Involvement of the epidermal growth factor receptor in the modulation of multidrug resistance in human hepatocellular carcinoma cells in vitro
- Katrin Hoffmann†1,
- Zhi Xiao†1,
- Clemens Franz1,
- Elvira Mohr1,
- Susanne Serba1,
- Markus W Büchler1 and
- Peter Schemmer1Email author
© Hoffmann et al; licensee BioMed Central Ltd. 2011
Received: 27 June 2011
Accepted: 17 November 2011
Published: 17 November 2011
Hepatocellular carcinoma (HCC) is a molecular complex tumor with high intrinsic drug resistance. Recent evidence suggests an involvement of the tyrosine kinase pathway in the regulation of ATP-binding cassette protein (ABC-transport protein) mediated multidrug resistance in cancer cells. The aim of this study was to examine whether EGFR inhibition sensitizes HCCs to chemotherapy and to elucidate its mechanism.
Chemotherapeutic treatment induces multidrug resistance and significantly increases ABC-transport protein expression and function in a time- and dose-dependent manner in HCC cells. Furthermore, cytostatic treatment increases the mRNA expression of tyrosine kinases and induces the phosphorylation of ERK. EGF activation of the tyrosine kinase pathway up-regulated the ABC-transport protein mRNA expression and enhanced the survival of resistant HCC cells. Consistent with these effects, inhibition of the EGFR using siRNA decreased the ABC-transport protein mRNA expression and inhibited the proliferation of resistant cells. Additional treatment with Gefitinib, a clinically approved EGFR inhibitor, caused a dose-dependent reversal of resistance to conventional chemotherapy.
The present study demonstrates that the multidrug resistance of HCC is modulated through the EGF-activated tyrosine kinase cascade. Consequentially, the restoration of chemosensitivity by EGFR inhibition may lead towards new tailored therapies in patients with highly resistant tumors.
Hepatocellular carcinoma (HCC) is a major health problem worldwide. Its incidence is increasing continuously in the Western world. In the United States and Europe the diagnosis of HCC has almost doubled during the last two decades . Despite recent improvements in surveillance programs and diagnostic tools, only 30-40% of HCC patients are eligible for liver resection or transplantation, the only curative treatment options to date . The tyrosine kinase inhibitor sorafenib is the current standard of care for palliative treatment; the partial response rate, however, is only about 10% . Conventional systemic chemotherapy has shown only minor effectiveness with response rates far below 10% . A substantial resistance against structurally and functionally unrelated cytostatic drugs develops through the destruction of vulnerable and negative chemoresistant tumor cell populations during hepatocarcinogenesis. An increased cellular extrusion of chemotherapeutics by multidrug resistance mediating ABC-transport proteins (MDR proteins) and consequently reduced cytostatic activity has been described . The expression of transmembrane ABC-transport proteins in HCC has been demonstrated in vitro and in vivo [6, 7]. However, the over-expression of MDR proteins is an independent prognostic factor, associated with increased vascular and lymphatic invasion, shorter disease-free survival as well as significantly reduced overall survival [8–10].
An association of the tyrosine kinase pathway with the development and regulation of multidrug resistance (MDR) has been discussed in various tumor entities [11–14]. The epidermal growth factor receptor (EGFR)-associated activation of the tyrosine kinase pathway plays a key role in the signal transduction of cell differentiation, motility and proliferation in HCC. Guan et al. have demonstrated an increased tyrosine kinase activity in resistant hepatoma cells . The induction of the tyrosine kinase activity by epidermal growth factor (EGF) and consequentially increased MDR gene expression has been discussed previously in breast cancer cells . However, the involvement of the EGFR in the development of MDR in HCC has not yet been elucidated.
We show for the first time that conventional cytostatics induce the EGF-activated tyrosine kinase pathway leading to the induction of ATP-binding cassette protein mediated multidrug resistance in HCC. Moreover, we present the evidence that EGFR inhibition is a potent sensitizer for chemotherapeutic treatment in cancer cells.
Material and methods
HCC cell line
The human HCC cell line HepG2 (Toni Lindl GmbH, Munich, Germany) was used for in vitro experiments, cultured in RPMI 1640/DMEM containing 10% fetal bovine serum (FBS) in 5% CO2 at 37°C . Cell culture reagents were obtained from Life Technologies Inc. (Gaithersburg, USA), unless indicated otherwise.
