Multidrug Resistance In Hepatocellular Carcinoma Cells Research Articles

NAP1L1 regulates BIRC2 ubiquitination modification via E3 ubiquitin ligase UBR4 and hence determines hepatocellular carcinoma progression

We have previously shown that nucleosome assembly protein 1-like 1 (NAP1L1) plays an important role in the abnormal proliferation of hepatocellular carcinoma (HCC) cells. However, the effects of NAP1L1 on the malignant behaviour of HCC cells, including cell migration, invasion and apoptosis, remain unclear. Baculoviral IAP repeat-containing 2 (BIRC2) plays a key role in initiating the abnormal proliferation, apoptotic escape and multidrug resistance of HCC cells; however, the mechanisms through which its stability is regulated in HCC remain elusive. Here, we found that knockdown of NAP1L1 inhibited the proliferation of HCC cells and activated apoptotic pathways but did not remarkably affect the migratory and invasive abilities of HCC cells. In addition, knockdown of NAP1L1 did not alter the expression of BIRC2 at the transcriptional level but substantially reduced its expression at the translational level, suggesting that NAP1L1 is involved in the post-translational modification (such as ubiquitination) of BIRC2. Furthermore, BIRC2 was highly expressed in human HCC tissues and promoted the proliferation and apoptotic escape of HCC cells. Co-immunoprecipitation (Co-IP) assay and mass spectrometry revealed that NAP1L1 and BIRC2 did not bind to each other; however, ubiquitin protein ligase E3 component n-recognin 4 (UBR4) was identified as an intermediate molecule associating NAP1L1 with BIRC2. Knockdown of NAP1L1 promoted the ubiquitin-mediated degradation of BIRC2 through the ubiquitin–protein junction of UBR4, which in turn inhibited the proliferation and apoptotic escape of HCC cells and exerted anti-tumour effects. In conclusion, this study reveals a novel mechanism through which NAP1L1 regulates the ubiquitination of BIRC2 through UBR4, thereby determining the progression of HCC. Based on this mechanism, suppression of NAP1L1 may inhibit tumour progression in patients with HCC with high protein expression of NAP1L1 or BIRC2.

Development of a DNA Aptamer against Multidrug-Resistant Hepatocellular Carcinoma for In Vivo Imaging.

Hepatocellular carcinoma (HCC) is a type of cancer that has high rates of recurrence and mortality. One of the most important factors that lead to treatment failure of HCC is the acquisition of multidrug resistance (MDR). Development of specific ligands for multidrug-resistant HCC will provide useful molecular tools for precise diagnosis and targeted theranostics. Herein, a multidrug-resistant HCC cell (HepG2/MDR)-specific aptamer was developed through Cell-SELEX (systematic evolution of ligands by exponential enrichment) technology. With dissociation constants lying in the nanomolar range, the molecularly designed PS-ZL-7c aptamer showed great selectivity to drug-resistant cancer cells. The in vivo imaging results illustrated that the PS-ZL-7c specifically accumulated in the drug-resistant tumors but not in drug-sensitive tumors and normal tissues, indicating that the PS-ZL-7c aptamer possessed excellent potential as a targeting ligand for precise diagnosis and target theranostics of multidrug-resistant HCC.

Inhibition of Polo-Like Kinase 1 by BI2536 Reverses the Multidrug Resistance of Human Hepatoma Cells In Vitro and In Vivo.

Multi Drug Resistance (MDR) is the phenomenon that cancers develop resistance to majority of chemotherapy drugs and is a serious obstacle to the treatment for Hepatocellular Carcinoma (HCC). Polo-Like Kinase 1 (PLK1) is a serine/threonine kinase associated with tumor growth and clinical prognosis in HCC and BI2536 is its potent inhibitor with IC50 of 0.83nM. To test whether the down-regulation of PLK1 by its inhibitor BI2536 would have beneficial effects on the reversal of MDR in HCC cells. The CCK-8 assay was used to determine the viability of HepG2/ADM and SMMC7721/ADM cells and their parental cells treated with BI2536. Then animal model studies were performed. Cell invasion assay and wound healing assay were used to determine the invasion ability and motility. Flow cytometric was used to test the apoptosis induced by BI2536. Western blot and quantitative real-time PCR were performed to test the change of expression of MDR and apoptosis-related gene. BI2536 down-regulated the expression of PLK1 protein and mRNA specifically. BI2536 can significantly reduce IC50 for ADM and other drugs in ADM-resistant HCC cells. Meanwhile, it inhibited cell viability, proliferation, and invasion, and induced cell cycle arrest and apoptosis in HCC cells with MDR. Our results suggest that PLK1 inhibitor BI2536 can re-sensitize HCC cancer cell with MDR through induction of apoptosis. Thus, PLK1 inhibitor BI2536 may act as an effective chemotherapeutic drug in the clinical treatment of HCC patients with MDR.

