Med Oncol (2011) 28:385390 DOI 10.

1007/s12032-010-9447-x

ORIGINAL PAPER

Suppression of Na+/H+ exchanger 1 by RNA interference or amiloride inhibits human hepatoma cell line SMMC-7721 cell invasion
Xuekang Yang Desheng Wang Wei Dong Zhenshun Song Kefeng Dou

Received: 10 December 2009 / Accepted: 1 February 2010 / Published online: 12 February 2010 Springer Science+Business Media, LLC 2010

Abstract Na?/H? exchanger 1 (NHE1), a primary regulator of intracellular pH (pHi) and extracellular pH (pHe), plays a signicant role in acidifying the tumor microenvironment, possibly resulting in their malignant potential. However, currently, very little is known about the roles of NHE1 in invasion of hepatocellular carcinoma (HCC) cells. We have recently shown that NHE1 is over-expressed in HCC tissues and that this increased expression is associated with HCC invasiveness. In this study, we also found that NHE1 is over-expressed in HCC cell lines. Subsequently, we silenced NHE1 expression in the human HCC cell line SMMC-7721 using RNA interference (RNAi) and examined the invasiveness and proliferation of NHE1-silenced SMMC-7721 cells and the matrix metalloproteinase-2 (MMP-2) activity. The knockdown of NHE1 expression signicantly inhibited the invasive ability of SMMC-7721 cells but had only a minor effect on the cellular proliferation rate. Moreover, NHE1 knockdown signicantly reduced the secretion of MMP-2. Further experiment using amiloride (an inhibitor of NHE1) conrmed the above result. Together, these ndings indicate that NHE1 has an important role in SMMC-7721 cell invasion and that NHE1 might be a new target of HCC treatment.

Keywords Hepatocellular carcinoma Tumor microenvironment Na?/H? exchanger 1 RNA interference Invasiveness

Introduction Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide and is currently the second leading cause of cancer death among men in China [1]. Now it is also increasing in United States and Europe [2, 3]. Despite diverse para-operative treatment and the clinical progress achieved in recent years, the overall outcome of HCC remains very poor, which is largely the result of a high ratio of recurrence or metastasis after operation [47]. Although much is known about the development and causes of HCC, there is no effective therapy for the vast majority of patients with HCC [8, 9]. Therefore, the development of novel therapeutic agents targeting the malignant behavior of HCC cells, especially their invasiveness, is important to improve the prognosis of patients. NHE1 is a ubiquitously expressed member of the Na?/H? exchanger family that catalyzes the extrusion of intracellular proton (H?) ions in exchange for extracellular sodium (Na?) ions [1012], thereby regulating intracellular pH (pHi) and extracellular pH (pHe). Studies have reported that NHE1 plays a signicant role in acidifying the tumor microenvironment, possibly resulting in their malignant potential [13, 14]. Other studies in human melanoma cells have demonstrated that NHE1 is involved in tumor cell migration [15]. In our previous study, we found that NHE1 is highly expressed in human HCC tumors compared with normal liver tissues and that the NHE1 expression level in human HCC is correlated with the level of HCC invasiveness. Therefore, NHE1 may play a key role in HCC

Xuekang Yang and Desheng Wang contributed equally to this work and should be considered as co-rst authors. X. Yang D. Wang W. Dong Z. Song K. Dou (&) Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xian, Shanxi Province 710032, Peoples Republic of China e-mail: doukefeng2009@yahoo.cn

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metastasis. However, little is known about the detailed molecular mechanisms of NHE1-mediated HCC cell invasion. In the present study, RNA interference (RNAi) and amiloride (an inhibitor of NHE1) were used to inhibit the NHE1 gene in HCC cells, and the effects mediated by deleting NHE1 gene were examined. Furthermore, the potential downstream molecular mechanisms of NHE1mediated invasion were investigated.

