Doxorubicin Resistance In Hepatocellular Carcinoma Research Articles

SPG21, a potential oncogene targeted by miR-128-3p, amplifies HBx-induced carcinogenesis and chemoresistance via activation of TRPM7-mediated JNK pathway in hepatocellular carcinoma.

Chronic hepatitis B virus (HBV) infection is the primary risk factor for the malignant progression of hepatocellular carcinoma (HCC). It has been reported that HBV X protein (HBx) possesses oncogenic properties, promoting hepatocarcinogenesis and chemoresistance. However, the detailed molecular mechanisms are not fully understood. Here, we aim to investigate the effects of miR-128-3p/SPG21 axis on HBx-induced hepatocarcinogenesis and chemoresistance. The expression of SPG21 in HCC was determined using bioinformatics analysis, quantitative real-time PCR (qRT-PCR), western blotting, and immunohistochemistry (IHC). The roles of SPG21 in HCC were elucidated through a series of in vitro and in vivo experiments, including real-time cellular analysis (RTCA), matrigel invasion assay, and xenograft mouse model. Pharmacologic treatment and flow cytometry were performed to demonstrate the potential mechanism of SPG21 in HCC. SPG21 expression was elevated in HCC tissues compared to adjacent non-tumor tissues (NTs). Moreover, higher SPG21 expression correlated with poor overall survival. Functional assays revealed that SPG21 fostered HCC tumorigenesis and invasion. MiR-128-3p, which targeted SPG21, was downregulated in HCC tissues. Subsequent analyses showed that HBx amplified TRPM7-mediated calcium influx via miR-128-3p/SPG21, thereby activating the c-Jun N-terminal kinase (JNK) pathway. Furthermore, HBx inhibited doxorubicin-induced apoptosis by engaging the JNK pathway through miR-128-3p/SPG21. The study suggested that SPG21, targeted by miR-128-3p, might be involved in enhancing HBx-induced carcinogenesis and doxorubicin resistance in HCC via the TRPM7/Ca2+/JNK signaling pathway. This insight suggested that SPG21 could be recognized as a potential oncogene, offering a novel perspective on its role as a prognostic factor and a therapeutic target in the context of HCC.

Knockdown of the long noncoding RNA PURPL induces apoptosis and sensitizes liver cancer cells to doxorubicin

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with increasing incidence in western countries. Most HCC patients have advanced cancer at the time of diagnosis due to the asymptomatic nature of early-stage HCC and do not qualify for potentially curative surgical treatment, thus, highlighting the need for new therapeutic strategies. Long noncoding RNAs (lncRNAs) comprise a large and heterogeneous group of non-protein coding transcripts that play important regulatory roles in numerous biological processes in cancer. In this study, we performed RNA sequencing of liver biopsies from ten HCC, ten hepatitis C virus-associated HCC, and four normal livers to identify dysregulated lncRNAs in HCC. We show that the lncRNA p53-upregulated-regulator-of-p53-levels (PURPL) is upregulated in HCC biopsies and that its expression is p53-dependent in liver cancer cell lines. In addition, antisense oligonucleotide-mediated knockdown of PURPL inhibited cell proliferation, induced apoptosis, and sensitized HepG2 human HCC cells to treatment with the chemotherapeutic agent doxorubicin. In summary, our findings suggest that PURPL could serve as a new therapeutic target for reversing doxorubicin resistance in HCC.

The lncARSR/PTEN/Akt/nuclear factor-kappa B feedback regulatory loop contributes to doxorubicin resistance in hepatocellular carcinoma.

Chemoresistance is a major obstacle to hepatocellular carcinoma (HCC) chemotherapy. Our previous study found that long noncoding RNA lncARSR (lncRNA Activated in RCC with Sunitinib Resistance) activated Akt signaling via repressing phosphatase and tensin homolog (PTEN) during doxorubicin resistance in HCC. The purpose of this study is to further explore lncARSR-mediated mechanisms and roles during doxorubicin resistance in HCC. The expression of lncARSR was detected by real-time quantitative polymerase chain reaction (qPCR). Nuclear factor-kappa B (NF-κB) activity was detected by NF-κB luciferase reporter assays, western blot, and NF-κB transcription factor assays. The effects of NF-κB on lncARSR were detected by chromatin immunoprecipitation assay, promoter luciferase reporter assay, and real-time qPCR. The effects of lncARSR/Akt/NF-κB on doxorubicin resistance were detected by Cell Counting Kit-8 assay, capsase-3 activity assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelingassay. lncARSR activated NF-κB signaling through activation of Akt. NF-κB transactivated lncARSR through directly binding lncARSR promoter and increasing lncARSR promoter activity. Akt transactivated lncARSR via activating NF-κB signaling. Thus, lncARSR, Akt, and NF-κB formed a positive feedback regulatory loop in HCC. Through this feedback loop, lncARSR auto-regulated its transcription. Drug sensitivity assays showed that the lncARSR/Akt/NF-κB feedback regulatory loop promoted doxorubicin resistance in HCC. These findings identified the lncARSR/Akt/NF-κB feedback regulatory loop in HCC, which represent potential therapeutic targets for improving doxorubicin sensitivity in HCC.

Linc‑ROR facilitates hepatocellular carcinoma resistance to doxorubicin by regulating TWIST1‑mediated epithelial‑mesenchymal transition.