Baicalein sensitizes hepatocellular carcinoma cells to 5-FU and Epirubicin by activating apoptosis and ameliorating P-glycoprotein activity

Hepatocellular carcinoma (HCC) has a dismal prognosis in part because of multi-drug resistance (MDR). Baicalein is a flavonoid extracted from Radix Scutellariae with anti-HCC activity. We tested the effects of Baicalein on multi-drug resistant HCC cells (Bel7402/5-FU) known to be resistant to the anticancer drugs 5-FU and Epirubicin. Flow cytometry analysis showed that treatment with 5 μg/ml and 10 μg/ml Baicalein resulted in increases in the intra-cellular concentrations of Rho123 and Epirubicin in the corresponding group of cells compared to untreated cells, illustrating that Baicalein reverses MDR in Bel7402/5-FU cells. Bel7402/5-FU cells displayed increased P-glycoprotein (P-gp)-mediated drug efflux. However, this efflux was inhibited in cells pre-incubated in Baicalein for 48 h. Moreover, Baicalein induced apoptosis and autophagy and decreased P-gp and Bcl-xl expression levels. All of these results indicate that Baicalein can reverse P-gp-mediated MDR in HCC and may thus be useful for the treatment of drug-resistant HCC.

Ubenimex suppresses Pim-3 kinase expression by targeting CD13 to reverse MDR in HCC cells.

Hepatocellular carcinoma (HCC) is one of the most serious cancers, with rapid progression and high mortality. However, chemotherapy of HCC is hindered by multi-drug resistance (MDR). It is urgent, therefore, to explore new approaches for overcoming MDR of HCC cells. Ubenimex, an inhibitor of CD13, has been used as an immuno-enhancer for treating hematological neoplasms and other solid tumors. Here, we demonstrate that Ubenimex can also reverse MDR in the HCC cell lines HepG2/5-FU and Bel7402/5-FU. Ubenimex inhibits the expression of the proto-oncogene, Pim-3, which is accompanied by decreased expression of BCL-2 and BCL-XL, decreased phosphorylation of Bad, and increased tumor apoptosis.Moreover, Ubenimex decreases expression of the MDR-associated proteins P-gp, MRP3 and MRP2 to enhance intracellular accumulation of Cisplatin, for which down-regulation of Pim-3 is essential. Our results reveal a previously uncharacterized function of Ubenimex in mediating drug resistance in HCC, which suggests that Ubenimex may provide a new strategy to reverse MDR and improve HCC sensitivity to chemotherapeutic drugs via its effects on Pim-3.

MEK inhibitor enhances sensitivity to chemotherapeutic drugs in multidrug resistant hepatocellular carcinoma cells.

The aim of the present study was to investigate the association between the mitogen-activated protein kinase (MAPK) signal transduction pathway and multidrug resistance in hepatocellular carcinoma cells. A Cell Counting Kit-8 assay was used to determine the drug sensitivity of HepG2 and HepG2/ADM hepatocellular carcinoma cell lines in combination with the MAPK/extracellular-signal-regulated kinase kinase (MEK) inhibitor U0126. Flow cytometry was used to analyze the rate of apoptosis. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) mRNA expression following treatment with various concentrations of U0126. P-gp and MRP1 expression levels were measured using Western blot analysis. The half-maximal inhibitory concentration was markedly decreased in combination with U0126. RT-qPCR results demonstrated that the expression of multidrug resistance 1 (MDR1) and MRP1 in HepG2/ADM cells was increased 5.37- and 6-14-fold compared with that in HepG2 cells. Furthermore, the expression levels in HepG2/ADM cells were decreased following U0126 treatment in a dose-dependent manner. The expression of P-gp and MRP1 in HepG2/ADM cells was increased 2.68- and 2.76-fold compared with that in HepG2 cells. Furthermore, the expression levels in HepG/ADM cells were decreased following U0126 treatment in a dose-dependent manner. The results of the present study indicate that the MEK inhibitor U0126 enhances sensitivity to chemotherapeutic drugs by downregulating P-gp and MRP1 expression in resistant hepatocellular carcinoma cells. The combination of MEK inhibitor and conventional chemotherapeutic drugs may provide novel therapeutic prospects for the treatment of drug-resistant hepatocellular carcinoma.