Materials and methods Cell lines and culture BEL7402, SMMC-7721, HepG2, HHCC, and Chang liver (transformed liver cell line) were obtained from American Type Culture Collection (Manassas, VA) and were cultivated in RPMI-1640 medium supplemented with 10% fetal calf serum (Sigma Chemical Co, St Louis, MO). Cells were maintained at 37C in a humidied chamber with 95% air and 5% CO2. Quantitative real-time RTPCR Total RNA was extracted using Trizol solution (Invitrogen, USA) according to the manufacturers protocol, and RNAsefree DNase was used to remove DNA contamination. Total RNA concentration and quantity were assessed using a DNA/Protein Analyzer (DU 530, Beckman, USA). cDNA was synthesized from RNA, using an PrimeScriptTM RT reagent Kit (TaKaRa). The cDNA specimens were amplied using an SYBR Premix Ex TaqTM II (TaKaRa). The polymerase chain reaction (PCR) primers used were as follows: 50 -CACTATCTCAAGCATCGTCCCG-30 (forward) and 50 AGACAGCCAGAACCGCCAC-30 (reverse) for NHE1; 50 -CGGGAAGCTTGTCATCAATGG-30 (forward) and 50 GGCAGTGATGGCATGGACTG-30 (reverse) for b-actin. PCR amplication was done on the ABI 7500 system (Applied Biosystems) using SYBR Green I (TAKARA). We used b-actin to normalize mRNA. Relative quantitation of mRNA expression levels was determined using the relative standard curve method according to the manufacturers instructions (Applied Biosystems). NHE1-RNAi and gene transfection The sequence 50 -TCATTCCGTCACTGATCAT-30 was used to inhibit NHE1 expression, and a non-specic sequence 50 -UUCUCCGAACGUGUCACGUTT-30 was used as a negative control. These oligonucleotides were custom-

synthesized by Genepharm (Shanghai, China) and then used for our in vitro gene transfection. Briey, the cells were trypsinized and seeded onto six-well plates at density of 1 9 105 cells/well with 1 ml of RPMI-1640 supplemented with 10% fetal calf serum without antibiotics for 24 h. Thereafter, 100 pM of the NHE1 RNAi oligonucleotide or the negative control oligonucleotide in 50 ll of Opti-MEM and 5 ll of Lipofectamine 2000 (Invitrogen, USA) in 50 ll of Opti-MEM were preincubated for 5 min at room temperature and then mixed together and incubated for additional 25 min at room temperature. After addition of 150 ll of Opti-MEM, the entire mixture was added to the well, and the cells were further cultivated for additional 13 days. NHE 1 expression levels were determined by Western blotting. Measurement of pHe levels The pHe was measured by phenolsulfonephthalein (phenol red) absorbance. Specically, cells were washed twice with Hanks balanced salt solution (HBSS; 138 mM NaCl, 5.4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 0.35 mM Na2HPO4) containing 0.03 mM phenol red. Cells were prewarmed at 37C, equilibrated with CO2 and then incubated with 1.5 ml of the same unbuffered solution without bicarbonate at 37C in a water-saturated atmosphere devoid of CO2. The ratio of 450/490 nm absorbance of phenol red was monitored by a spectrophotometer and converted to pH using the following equation: pH = log ((R - Rmin)/(Rmax - R)) ? pKa, where R is the experimentally derived ratio of absorbance; Rmax and Rmin indicate the limiting values of R; and pKa indicates the negative log of the dissociation constant (pKa = 7.5 for phenol red). Rmax and Rmin values were calculated from a standard curve for each experiment. Protein extraction and Western blotting Total cellular protein from the cultured tumor cells was extracted using a lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, and 0.5% NP40. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Samples containing 50 g of protein were separated on polyacrylamide SDS PAGE gels and then transferred electrophoretically to a Hybond-C nitrocellulose membrane (GE-Healthcare, Arlington Heights, IL). The membranes were incubated overnight. The next day, the membranes were incubated with a monoclonal antibody or anti-b-actin antibody. After washing three times with TBS, the membrane was incubated with a goat anti-mouse IgG antibody. The membranes were then incubated with enhanced