Gemcitabine (Lilly, Indianapolis, USA) and doxorubicin (Sandoz Pharmaceuticals GmbH, Holzkirchen, Germany) were prepared according to the manufacturer's instructions in the Pharmacy of the university hospital of Heidelberg. Gefitinib (AstraZeneca, London, UK) were prepared according to the manufacturer's instruction by dissolving in DMSO. Cells were treated as follows for induction of drug resistance: untreated controls, twice weekly, gemcitabine or doxorubicin at two different concentrations (11.4 μg/ml and 114 μg/ml or 0.15 μg/ml and 1.5 μg/ml, respectively). EGF effects were assessed in the following groups: untreated controls, EGF 500 ng/ml (Biomol, Hamburg, Germany) for 24 hours, gemcitabine in the above-mentioned doses either in combination with EGF or alone. Effects of gefitinib were analysed in the following groups: untreated controls, gefitinib 10 μg/ml, gemcitabine or doxorubicin in the above-mentioned doses in combination with gefitinib or alone.
Cells at 2 × 103 were seeded into 96-well plates and cultured in 100 μl medium. Cells were treated as mentioned above, and 24, 48, 72 and 96 hours after treatment, MTT [3-(4, 4- dimeththiazol-2-yl)2, 5-diphenylterazolium bromide] in PBS was added to each well, incubated for 4 hours at 37°C and dissolved in 100 μl of propanol-2. The absorbance was recorded at 570 nm on a photometer (Eppendorf, Germany). A minimum of four independent experiments were performed in each treatment group.
HepG2 cells were lysed and 40 μg of total protein was separated by electrophoresis on a SDS-PAGE and transferred to PVDF membranes using an XCell IITM Blot Module (Invitrogen, Carlsbad, USA). Blotting was performed with PBS containing 0.05% Tween 20 plus 5% BSA and incubated overnight with primary antibodies (PGP, MRP1, MRP2, MRP3, ERK all Santa Cruz Biotechnology Inc., Santa Cruz, USA, EGFR and pERK Cell signalling Technology, Danvers, MA, USA, Actin Sigma Aldrich, Munich, Germany) at 4°C. The horseradish-peroxidase conjugated secondary antibody (Santa Cruz) was used for protein detection at room temperature for 2 hours, followed by chemiluminescence detection (ECL Western blot Analysis System, GE Healthcare, Munich, Germany). The results of Western blot were analyzed with the analysis software QUANTITY ONE (BIO-RAD Laboratories, Hercules, CA, USA).
EGFR small interfering RNA (siRNA) duplexes that target the sequences 5'-TACGAATATTAAACACTTCAA-3' and high-purity positive control siRNA oligo-nucleotides were used for siRNA experiments following the manufacturer's instructions (Qiagen, Hilden, Germany). Cells at 4 × 104 were seeded in the 6-well plates and transfection was performed using 12.5 nM siRNA and 12 μl transfection reagent according to the manufacturer's protocol (HiPerfect Transfection Reagent, Qiagen). After 48 hours, total RNA was extracted from cells and RT-PCR performed as described above.
Rhodamine uptake assay
Rhodamine uptake assay was performed to evaluate the PGP transport function. Cells were treated as mentioned above and incubated with PBS, Verapamil or Rhodamine 123. Rhodamine uptake was measured with FACS Canto II Flow Cytometry System (Becton Dickinson, New York, USA) and analyzed by FACS Diva 6.0 software as described by Huet et al. .
A one-way Anova test was carried out to reveal significant differences in the mRNA expression. A value of p ≤ 0.05 was defined as the level of significance. All statistical analyses were performed with SigmaStat 1.0 software (Jandel Scientific, Sanrafael, CA, USA).
Induction of multidrug resistance after chemotherapy
Chemotherapy-induced effects on the tyrosine kinase pathway
Effects of EGF on multidrug resistance proteins
Involvement of the EGFR in the regulation of multidrug resistance
Effects of the specific EGFR inhibitor gefitinib on multidrug resistance
Hepatocellular carcinoma is a molecular complex tumor with high intrinsic drug resistance . New approaches to overcome this resistance and offer patients tailored treatment strategies are urgently required . In this study we investigated the ability of tyrosine kinase inhibition to restore chemosensitivity in HCC. We demonstrate for the first time that EGFR inhibition sensitizes HCC cells to conventional chemotherapy. Furthermore, we provide evidence that EGFR-activated signal transduction via the tyrosine kinase pathway is involved in the development of MDR in HCC.
