessing drug sensitivity. The results showed that the synergistic effect of paclitaxel and curcumin was inhibited in HeLa-IB DM cells and the NF-B inactivation itself sensitized the cells to the paclitaxel. 5.2.1. Regulatory role of NF-B on the synergistic effect of paclitaxel and curcumin To confirm the role of NF-B on the synergistic effect of paclitaxel and curcumin, we inactivated NF-B by stably transfecting HeLa cells with pcDNA3-IB, a double mutant of IB which lacks the serine residues that are essential for IB phosphorylati on and NF-B activation. HeLa cells transfected with empty vector pcDNA3 (HeLa-Neo) were used as control. HeLa-Neo and HeLa-IB-DM cells were treated with curcumin and /or paclitaxel as described in Materials and Methods and the cells were incubated for 24 hours and the cell viability was determined by MTT assay. It is observed that in HeLa-Neo cells, there is a

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significant reduction in the relative cell viability when curcumin and paclitaxel treatment in combination compared to the treatment with paclitaxel alone. But in HeLa-IB-DM cells there was no such synergistic effect was observed. The treatment of curcumin alone can produce no significant result in both cells. It is clear that the inactivation of NF-B in these cells inhibited the synergistic effect of curcumin and paclitaxel. Therefore it can be assumed that curcumin potentiates taxol induced apoptosis through the down regulation of NF-B. However, IB transfection itself sensitized the cells to taxol induced apoptosis. Therefore it is evident that NF-B plays a major role in the development of chemoresistence HeLa cells against paclitaxel.

5.3. WESTERN BLOT

Paclitaxel induced up-regulation of cox-2 in HeLa cells is evident from previous studies. Inorder to examine NF-Bs role in the upregulation process, both HeLa-Neo and HeLa-IB-DM cells were treated with paclitaxel. Untreated cells were used as control and actin was used as the loading control. The whole cell lysate from the control and treated cells of HeLa-Neo-IB-DM cells were obtained and immunobloting was performed. The immunoblot result showed an expression of cox-2 in paclitaxel treated HeLa-Neo cells, whereas in paclitaxel treated NF-B inactivated cells (HeLa-Neo-IB-DM), the cox-2 over expression was completely inhibited. This suggest that by inactivating NF-B, the cox-2 expression was completely inhibited in HeLa, which is a key enzyme in the conversion of arachodonic acid to prostaglandins which has been shown to have particular importance in the progression of several malignancies. 5.3.1. Regulatory role of Nf-B in the expression of Cox2 in response to paclitaxel treatment in HeLa cells The expression level of Cox2 proteins in response to paclitaxel was analyzed by using western blot. There was an increase in the expression level of Cox2 in response to paclitaxel in HeLa-Neo cells. These results showed that paclitaxel cause an increase in the expression of cox2 and this may lead to chemoresistence.

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In HeLa-IB-Dm cells there were no expression of Cox2 in response to paclitaxel. This indicates that Nf-B plays a major role in the paclitaxel induced upregulation of Cox2. The inactivation of Nf-B prevents the paclitaxel induced upregulation of Cox2.

CLONE SELECTION

Expression level of IB in HeLa-IB-DM clones as assessed by Western blotting

CELL VIABILITY ASSAY (MTT)

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Synergistic Effect of Paclitaxel and Curcumin was assessed using MTT Assay.HeLa Neo and HeLa-IB-DM cells were subjected to Paclitaxel and Curcumin treatment individually and or in Combination and incubated for72 hours. Relative cell viability was observed. Paclitaxel (5nM) Curcumin (5M) 92 90 Paclitaxel (5nM)+Curcumin (5M) 67 64

HeLa Neo HeLa IB DM

93 64

HeLa-IB-DM

Effect of Nf-B on the paclitaxel induced upregulation of Cox 2 The HeLa-IB-DM cells were treated with 5nM of Paclitaxel for 24 hours and the Whole cell lysates prepared were western blot against Cox2 specific antibody. -actin (43kDa) was used as loading control.