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chemiluminescence solution (GE-Healthcare) and protein levels quantied by the autoradiogram densitometry. Relative protein expression was then normalized to b-actin levels in each sample. MTT assay The cells were seeded onto 24-well plates at a density of 5 9 104 cells/well and grown for up to 72 h. Cell viability was assessed using the 3-(4, 5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay (Sigma Chemicals Co, St. Louis, MO) according to the manufacturers protocol. Each experiment contained six replicates and was repeated at least twice. The data were summarized as mean SD. Invasion determinations Cells were grown and transfected with NHE1 RNAi or treated with amiloride. Cell invasion was determined with Matrigel-coated transwell cell culture chambers (8 lmpore size) (Millipore, Billerica,MA, USA). Cells (5 9 104cells/ well) were transferred in 350 ll of serum-free medium to the upper chamber and then incubated in the bottom chamber. After incubation for 48 h, cells on the upper side of the lters were mechanically removed, and those migrated on the lower side were xed with 4% formaldehyde, then stained with 0.5% crystal violet for 10 min. Finally, invaded cells were counted at 2009 magnication in 10 different elds of each lter. ELISA assay Substrate-linked enzyme-linked immunosorbent assay (ELISA) techniques (Amersham, Buckinghamshire, UK) were used to quantify activity of individual MMP-2. The samples were thawed on ice, and all reagents needed for the assay were brought to room temperature. The MMP-2 activities were performed according to the manufacturers instructions. Statistical analysis Intensities of the target bands from Western blots were quantied using Quantity One software (Bio-Rad). Relative protein levels were calculated as a percentage of b-actin. All data were summarized as mean SD. The difference between means was statistically analyzed using the t-test. All statistical analyses were performed using SPSS 11.0 software (Chicago, IL). P \ 0.05 was considered as statistically signicant.

Results NHE1 expression levels in HCC cells NHE1 expression in four HCC cell lines was examined to assess the biological activities by Real-Time RTPCR. Chang liver was used as references for NHE1 expression. Our data indicate that compared with the Chang liver cells, the expression of NHE1 was signicantly increased in all four HCC cell lines (Fig. 1a). To determine the role of NHE1 in HCC, we designed a small inhibitory RNA (siRNA) oligonucleotide to target NHE1 mRNA. The NHE1-siRNA and a negative control oligonucleotide were transiently transfected into SMMC-7721 cells. Our NHE1siRNA signicantly decreased NHE1 protein expression 48 h after transfection compared to the control (Fig. 1b). Together, these results suggested that NHE1 was over-expressed in HCC cells, and transfection of the SMMC-7721 cells with the NHE1-siRNA strongly inhibited the expression of NHE1 in these cells. Effect of NHE1 down-regulation on extracellular pH (pHe) of SMMC-7721 cells We next investigated the extracellular pH (pHe) of SMMC7721 cells after NHE1 was knockdown. As shown in Fig. 2, we found that NHE1-siRNA signicantly increased the pHe levels in SMMC-7721 cell compared to the control oligonucleotide (Fig. 2). The data indicate that the downregulation of NHE1 gene could inhibit extracellular acidication of SMMC-7721 cells.

Fig. 1 Expression of NHE1 in HCC cells. a Using quantitative realtime RTPCR, NHE1 mRNA were over-expressed in all selected cell lines (BEL7402, SMMC-7721, HepG2, HHCC). * P \ 0.05 compared to the control. b Deletion of NHE1 using RNA interference signicantly inhibits the expression of NHE1 in SMMC-7721 cells

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7721 cells with NHE1-siRNA reduced MMP-2 protein expression. We further conrmed the inhibition of NHE1siRNA on MMP-2 by ELISA assay. As shown in Fig. 4b, transfection of SMMC-7721 cells with NHE1-siRNA signicantly inhibited MMP-2 activity. The data indicate that the invasion effect of NHE1 could be partly mediated by the MMP-2.
Fig. 2 The change on extracellular pH (pHe) of SMMC-7721 cells after NHE1 was knockdown. The pHe values of cells transfected with NHE1-siRNA were signicantly higher than that of control. * P \ 0.05 compared to the control

Effect of amiloride on pHe, invasion, and MMP-2 activity of SMMC-7721 cells Next, amiloride was used to conrm the role of NHE1 in SMMC-7721 cells. We treated SMMC-7721 cells with different doses of amiloride (10, 30 lM) and found that amiloride treatment increased pHe levels and inhibited invasion of SMMC-7721 cells in a dose-dependent manner compared with untreated cells (Fig. 5a and b). Amiloride did not affect cell viability at the indicated concentrations (Fig. 5c). Furthermore, using both Western blot and ELISA assay, different doses of amiloride signicantly inhibited MMP-2 activity (Fig. 5d and e).