Indeed, data presented in this study clearly show that standard chemotherapy dramatically induces MDR in both of the investigated HCC cells. Both gemcitabine and doxorubicin treatment significantly increased the ABC-transport protein expression and mRNA levels in a time- and dose dependent manner. Additionally, cytostatic treatment enhanced the PGP activity. Thus the survival of drug resistant cells was significantly prolonged compared to chemo-sensitive cells. This is in line with previous reports, demonstrating an up-regulation of ABC-transport proteins in HepG2 cells as well as in patients with HCC after chemotherapy [15, 22]. The over-expression of drug-resistance proteins is an independent prognostic factor for the impaired survival of HCC patients and conventional chemotherapy has shown only minor effectiveness, with low response rates of 5-10% [4, 8–10].
There is upcoming evidence of a potential link between the tyrosine kinase pathway and ABC-transport proteins. Previously, cisplatin-induced ERK activation was described in human cervical carcinoma cells . However, several factors may be responsible for the modulation of the drug-resistance phenotype and the regulatory mechanisms involved have yet not been identified . Up to now an increased phosphorylation of ABC transporters by activation of the EGFR-RAS-MAPK cascade or modulation of the MDR transporter ATPase activity due to tyrosine kinase inhibition have been discussed [12, 24]. In the present study, we found that chemotherapeutic treatment influenced the gene expression of tyrosine kinases. The mRNA levels of RAF1, ERK, MAPK14 and the EGFR increased in a dose-dependent manner after treatment with gemcitabine or doxorubicin. Furthermore, chemotherapy enhanced the activity of ERK and increased the protein expression of its phosphorylated form in a dose-dependent manner which is in line with a previous report of Wang et al. .
To test the hypothesis of an interaction between the tyrosine kinase pathway and MDR we activated the EGFR-RAS-MAPK cascade by EGF. A simultaneous increase of MDR protein mRNA expression was found after EGF treatment in both of he investigated HCC cell line, with dramatically increased gene expression levels of PGP, MRP2 and MRP3 mRNA. In line with this, PGP efflux activity was enhanced and the cellular survival significantly increased in a time-dependent manner. Simultaneously, the gene expression of EGF-activated tyrosine kinases increased. These observations are consistent with previous reports on a potential influence of EGF on PGP and MRP1 expression [16, 25, 26]. An EGF-stimulated activation of the EGFR and increased PGP protein expression were described in colorectal cancer cells by Katayama et al. . Furthermore, enhanced MRP1 gene expression and a high MRP1 promoter activity have been detected in the presence of EGF in MCF-7 breast cancer cells .
Since our data indicate an involvement of the EGF-mediated downstream activation of tyrosine kinases in the regulation of ABC-transport proteins, we inhibited the EGFR using siRNA. Consequentially, an increased cytotoxicity of conventional chemotherapy and reduced survival of resistant cells was detectable. The ABC-transport protein gene expression was found to be significantly lower after EGFR inhibition in these cells. This supports the report of Garcia et al., who described a decreased MRP1 expression after inhibition of the EGFR in breast cancer cells for the first time . In addition, since the EGFR is over-expressed in several highly resistant tumor entities and restoration of chemosensitivity might have a significant therapeutic impact, we evaluated the effects of gefitinib as a commercially available EGFR inhibitor on the drug-resistance phenotype [27–29]. Gefitinib is FDA approved for the treatment of advanced non-small cell lung cancer and attaches to the ATP-binding site of the EGFR. This study clearly demonstrates considerable chemosensitizing effects of combinative treatment with gefitinib in resistant hepatocellular carcinoma cells. The ABC-transport protein gene expression levels dropped by up to ten-fold after addition of gefitinib to gemcitabine or doxorubicin treatment. In line with this, increased growth inhibitory activity was detected and the cellular efflux function of PGP was reduced. Recently, a dose-dependent reversal of drug resistance in breast and lung carcinoma cell lines after simultaneous treatment with clinically relevant doses of gefitinib has been shown . Furthermore, Gaikwad et al. detected decreased PGP-mRNA levels after combinative treatment with gefitinib and cisplatin in endometrial cancer cells . Nevertheless, synergistic effects of gefitinib and chemotherapeutic agents have yet not been observed in clinical trials [32, 33].
In conclusion, the EGF-activated tyrosine kinase pathway seems to be involved in the regulation of MDR in HCC. The tyrosine kinase mRNA expression and phosphorylation is up-regulated in resistant HCC cells. Furthermore, the gene expression and function of ABC-transport proteins can be induced by EGFR activation. In contrast, the inhibition of the EGFR restores the chemosensitivity of drug-resistant HCC cells. In terms of a clinical perspective, the combination of EGFR inhibitor and selected conventional chemotherapeutic agents may be a novel strategy to improve the treatment efficacy of tailored therapies in a variety of patients with highly resistant tumors.