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6. DISCUSSION
Tumor cells often escape apoptosis by over-expressing anti-apoptotic proteins, which give them survival advantage. Some conventional chemotherapeutic drugs in low concentration cause up-regulation of survival signals, thereby necessitating increment of the effective dose of treatment. Several studies have shown that paclitaxel activates NF-B which has critical role in regulating cell survival, proliferation, invasion, and metastasis (Huang and Fan, 2000; Bava et al., 2011; Mabuchi et al., 2002). On these contrary, curcumin, the natural polyphenolic compound promotes apoptosis by interfering in various survival signaling pathways including NF-B (Anto et al., 2002; Aggarwal et al., 2006). Earlier findings (Bava et al., 2011) have clearly shown that paclitaxel induced activation of NF-B in HeLa cells involves phosphorylation of IKK and its down regulation by curcumin, which contributes to the sensitization of HeLa cells to the paclitaxel-induced apoptosis, involves inhibition of the same. Early reports have shown that, curcumin inhibits NF-B activation induced by various agents (Anto et al., 2000). In the current study, we observed a complete inhibition of the synergism of paclitaxel and curcumin upon activation of NF-B suggesting that NF-B plays a major regulatory role in the sensitization of paclitaxel treated tumor cells by curcumin and hence on their synergistic effect. These results demonstrate that curcumin potentiates Taxol induced apoptosis through the disruption of cell survival signaling pathways involving NF-B in HeLa cells. Accumulating evidence also suggest that curcumin is a compound that possess potent antiproliferative, antiangiogenic, antimetastatic and proapoptotic properties in vitro as well as in vivo (Anto et al., 2002; Li et al., 2000 ; Aggarwal et al., 2006). So, in this study, to determine the effect of NF-B activation in the synergism of paclitaxel and curcumin, HeLaNeo & HeLa-IB DM cells were taken. They were treated with paclitaxel and curcumin and curcumin and untreated cells kept as control. The result observed showed that the relative cell viability decreased to a greater range in HeLa-Neo cells treated with both paclitaxel (5nm) & curcumin (5m), showing the synergistic effect. But in HeLa-IB-DM cells, the activation of NF-B sensitized the cells to paclitaxel but the synergism was not obtained.

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The major disadvantage of Taxol chemotherapy is that tumors tend to acquire chemoresistence against Taxol. Taxol has been implicated in regulating targeted cellular proteins that promote cell survival and block apoptosis. Another suggestive mechanism involves the development of chemoresistence by cytotoxic drugs involves elevated levels of phosphorylated PKB/Akt. We have observed that elevated levels IB transfection itself sensitized the cells to taxol induced apoptosis. This indicates that NF-B plays a major role in the development of chemoresistence in HeLa cells against paclitaxel. NF-B signaling pathways has been considered as a highly attractive target for the development of chemotherapeutic drug and several compounds which inhibit this pathway are currently under preclinical or clinical trials. Several of the general inhibitors of NF-B activation, some inhibit target specific steps and others target multiple steps in NF-B pathway, however, none of them has been approved for human use because; most them lack specificity and thus interfere with the physiological role of NF-B in maintaining cellular homeostasis. Hence the most important challenge is to develop NF-B inhibitors based on their ability to specifically inhibit pathways leading to carcinogenesis, so that the risk of undesired side effects can be avoided. Paclitaxel is a very effective chemotherapeutic drug which acts mainly through induction of tubulin polymerization and cell cycle arrest. However studies have shown that paclitaxel induce apoptotic cell death also via pathway independent of mitotic arrest (Huang et al., 2000). Since lower concentration of paclitaxel leads to the concomitant activation of the survival signals, if fails to induce apoptosis at these concentrations necessitating a higher concentration of the drug of this purpose, which in turn becomes the main reason for its toxicity. Hence compounds like curcumin, which can counteract these survival signals can be therapeutic benefit when used in combination with paclitaxel. Taken together, the results from the present study denote that curcumin mediated sensitization of cervical cancer cells to paclitaxel is dependent on NF-B. Results indicate that NF-B is the central player in the synergism of paclitaxel and curcumin.