Inhibition of SMMC-7721 cell invasion by down-regulation of NHE1 expression The invasive potential of SMMC-7721 cells was tested after NHE1 was knockdown. As shown in Fig. 3a, transfection of SMMC-7721 cells with NHE1-siRNA signicantly reduced their ability to invade through the Matrigel coating. The effect of NHE1 down-regulation on the proliferation of SMMC-7721 cells was further analyzed. In SMMC-7721 cells transfected with NHE1-siRNA, there was a small reduction in proliferation compared with that of control. However, this difference was not signicant (Fig. 3b), indicating that a relatively minor effect of downregulation of NHE1 expression on SMMC-7721 cell growth. Loss of NHE1 expression correlates with inhibition of MMP-2 activity To determine whether the reduced invasion ability of NHE1-silenced SMMC-7721 cell was due to the regulation of MMP-2, Western blot was rst used to detect MMP-2 expression. As shown in Fig. 4a, transfection of SMMC-

Discussion We have observed previously that NHE1 is up-regulated in HCC tissues compared with normal liver tissues and that the NHE1 expression level was associated with increased tumor size, venous invasion, and advanced pTNM stage, indicating that NHE1 may play a role in HCC invasion. Our present study found that NHE1 was also overexpressed in all selected HCC cell lines (BEL7402, SMMC-7721, HepG2, HHCC), detected by RTPCR. Meanwhile, inhibition of NHE1 using either siRNA or amiloride treatment signicantly suppressed the invasion

Fig. 3 Down-regulation of NHE1 expression inhibited the invasive abilities of SMMC-7721 cells. a The cells that invaded through the Matrigel-coated inserts were stained and counted. Transfecting NHE1 siRNA into SMMC-7721 cells achieved signicantly greater inhibition of SMMC-7721 cell invasiveness. * P \ 0.05 compared to the control. b NHE1 knockdown had no signicant effect on SMMC7721 cell growth

Fig. 4 Down-regulation of NHE1 expression inhibited MMP-2 expression and activity of SMMC-7721 cells. a Using Western blot, MMP-2 protein expression of SMMC-7721 cells transfected with NHE1-siRNA was lower than that of control. b Using ELISA assay, MMP-2 proteolytic activity of SMMC-7721 cells transfected with NHE1-siRNA was lower than that of control. * P \ 0.05 compared to the control

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Med Oncol (2011) 28:385390 Fig. 5 Effect of amiloride on pHe, cell invasion, and MMP-2 activity. SMMC-7721 cells were treated with various concentrations (0, 10, 30 lM) of amiloride. Then, each conditioned medium was collected for analysis of extracellular pH (a), cell invasion (b), cell growth (c), MMP-2 protein expression (d), and proteolytic activity (e). The data were summarized as mean SD, * P \ 0.05 compared to the control

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of HCC cells. These data indicate that inhibition of NHE1 might be used therapeutically to suppress invasion and metastasis of HCC. Recent research has highlighted the fundamental role of the tumors extracellular metabolic microenvironment in malignant invasion. One of the principal hallmarks in vivo tumor microenvironment is the presence of low extracellular pH (pHe) [16, 17]. In fact, numerous studies have consistently shown that acidication of local tumor microenvironments of both human and animal tumors can reach a pHe as low as 6.0 [18, 19]. Moreover, under some conditions, acidic pHe has been shown to stimulate in vitro invasion [20] and in vivo metastasis [21]. In the current study, we rst showed that an increase in pHe levels cells by inhibition of NHE1 resulted in suppression of SMMC7721 cell invasion. Furthermore, our experiments also show that down-regulation of NHE1 had only a small effect on proliferation of SMMC-7721 cells, which indicates that NHE1 may promote HCC metastasis through mechanisms other than increasing tumor cell proliferation. These data reveal that inhibition of NHE1 might be used to control the acidic extracellular pH of tumor microenvironment and suppress invasion and metastasis of HCC. Several researchers have investigated the role of NHE1 gene in tumor metastasis. Klein et al. [22] reported that NHE1 inhibition could lead to a reduction in the migration rate of MDCK-F cells. Moreover, Reshkin et al. [14] revealed that NHE1 activation augments the motility and the invasiveness of human breast carcinoma cells. Thus, all of these studies, including ours, also suggest that NHE1 might play a role on advancement of malignant neoplasms. Further research is therefore critical.