USP22 mediates the multidrug resistance of hepatocellular carcinoma via the SIRT1/AKT/MRP1 signaling pathway.

Drug treatments for hepatocellular carcinoma (HCC) often fail because of multidrug resistance (MDR). The mechanisms of MDR are complex but cancer stem cells (CSCs), which are able to self‐renew and differentiate, have recently been shown to be involved. The deubiquitinating enzyme ubiquitin‐specific protease 22 (USP22) is a marker for CSCs. This study aimed to elucidate the role of USP22 in MDR of HCC and the underlying mechanisms. Using in vitro and in vivo assays, we found that modified USP22 levels were responsible for the altered drug‐resistant phenotype of BEL7402 and BEL/FU cells. Downregulation of USP22 dramatically inhibited the expression of ABCC1 (encoding MRP1) but weakly influenced ABCB1 (encoding P‐glycoprotein). Sirtuin 1 (SIRT1) was reported previously as a functional mediator of USP22 that could promote HCC cell proliferation and enhance resistance to chemotherapy. In this study, USP22 directly interacted with SIRT1 and positively regulated SIRT1 protein expression. Regulation of the expression of both USP22 and SIRT1 markedly affected the AKT pathway and MRP1 expression. Inhibition of the AKT pathway by its specific inhibitor LY294002 resulted in downregulation of MRP1. USP22 and MRP1 expression was detected in 168 clinical HCC samples by immunohistochemical staining, and a firm relationship between USP22 and MRP1 was identified. Together, these results indicate that USP22 could promote the MDR in HCC cells by activating the SIRT1/AKT/MRP1 pathway. USP22 might be a potential target, through which the MDR of HCC in clinical setting could be reversed.

Regulation of Multi-drug Resistance in hepatocellular carcinoma cells is TRPC6/Calcium Dependent.

Hepatocellular carcinoma (HCC) is notoriously refractory to chemotherapy because of its tendency to develop multi-drug resistance (MDR), whose various underlying mechanisms make it difficult to target. The calcium signalling pathway is associated with many cellular biological activities, and is also a critical player in cancer. However, its role in modulating tumour MDR remains unclear. In this study, stimulation by doxorubicin, hypoxia and ionizing radiation was used to induce MDR in HCC cells. A sustained aggregation of intracellular calcium was observed upon these stimuli, while inhibition of calcium signalling enhanced the cells’ sensitivity to various drugs by attenuating epithelial-mesenchymal transition (EMT), Hif1-α signalling and DNA damage repair. The effect of calcium signalling is mediated via transient receptor potential canonical 6 (TRPC6), a subtype of calcium-permeable channel. An in vivo xenograft model of HCC further confirmed that inhibiting TRPC6 enhanced the efficacy of doxorubicin. In addition, we deduced that STAT3 activation is a downstream signalling pathway in MDR. Collectively, this study demonstrated that the various mechanisms regulating MDR in HCC cells are calcium dependent through the TRPC6/calcium/STAT3 pathway. We propose that targeting TRPC6 in HCC may be a novel antineoplastic strategy, especially combined with chemotherapy.

Reversal of stemness in multidrug-resistant hepatocellular carcinoma cells by SIS3