Acknowledgements and Funding
Katrin Hoffmann was supported by the Olympia-Morata-Grant of the University of Heidelberg. Xiao Zhi was supported by the China Scholarship Council. We thank Katherine Hughes for language editing.
- El-Serag HB: Hepatocellular carcinoma and hepatitis C in the United States. Hepatology. 2002, 36: S74-S83.View ArticlePubMedGoogle Scholar
- Llovet JM, Bruix J: Hepatocellular carcinoma. The Lancet. 2003, 362: 1907-1917. 10.1016/S0140-6736(03)14964-1.View ArticleGoogle Scholar
- Abou-Alfa GK, Schwartz L, Ricci S, Amadori D, Santoro A, Figer A: Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol. 2006, 24: 4293-4300. 10.1200/JCO.2005.01.3441.View ArticlePubMedGoogle Scholar
- Ganne-Carrie N, Trinchet JC: Systemic treatment of hepatocellular carcinoma. European Journal of Gastroenterology and Hepatology. 2004, 16: 275-281.View ArticlePubMedGoogle Scholar
- Gottesman MM: Mechanisms of cancer drug resistance. Annu Rev Med. 2002, 53: 615-627. 10.1146/annurev.med.53.082901.103929.View ArticlePubMedGoogle Scholar
- Nies AT, Konig J, Pfannschmidt M, Klar E, Hofmann WJ, Keppler D: Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma. Int J Cancer. 2001, 94: 492-499. 10.1002/ijc.1498.View ArticlePubMedGoogle Scholar
- Li B, Ye T, Zhao L, Li DH, Gou XH, Zhao LY: Effects of multidrug resistance, antisense RNA on the chemosensitivity of hepatocellular carcinoma cells. Hepatobiliary Pancreat Dis Int. 2006, 5: 552-559.PubMedGoogle Scholar
- Soini Y, Virkajarvi N, Raunio H, Paako P: Expression of P-glycoprotein in hepatocellular carcinoma: a potential marker of prognosis. J Clin Pathol. 1996, 49: 470-473. 10.1136/jcp.49.6.470.PubMed CentralView ArticlePubMedGoogle Scholar
- Kato A, Miyazaki M, Ambiru S, Yoshitomi H, Ito H, Nakagawa K: Multidrug resistance gene (MDR-1) expression as a useful prognostic factor in patients with human hepatocellular carcinoma after surgical resection. J Surg Oncol. 2001, 78: 110-115. 10.1002/jso.1129.View ArticlePubMedGoogle Scholar
- Vander BS, Komuta M, Libbrecht L, Katoonizadeh A, Aerts R, Dymarkowski S: Expression of multidrug resistance-associated protein 1 in hepatocellular carcinoma is associated with a more aggressive tumour phenotype and may reflect a progenitor cell origin. Liver Int. 2008, 28: 1370-1380. 10.1111/j.1478-3231.2008.01889.x.View ArticleGoogle Scholar
- Barancik M, Bohacova V, Kvackajova J, Hudecova S, Krizanova O, Breier A: SB203580, a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance. Eur J Pharm Sci. 2001, 14: 29-36. 10.1016/S0928-0987(01)00139-7.View ArticlePubMedGoogle Scholar
- Yang JM, Sullivan GF, Hait WN: Regulation of the function of P-glycoprotein by epidermal growth factor through phospholipase C. Biochem Pharmacol. 1997, 53: 1597-1604. 10.1016/S0006-2952(97)82451-3.View ArticlePubMedGoogle Scholar
- Yang JM, Vassil AD, Hait WN: Activation of phospholipase C induces the expression of the multidrug resistance (MDR1) gene through the Raf-MAPK pathway. Mol Pharmacol. 2001, 60: 674-680.PubMedGoogle Scholar
- Kuo TC, Lu HP, Chao CC: The tyrosine kinase inhibitor sorafenib sensitizes hepatocellular carcinoma cells to taxol by suppressing the HURP protein. Biochem Pharmacol. 2011, 82 (2): 184-94. 10.1016/j.bcp.2011.04.008.View ArticlePubMedGoogle Scholar
- Guan J, Chen XP, Zhu H, Luo SF, Cao B, Ding L: Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line. World J Gastroenterol. 2004, 10: 3522-3527.PubMed CentralView ArticlePubMedGoogle Scholar
- Garcia R, Franklin RA, McCubrey JA: EGF induces cell motility and multi-drug resistance gene expression in breast cancer cells. Cell Cycle. 2006, 5: 2820-2826. 10.4161/cc.5.23.3535.View ArticlePubMedGoogle Scholar
- Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J: Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982, 42: 3858-3863.PubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Huet S, Marie JP, Gualde N, Robert J: Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: a method validated by the laboratories of the French Drug Resistance Network. Cytometry. 1998, 34: 248-256. 10.1002/(SICI)1097-0320(19981215)34:6<248::AID-CYTO2>3.0.CO;2-X.View ArticlePubMedGoogle Scholar
- Herr I, Schemmer P, Buchler MW: On the TRAIL to therapeutic intervention in liver disease. Hepatology. 2007, 46: 266-274.View ArticlePubMedGoogle Scholar
- Herr I, Groth A, Schemmer P, Buchler MW: Adult stem cells in progression and therapy of hepatocellular carcinoma. Int J Cancer. 2007, 121: 1875-1882. 10.1002/ijc.23041.View ArticlePubMedGoogle Scholar
- Ng KK, Vauthey JN, Pawlik TM, Lauwers GY, Regimbeau JM, Belghiti J: Is hepatic resection for large or multinodular hepatocellular carcinoma justified? Results from a multi-institutional database. Ann Surg Oncol. 2005, 12: 364-373. 10.1245/ASO.2005.06.004.View ArticlePubMedGoogle Scholar
- Wang X, Martindale JL, Holbrook NJ: Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem. 2000, 275: 39435-39443. 10.1074/jbc.M004583200.View ArticlePubMedGoogle Scholar
- Ozvegy-Laczka C, Hegedus T, Varady G, Ujhelly O, Schuetz JD, Varadi A: High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter. Mol Pharmacol. 2004, 65: 1485-1495. 10.1124/mol.65.6.1485.View ArticlePubMedGoogle Scholar
- Katayama K, Yoshioka S, Tsukahara S, Mitsuhashi J, Sugimoto Y: Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein. Mol Cancer Ther. 2007, 6: 2092-2102. 10.1158/1535-7163.MCT-07-0148.View ArticlePubMedGoogle Scholar
- Hirsch-Ernst KI, Ziemann C, Schmitz-Salue C, Foth H, Kahl GF: Modulation of P-glycoprotein and mdr1b mRNA expression by growth factors in primary rat hepatocyte culture. Biochem Biophys Res Commun. 1995, 215: 179-185. 10.1006/bbrc.1995.2450.View ArticlePubMedGoogle Scholar
- Dickstein B, Valverius EM, Wosikowski K, Saceda M, Pearson JW, Martin MB: Increased epidermal growth factor receptor in an estrogen-responsive, adriamycin-resistant MCF-7 cell line. J Cell Physiol. 1993, 157: 110-118. 10.1002/jcp.1041570115.View ArticlePubMedGoogle Scholar
- Chin KV, Ueda K, Pastan I, Gottesman MM: Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Science. 1992, 255: 459-462. 10.1126/science.1346476.View ArticlePubMedGoogle Scholar
- Meyers MB, Shen WP, Spengler BA, Ciccarone V, O'Brien JP, Donner DB: Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells. J Cell Biochem. 1988, 38: 87-97. 10.1002/jcb.240380203.View ArticlePubMedGoogle Scholar
- Yang CH, Huang CJ, Yang CS, Chu YC, Cheng AL, Whang-Peng J: Gefitinib reverses chemotherapy resistance in gefitinib-insensitive multidrug resistant cancer cells expressing ATP-binding cassette family protein. Cancer Res. 2005, 65: 6943-6949. 10.1158/0008-5472.CAN-05-0641.View ArticlePubMedGoogle Scholar
- Gaikwad A, Wolf JK, Brown J, Ramondetta LM, Smith JA: In vitro evaluation of the effects of gefitinib on the cytotoxic activity of selected anticancer agents in a panel of human endometrial cancer cell lines. J Oncol Pharm Practice. 2009, 15: 35-44.View ArticleGoogle Scholar
- Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V: Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial--INTACT 1. J Clin Oncol. 2004, 22: 777-784. 10.1200/JCO.2004.08.001.View ArticlePubMedGoogle Scholar
- Herbst RS, Giaccone G, Schiller JH, Natale RB, Miller V, Manegold C: Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial--INTACT 2. J Clin Oncol. 2004, 22: 785-794. 10.1200/JCO.2004.07.215.View ArticlePubMedGoogle Scholar
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