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7. SUMMARY AND CONCLUSIONs

Paclitaxel is a microtubule targeted agent widely used in cancer therapy. Recent studies showed that paclitaxel is able to induce early reactive oxygen species (ROS) production and hydrogen peroxide (H2O2) in cancer cells and involved in paclitaxel-induced cancer cell death in vitro and in vivo. Its high cost and dose limiting toxicity are its major disadvantages. The sensitivity of cells to chemotherapeutic drug-induced apoptosis seems to depend on the balance between proapoptotic and antiapoptotic signals. The invitro and invivo studies reported till date have shown that the anti-tumor effects of chemotherapeutic agents could be enhanced by combination treatment with chemopreventive agents. It has been proposed that activation of NF-B induce the expression of genes that counteract apoptotic signals and prevent cell death. Activation of NF-B has been observed in many cancers, including but not limited to breast cancer, melanoma, lung cancer, colon cancer, multiple myeloma, pancreatic cancer, esophageal adenocarcinoma, and various types of leukemia and lymphoma. Curcumin (diferuloyl methane), the major yellow pigment from the rhizomes of turmeric (Curcuma longa Linn), has anticancer properties. Curcumin also down regulated the expression of cox-2, a gene regulated by NF-B. Our aim was to determine the regulatory role of NF-B in the synergism of paclitaxel and curcumin in HeLa cells and also its role in paclitaxel-induced up-regulation of cyclooxygenase-2. The results showed that inactivation of NF-B has inhibit the synergistic effect of paclitaxel and curcumin in HeLa cell line completely and inactivation itself suppressed the paclitaxel-induced upregulation of NF-B activation and NF-B regulated gene expression cox-2 in cervical cancer cell line HeLa. The present study concludes that, NF-B plays a major role in regulating the synergistic effect of paclitaxel and curcumin in cervical cancer cells. Paclitaxel induces upregulation of cox2 in the cervical cancer cell line HeLa. The paclitaxel induced upregulation of cox2 is through an NF-B mediated signaling pathway in HeLa cells.

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9. INTERNET REFERENCES

1. http://www.21cecpharm.com/px/ 2. http://www.avaplant.com 3. http://www.wildflowerfinder.org 4. http://www.curcuminresearch.org/

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10. APPENDIX
REAGENTS FOR EXPERIMENT
DULBECCOS MODIFIED EAGLES MEDIUM (DMEM) REAGENTS DMEM SODIUM BICARBONATE HEPES STREPTOMYCIN SULPHATE PENCILIN DISTILLED WATER pH AMOUNT 13.37g 3.75g 5.95g 100mg 500l 1000ml 7.4

PBS-EDTA REAGENTS NaCl KCl NaHPO4 KH2PO4 EDTA Distilled Water pH AMOUNT 8g 0.2g 1.44g 0.24g 0.2g 1000ml 7.4

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0.25% TRYPSIN-EDTA REAGENTS Trypsin Glucose PBS-EDTA AMOUNT 0.7g 0.07g 350ml

10 X PBS REAGENTS NaCl KCl Na2PO4 KH2PO4 Distilled Water pH AMOUNT 8g 0.2g 1.44g 0.2g 1000ml 7.4

CURCUMIN STOCK PREPARATION (20mM) REAGENTS Curcumin DMSO AMOUNT 7.368g 1ml

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TAXOL STOCK PREPARATION REAGENT Taxol DMSO AMOUNT 10g 1ml

MTT ASSAY 1. LYSIS BUFFER 20%SDS dissolved in 50%Dimethyl Formamide MTT 250mg MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) in 50ml 1 X PBS.

2.