To investigate whether SIS3, a specific inhibitor of Smad3 phosphorylation, can reverse the stemness of multidrug-resistant(MDR) hepatocellular carcinoma cells. MDR HCC Huh7.5.1/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. CCK-8 assay was used to determine the cellular sensitivity of various anticancer drugs. Flow cytometry (FCM) was used to analyze the expression level of cancer stem cell marker CD133. Clone formation assay and mouse subcutaneous xenograft tumors were used to investigate the tumorigenicity in vitro and in vivo. Western blotting (WB) was used to analyze the changes of expressions of CD133, Smad3, Bcl-2, Bax and p-Smad3 in different conditions. ADM treatment of HCC cells in vitro resulted in a development of subline, Huh7.5.1/ADM cells, with CSC phenotypes: stable MDR phenotype (besides ADMc Huh7.5.1/ADM cells were also more resistant to some other anticancer drugs including VCR, MMC and CTX ) (IC50: 0.215 ± 0.018 vs. 0.123 ± 0.004, 0.145 ± 0.009 vs. 0.014 ± 0.002, 1.021 ± 0.119 vs. 0.071 ± 0.006, 27.007 ± 1.606 vs. 1.919 ± 0.032) (unit: µg/ml) (P<0.05). Huh7.5.1/ADM cells enriched the cancer stem-like cell fraction (CD133-positive subpopulation) (76.06 ± 2.948% vs. 25.38 ± 4.349%) (P<0.05), had stronger tumorigenicity in vivo and colony formation ability, and activated the Smad3 activity. Inhibition of Smad3 activity by SIS3 decreased stemness of the Huh7.5.1/ADM cells: CD133-positive subpopulation (48.49 ± 2.304% vs. 76.06 ± 2.948%) (P<0.05); ADM IC50: (0.112 ± 0.019 vs. 0.215 ± 0.018), VCR IC50 (0.065 ± 0.013 vs. 0.145±0.009), MMC IC₅₀ (0.749 ± 0.121 vs. 1.021 ± 0.119), CTX IC50 (10.576 ± 1.248 vs. 27.007 ± 1.606) (unit: µg/ml) (P<0.05), and decreased tumorigenicity and colony formation ability. SIS3 as a specific inhibitor of Smad3 signal is involved in the stemness of multidrug resistant hepatocellular carcinoma cells.

HtrA1 resensitizes multidrug-resistant hepatocellular carcinoma cells by targeting XIAP

The study aims to clarify the relation between chemosensitivity and HtrA1 expression, and the possible way HtrA1 works. Drug-resistant cell line HepG2/ADM was induced by increasing adriamycin (ADM), and eukaryotic expression vector pEGFP-N1-HtrA1 was constructed using BamHI and EcoRI restriction enzymes, after which, HepG2/ADM was transfected with pEGFP-N1-HtrA1. Resistance index (RI) of the hepatoma cell lines to different anti-cancer drugs (ADM, 5-Fu, MMC, L-OHP and VCR) was determined by MTT assay before and after HtrA1 high expression. After an HtrA1 inhibitor, NVP-LEB748 was adopted in the HtrA1 overexpressing cells, expression of proteins P-gp, MRP and XIAP (X-linked inhibitor of apoptosis protein) in HepG2/ADM cells were analyzed by western blot, and the activities of caspases 3, 7 and 9 were respectively measured using activity assay kits. The results showed that RI was negatively correlated with the expression of HtrA1, upregulated XIAP expression was resulted from the HtrA1 inhibitor, and variance of activities of caspases 3, 7 and 9 were remarkably descended with its increasing concentration. It was concluded that high expression of HtrA1 could significantly reverse multidrug resistance of hepatoma cells by targeting XIAP. HtrA1 is therefore expected to be an effective tool in the therapy of hepatocellular carcinoma.

Effects of mitochondrial translocation of telomerase on drug resistance in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) cells exhibit multidrug resistance (MDR), but the underlying mechanisms remain unclear. Cancer cells that overexpress telomerase are resistant to chemotherapeutic drugs. This study aimed to determine the effects of mitochondrial translocation of telomerase on MDR in HCC cells. HepG2 cells were transfected with negative plasmid and PTPN11 (Shp-2) short hairpin RNA (ShRNA) plasmid to establish HepG2-negative (HepG2 transfected with negative plasmid) and HepG2-ShShp-2 (HepG2 transfected with Shp-2 ShRNA plasmid) cells. Sensitivity to chemotherapeutic drugs was assessed by Cell Counting Kit-8 (CCK-8) assays. Distribution of human telomerase reverse transcriptase (hTERT) within mitochondria was detected by western blotting and immunofluorescence combined with laser scanning confocal microscopy. Mitochondrial reactive oxygen species (ROS) generation was demonstrated by flow cytometry with the mitochondrial superoxide (Mito-Sox) indicator. The frequency of damaged mitochondrial DNA (mtDNA) was illustrated by quantitative real-time polymerase chain reaction (Q-PCR). Expression of mitochondrial respiratory chain complex subunits ND1 and COXII were also demonstrated by western blotting. Knockdown of Shp-2 in HepG2 cells resulted in upregulation of mitochondrial TERT expression and increased resistance to cisplatin (CDDP) and 5-fluorouracil (5-FU) (resistance indices, 2.094 and 1.863, respectively). In addition, both the mitochondrial ROS and the frequency of mtDNA damage were decreased, and COXII expression was upregulated. Our results suggest that Mitochondrial translocation of hTERT may lead to chemotherapeutic resistance in HCC cells. Mitochondrial hTERT contributes to the drug resistance of tumor cells by reducing ROS production and mtDNA damage, and exerting a protective effect on the mitochondrial respiratory chain.