MMP-2, which belongs to the gelatinase MMP subclass, was thought to have a key role in degradation of the extracellular matrix (ECM), leading to increased tumor invasion ability during metastasis [23]. It has previously been shown that MMP-2 activity is necessary for Matrigel invasion of HCC cells [24]. Because acidic pHe has been shown to cause up-regulation of the MMP-2 (gelatinase A) [25], we hypothesized that this metastasis-promoting protein was involved in NHE1-mediated invasion. To test this hypothesis, we subsequently investigated the possible molecular mechanisms by which NHE1 might enhance tumor invasion by assessing the expression and proteolytic activities of MMP-2. In this study, down-regulation of NHE1 by either siRNA or amiloride treatment inhibited MMP-2 expression and proteolytic activity, which indicates that the NHE1-mediated invasive behavior of SMMC-7721 cells might be associated with MMP-2. Nevertheless, it is clear that the mechanisms responsible for NHE1-mediated tumor metastasis are quite complex, involving a number of biochemical and cellular events. Bourguignon LY [26] indicated that NHE1 could promote intra-endosomal/lysosomal pH changes and extracellular acidication leading to a concomitant activation of at least two low pH-dependent enzymes, Hyal-2 and cathepsin B, required for ECM degradation, hyaluronan modication, and tumor cell invasion. Moreover, Lagana A [27] suggested that NHE1 is involved in the formation of tumor cell pseudopodia, and NHE1 inhibition can also inhibit cell motility in vitro by preventing pseudopodial protrusion. To date, the precise mechanism by which NHE1 facilitates HCC cell invasion is not clear and needs further investigation.

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Med Oncol (2011) 28:385390 13. Boyer MJ, Tannock IF. Regulation of intracellular pH in tumor cell lines: inuence of microenvironmental conditions. Cancer Res. 1992;52:44417. 14. Reshkin SJ, et al. Phosphoinositide 3-kinase is involved in the tumor-specic activation of human breast cancer cell Na?/H? exchange, motility, and invasion induced by serum deprivation. J Biol Chem. 2000;275:53619. 15. Stock C, et al. Migration of human melanoma cells depends on extracellular pH and Na?/H? exchange. J Physiol. 2005;225: 22538. 16. Rofstad EK. Microenvironment-induced cancer metastasis. Int J Radiat Biol. 2000;76:589605. 17. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 2004;4:8919. 18. Raghunand N, Gatenby RA, Gillies RJ. Microenvironmental and cellular consequences of altered blood ow in tumours. Br J Radiol. 2003;76:1122. 19. Gillies RJ, Raghunand N, Karczmar G, Bhujwalla ZM. MRI of the tumor microenvironment. J Magn Reson Imaging. 2002;16: 43050. 20. Martinez-Zaguilan R, et al. Acidic pH enhances the invasive behavior of human melanoma cells. Clin Exp Metastasis. 1996;14: 17686. 21. Rofstad EK, Mathiesen B, Kindem K, Galappathi K. Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res. 2006;66: 6699707. 22. Klein M, Seeger P, Schuricht B, Alper SL, Schwab A. Polarization of Na?/H? and Cl-/HCO3- exchangers in migrating renal epithelial cells. J Gen Physiol. 2000;115:599607. 23. Zeng ZS, Cohen AM, Guillem JG. Loss of basement membrane type IV collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis. Carcinogenesis. 1999;20:74955. 24. Giannelli G, et al. Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion. Lab Invest. 2001;81:61327. 25. Kato Y, Nakayama Y, Umeda M, Miyazaki K. Induction of a 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines. J Biol Chem. 1992;267:1142430. 26. Bourguignon LY, Singleton PA, Diedrich F, Stern R, Gilad E. CD44 interaction with Na?/H? exchanger (NHE1) creates acidic microenvironments leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion. J Biol Chem. 2004; 279:269917007. 27. Lagana A, et al. Regulation of the formation of tumor cell pseudopodia by the Na?/H? exchanger NHE1. J Cell Sci. 2000; 113:364962.

In summary, in the present study, we have shown for the rst time that the inhibition of NHE1 gene using either siRNA or amiloride effectively controlled the invasiveness of human HCC cells. Our results showed that NHE1 gene has an important role in HCC cell invasion, which might be mediated by MMP-2. NHE1 might be a new therapeutic target of HCC. However, related reports are limited, and more studies still should be done.
Acknowledgments We thank all other members of our laboratory for their insight and technical support. This work was supported by grants from the National Natural Science Foundation of China (No. 30872480) and Shanxi Province Natural Science Foundation of China (No. 2008K09-05).

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