WHOLE CELL LYSATE REAGENT Tris pH 7.4 NaCl 5M EDTA 0.5M Triton 10% Dithiothreitol (DTT) 0.1M Aprotinin Leupeptin Phenyl methyl sulphonyl fluoride (PMSF) Sodium Vanadate (Na3VO4) 1M Distilled water AMOUNT 20l 50 l 4 l 10 l 10 l 5 l 5 l 5 l 4 l 916 l

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BRADFORDS REAGENT REAGENT Coomassive Brilliant Blue 95% Ethanol 85% Phosphoric acid Distilled water AMOUNT 10mg 5ml 10ml 85ml

SDS-PAGE COMPOSITION OF LOWER GEL REAGENT Distilled Water 30% Acrylamide Lower Tris (pH 8.8) 10% SDS 10% APS TEMED AMOUNT 4.6ml 2.7ml 2.5ml 100l 100 l 6 l

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COMPOSITION OF UPPER GEL (STACKING GEL) REAGENT Distilled Water 30% Acrylamide Upper Tris (pH 6.8) 10% SDS 10% APS TEMED AMOUNT 1.7ml 415 l 315 l 25 l 25 l 2.5 l

WESTERN BLOTTING 5 X LOADING REAGENTS Distilled Water 0.5M Tris HCl (pH 6.8) Glycerol 10% SDS -Mercaptoethanol 0.005% Bromophenol Blue AMOUNT 4ml 1ml 0.8ml 1.6ml 0.4 ml 0.2 ml

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8 X ELECTRO BUFFER REAGENTS 0.1M Tris Glycine Distilled Water pH AMOUNT 12g 57.6g 500ml 8.3

1 X ELECTRO BUFFER REAGENTS 8 X ELECTRO BUFFER 10% SDS Distilled Water AMOUNT 40ml 4 ml 456 ml

RUNNING BUFFER To 1X Electrode buffer

TRANSFER BUFFER/ TOWBINS BUFFER REAGENTS Tris Glysine Methanol Distilled Water AMOUNT 1.51g 7.2g 100ml 500ml

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TBS (10 X) REAGENT Tris Base NaCl Distilled water pH AMOUNT 12.1g 40g 500ml 7.6

TBS-T REAGENTS 1X TBS Tween 20 AMOUNT 500ml 2ml

PONCEAUS STAINING REAGENT REAGENT Ponceau Acetic Acid Distilled Water AMOUNT 0.2% w/v 10% w/v 100 ml

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BLOCKING SOLUTION REAGENTS Milk Powder TBST AMOUNT 1.5g 30ml

PREPARATION OF 3% BOVINE SERUM ALBUMIN (BSA) REAGENT BSA TBS-T AMOUNT 450mg 15ml

PREPARATION OF PRIMARY ANTIBODY (1: 1000) REAGENT Anti-COX2 3% BSA AMOUNT 15 l 15ml

PREPARATION OF SECONDARY ANTIBODY (1: 5000) REAGENT Antimouse HRP 5% non fat milk solution AMOUNT 7.5 l 15ml

PREPARATION OF PRIMARY ANTIBODY (1: 1000) REAGENT Anti-Cyclin D1 3% BSA AMOUNT 15 l 15ml

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PREPARATION OF SECONDARY ANTIBODY (1: 5000) REAGENTS Antirabbit HRP 5% non fat milk solution AMOUNT 7.5 l 15ml

PREPARATION OF PRIMARY ANTIBODY (1: 1000) REAGENTS Anti- actin 3% BSA AMOUNT 15 l 15ml

PREPARATION OF SECONDARY ANTIBODY (1: 5000) REAGENTS Antimouse HRP 5% non fat milk solution AMOUNT 7.5 l 15 ml

PREPARATION OF PRIMARY ANTIBODY (1: 1000) REAGENT Anti-IB 3% BSA AMOUNT 15 l 15ml

PREPARATION OF SECONDARY ANTIBODY (1: 5000) REAGENT Antimouse HRP 5% non fat milk solution AMOUNT 7.5 l 15ml