English

Chemotherapy is a main treatment for cancer, while multidrug-resistance is the main reason for chemotherapy failure, and tumor relapse and metastasis. Cancer stem cells or cancer stem-like cells (CSCs) are a small subset of cancer cells, which may be inherently resistant to the cytotoxic effect of chemotherapy. Some studies suggest that CSCs could be enriched by chemotherapy. However, the mechanism of chemotherapy regulating CSCs remains unknown. Therefore, we investigated whether drug treatment could enrich CSCs in hepatocellular carcinoma (HCC) cells and the molecular mechanism of chemotherapy regulating the expression of CSCs markers. In the present study, a multidrug-resistant (MDR) human HCC subline, Huh7.5.1/PTX, was developed by exposing parental cells to paclitaxel (PTX) repeatedly at a single high concentration. The cell counting kit-8 (CCK-8) assay was used to determine cellular sensitivity of various anticancer drugs. Flow cytometry (FCM) was used to analyze the CSCs markers expression level. Western blotting (WB) was used to analyze the changes of TGF-β1/Smads signaling. Our results show that PTX treatment of HCC cells in vitro resulted in a development of subline six months later, and Huh7.5.1/PTX, with stable MDR phenotype. Huh7.5.1/PTX cells enriched CSCs fraction and strongly activated the TGF-β1/Smad3 signaling. Activation of TGF-β1/Smad3 signaling resulted in enrichment of the CSCs population (CD133 + cells), while inhibition of this pathway activity attenuated the percentage of these cells. Taken together, our results suggest that MDR HCC cells are enriched with CSCs, which is partially dependent on TGF-β1/Smad3 pathway. Inhibition of TGF-β1/Smad3 pathway may be useful for targeting CSCs to develop more effective treatments for HCC. Keywords: Hepatocellular carcinoma (HCC) CD133, chemotherapy, cancer stem cells, TGF-β1, Smad3. African Journal of Biotechnology , Vol 13(31) 3538-3546

MiR-27a modulates the MDR1/P-glycoprotein expression by inhibiting FZD7/β-catenin pathway in hepatocellular carcinoma cells

Chemotherapy has been widely used to treat cancer, however, the appearance of multiple drug resistance (MDR) in cancer patients is regarded as a major clinical obstacle to successful chemotherapy. MicroRNAs (miRNAs) are evolutionary conserved small RNAs that regulate gene expression at the post-transcriptional level and have been shown to regulate cell differentiation, development, proliferation and apoptosis. Nevertheless, the involvement of miRNAs and their roles in the development of MDR in liver cancer are not fully understood. Our study found that the expression of miR-27a was down-regulated in the multidrug-resistant hepatocellular carcinoma cell line BEL-7402/5-fluorouracil (BEL/5-FU) compared with its parental BEL-7402 cell line, while the MDR1/P-glycoprotein expression was elevated. Overexpression of miR-27a by transfecting with miR-27a mimics in the BEL/5-FU cells could reduce the MDR1/P-glycoprotein and β-catenin expressions, enhance the sensitivity of these cells to 5-fluorouracil and 5-fluorouracil-induced apoptosis. Moreover, up-regulation of miR-27a did not decrease the FZD7 mRNA level, but significantly reduce its protein expression in BEL/5-FU cells. It was also confirmed that reduction of FZD7 by RNA interference induced inhibitory effects on the expression of MDR1/P-glycoprotein and β-catenin, similar to miR-27a. Taken together, our findings suggest that miR-27a could function as a novel regulator to reverse MDR in hepatocellular carcinoma cells by inhibiting the FZD7/β-catenin pathway.

Evaluation of Hydro-Alcoholic Extract of Eclipta alba for its Multidrug Resistance Reversal Potential: An In Vitro Study

The development of multidrug resistance (MDR) causes problems in the chemotherapy of human cancer. The present study was designed to evaluate and establish the role of Eclipta alba as MDR reversal agent using multidrug resistant hepatocellular carcinoma cell line (DR-HepG2). To develop DR-HepG2, hepatocellular carcinoma cell line (HepG2) was transfected with 2-Acetylaminofluorene (AAF) and Aflatoxin B1 (AFB). Cytotoxic effects of the Eclipta alba hydroalcoholic extract (EAE) and standard anti-ancer drug Doxorubicin (DOX) were determined in DR-HepG2 and the parental cells HepG2 using MTT assay. The expression level of MDR1 gene and P-glycoprotein (P-gp) level was analyzed by RT-PCR and western blotting. From the present investigation, it was found that EAE (10 and 20 μg/ml) could significantly inhibit cell proliferation in DR-HepG2 whereas DOX (0.5 μg/ml) could not because of enhancement effect of MDR1/P-gp. This study demonstrated for the first time the antiproliferative activities of EAE in multidrug resistant DR-HepG2 cells. The findings revealed that Eclipta alba components are effective inhibitors of MDR1/P-gp.

Steroidal saponin of Trillium tschonoskii. Reverses multidrug resistance of hepatocellular carcinoma

Combating with multidrug resistance (MDR) is a major part of hepatocellular carcinoma (HCC) chemotherapy. Steroidal saponin from Trillium tschonoskii (TTS) could be a potential weapon. We found TTS could reverse the MDR in HCC cells and significantly enhance chemosensitization. TTS inhibited HepG2 and R-HepG2 cells survival in a dose-dependent manner by 75% and 76%, respectively (p<0.01), as well as colony formation 77% and 81% (p<0.01). Moreover, TTS induced sensitization of R-HepG2 to anti-cancer drugs, indicated by significantly reduced IC50. On the other hand, TTS suppressed expression of P-glucoprotein in MDR HCC cells, and thereby increased accumulation of doxorubicin from 126ng/105cells to 752ng/105cells (p<0.01). TTS also repressed expression of many other MDR genes, such as MRP1, MRP2, MRP3, MRP5, MVP and GST-π. In vivo, TTS dose-dependently reduced R-HepG2 cells xenografts tumour formation by inhibiting tumour cells proliferation in mice. Consistence with in vitro finding, TTS induced R-HepG2 sensitization to doxorubicin and therefore reduced tumour formation in vivo.

RNAi-mediated EZH2 depletion decreases MDR1 expression and sensitizes multidrug-resistant hepatocellular carcinoma cells to chemotherapy

The histone-lysine N-methyltransferase, enhancer of zeste homologue2 (EZH2), functions as a transcriptional repressor and plays an important role in the development of various types of cancer. In this study, we observed the increased EZH2 expression in the Bel/FU multidrug-resistant hepatocellular carcinoma (HCC) cell line. The RNA interference (RNAi)-mediated depletion of EZH2 expression in the Bel/FU cells led to decreased multidrug resistance protein1 (MDR1) expression, which resulted in increased apoptosis and sustained G1/Sphase arrest. Moreover, siRNA targeting EZH2 sensitized Bel/FU cells to 5-fluorouracil treatment. These findings suggest that EZH2 plays a role in the development of multidrug resistance and may represent a novel therapeutic target for multidrug-resistant HCC.

P-0078 Reversing Drug Resistance of Bel-7402/5fu to 5-Fluorouracil Via Suppression of Nrf2 by the Noncytotoxic Dose of Sorafenib

IntroductionA growing body of evidence suggests that chemoresistance is partially caused by the constitutive activation of the NF-E2-related factor 2 (Nrf2)-mediated antioxidant defense system in cancer cells. Here, we investigated the role of Nrf2 in a noncytotoxic dose of sorafenib induced reversal of multidrug resistance of hepatocellular carcinoma (HCC) cells. MethodsMTT colorimetric assay was used to select a noncytotoxic dose of sorafenib, and to detect the reverse-drug-resistance ability of sorafenib. To investigate the role of Nrf2 in the effect of reversing drug resistance by sorafenib, we measured the expression levels of Nrf2 by Western blots and RT-PCR assays, and analyzed the subcellular localization of Nrf2 by immunofluorescent assay. Furthermore, we assessed how the Nrf2 knockdown by RNA interference technology affected susceptibility to chemotherapeutic drugs. ResultsSorafenib at the concentration of 2 micromol/L efficiently reversed the multidrug resistance of Bel-7402/5fu cells with 2.69% cell inhibition rate. At the concentration of 2 micromol/L, sorafenib partially reversed the drug resistance of Bel-7402/5fu cells to 5-fluorouracil (5-fu) with reversal indexes of 2.96. Moreover, we demonstrated that the expression of Nrf2 in Bel-7402/5fu cells was not different from that in their parent cells Bel-7402. However, treated of cells with 5-fu, it was shown that 5-fu caused more pronounced up-regulated expression of Nrf2 in Bel-7402/5fu cells in comparison with Bel-7402 cells. The 5-fu induced up-regulation of Nrf2 could be significantly abolished by 2 micromol/L sorafenib. In addition, treatment of 5-fu induced Nrf2 transfer the cytoplasm into the nucleus, and this translocation could also be attenuated by sorafenib. Last, we selected two small interfering RNA (siRNA) to block the expression of Nrf2 specifically by Western blots and RT-PCR assays. The results showed that Nrf2-siRNA also effectively reversed the drug resistance of Bel-7402/5fu cells to 5-fu with reversal indexes of 2.45, and suppressed the translocation of Nrf2 induced by 5-fu. ConclusionOur findings provide evidence that the noncytotoxic dose of sorafenib can reverse the multidrug resistance of HCC cells by suppressing chemotherapy drug-induced Nrf2 activation, which can be applied as an adjunct to traditional chemotherapy in the treatment of HCC.

Epigenetic loss of CDH1 correlates with multidrug resistance in human hepatocellular carcinoma cells

Promoter CpG hypermethylation of tumor suppressor genes is an essential step in cancer progression but little is known about its effect on cancer multidrug resistance. In this study, we showed that CDH1 promoter was hypermethylated in drug resistance of a doxorubicin-induced multidrug resistant hepatocellular carcinoma cell line R-HepG2. Transfection of CDH1 cDNA into R-HepG2 cells led to increased amount of doxorubicin uptake, decreased cell viability, decreased P-glycoprotein expression and increased apoptotic population of cells exposed to doxorubicin. Proto-oncogene tyrosine-protein kinase FYN was over-expressed in R-HepG2 cells which displayed a negative correlation with the expression of CDH1. FYN was knocked down in R-HepG2 cells, leading to less drug resistance by increased cell viability, increased doxorubicin uptake and attenuated P-glycoprotein expression. Our findings identified epigenetic silencing of CDH1 in cancer cells might be a new molecular event of multidrug resistance.

Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A, a compound isolated from Hedyotis corymbosa

Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study.The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [3H]-thymidine incorporation assay.Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC50 = 43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC50 > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells.Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor.

Suppression of BCRP Expression and Restoration of Sensitivity to Chemotherapy in Multidrug-Resistant Hepatocellular Carcinoma Cell Line HEPG2/ADM by RNA Interference

Breast cancer resistance protein (BCRP) is an ATP-binding cassette multidrug transporter that confers resistance to various anticancer drugs like Adriamycin. Overexpression of BCRP confers multidrug resistance (MDR) in hepatocellular carcinoma cells and is a frequent impediment to successful chemotherapy. We evaluated a new approach using RNA interference for the specific knockdown of BCRP in hepatocellular carcinoma cells. To overcome the BCRP-mediated atypical multidrug drug resistance, one small interfering RNA construct (RNAi) targeting one special region of BCRP gene were designed to inhibit the atypical MDR expression by transfecting them into HepG2/ADM cell lines. We found that the overexpression of BCRP gene was suppressed efficiently by the introduction of small interfering RNA, which caused sequence specific gene silence. The level of BCRP mRNA reduced to 22.55% after transfected by pSUPER-BCRPs compared with those of the controls. Similarly, the level of BCRP decreased too. Furthermore, the sensitivity to Adriamycin of pSUPER-BCRPs-treated HEPG2/ADM cells is increased 3.55-fold compared to their control (p<0.05). The relative reverse rate of HepG2/ADM cell to Adriamycin is 72.2%. These data indicated that pSUPER-BCRPs could modulate MDR and may present a new approach to overcome BCRP-mediated drug resistance in HEPG2/ADM cells. It may reverse multidrug resistance phenotype and therefore provide promising therapeutic modalities in the treatment of hepatocellular carcinoma.