Long non-coding RNAs are associated with cancer progression. Long intergenic non-protein coding RNA (linc)-regulator of reprogramming (ROR) enhances tumor development in hepatocellular carcinoma (HCC). However, the effect of chemoresistance and its underlying mechanisms in HCC are not completely understood. The present study aimed to identify the effect of ROR on sensitivity to doxorubicin (DOX) in HCC cells. In the present study, Cell Counting Kit-8 and EdU assays were performed to assess cell viability and proliferation, respectively. In addition, E-cadherin and vimentin protein expression levels were assessed via western blotting and immunofluorescence. The results of the present study demonstrated that HCC cells with high linc-ROR expression levels were more resistant to DOX, and linc-ROR knockdown increased HCC cell DOX sensitivity compared with the control group. The results indicated that compared with the NC siRNA group, linc-ROR knockdown notably suppressed epithelial-mesenchymal transition by downregulating twist family bHLH transcription factor 1 (TWIST1) expression. TWIST1 knockdown displayed a similar effect on HCC cell DOX sensitivity to linc-ROR knockdown. Moreover, linc-ROR knockdown-induced HCC cell DOX sensitivity was inhibited by TWIST1 overexpression. The present study provided evidence that linc-ROR promoted HCC resistance to DOX by inducing EMT via interacting with TWIST1. Therefore, linc-ROR might serve as a therapeutic target for reducing DOX resistance in HCC.

Circ_0003998 enhances doxorubicin resistance in hepatocellular carcinoma by regulating miR-218-5p/EIF5A2 pathway

BackgroundThe involvement of circular RNAs (circRNAs) in chemoresistance of tumors has been identified. Herein, this study aims to investigate the role and the underlying mechanism of circ_0003998 in doxorubicin (DOX) resistance in hepatocellular carcinoma (HCC).MethodsThe expression of circ_0003998 and microRNA (miR)-218-5p and eukaryotic translation initiation factor 5A-2 (EIF5A2) mRNA was detected using quantitative real-time polymerase chain reaction. Cell viability, migration and invasion were analyzed using cell counting kit-8, colony formation and transwell assay, respectively. The levels of matrix metallopeptidase 9 (MMP-9), E-cadherin, Vimentin, N-cadherin and EIF5A2 protein were detected using western blot. The interaction between miR-218-5p and circ_0003998 or EIF5A2 was confirmed by dual-luciferase reporter assay. In vivo experiments were performed using murine xenograft models.ResultsCirc_0003998 was elevated in HCC tissues, DOX-resistant tissues and cells, and circ_0003998 knockdown promoted DOX-sensitivity in HCC by inhibiting resistant cell viability, migration, invasion and EMT in vitro and enhanced DOX cytotoxicity in vivo. Bioinformatics analysis revealed circ_0003998 inhibited miR-218-5p expression, which was clarified to be a target of circ_0003998, and circ_0003998 knockdown sensitized HCC cell to DOX by sponging miR-218-5p. EIF5A2 was a target of miR-218-5p, and miR-218-5p mitigated DOX resistance in HCC cells through modulating EIF5A2 expression. Additionally, circ_0003998 served as a competing endogenous RNA for miR-218-5p to regulate EIF5A2 expression.ConclusionCirc_0003998 knockdown sensitized HCC cell to DOX by regulating miR-218-5p/EIF5A2 axis, indicating new markers of poor response to DOX and potential therapeutic strategies for the chemotherapy of HCC.

Knockdown of eukaryotic translation initiation factor 5A2 enhances the therapeutic efficiency of doxorubicin in hepatocellular carcinoma cells by triggering lethal autophagy.

Hepatocellular carcinoma (HCC) is an invasive malignant neoplasm with a poor prognosis. The development of chemoresistance severely obstructs the chemotherapeutic efficiency of HCC treatment. Therefore, understanding the mechanisms of chemoresistance is important for improving the outcomes of patients with HCC. Eukaryotic translation initiation factor 5A2 (eIF5A2), which is considered to be an oncogene, has been reported to mediate chemoresistance in various types of cancer; however, its precise role in HCC remains unclear. Accumulating evidence has suggested that autophagy serves a dual role in cancer chemotherapy. The present study aimed to investigate the role of autophagy in eIF5A2-mediated doxorubicin resistance in HCC. High expression levels of eIF5A2 in human HCC tissues were observed by immunohistochemistry using a tissue microarray, which was consistent with the results of reverse transcription-quantitative PCR analysis in paired HCC and adjacent healthy tissues. HCC patient-derived tumor xenograft mouse model was used for the in vivo study, and knockdown of eIF5A2 effectively enhanced the efficacy of doxorubicin chemotherapy compared with that in the control group. Notably, eIF5A2 served as a repressor in regulating autophagy under chemotherapy. Silencing of eIF5A2 induced doxorubicin sensitivity in HCC cells by triggering lethal autophagy. In addition, 5-ethynyl-2′-deoxyuridine, lactate dehydrogenase release assay and calcein-AM/PI staining were used to determine the enhanced autophagic cell death induced by the silencing of eIF5A2 under doxorubicin treatment. Suppression of autophagy attenuated the sensitivity of HCC cells to doxorubicin induced by eIF5A2 silencing. The results also demonstrated that knockdown of the Beclin 1 gene, which is an autophagy regulator, reversed the enhanced autophagic cell death and doxorubicin sensitivity induced by eIF5A2 silencing. Taken together, these results suggested eIF5A2 may mediate the chemoresistance of HCC cells by suppressing autophagic cell death under chemotherapy through a Beclin 1-dependent pathway, and that eIF5A2 may be a novel potential therapeutic target for HCC treatment.

A stimuli-responsive combination therapy for recovering p53-inactivation associated drug resistance.

Drug resistance is a major hindrance in the anticancer treatment, which encourages the development of effective therapeutic strategies. For the first time, MDM2-mediated p53 degradation was identified as a critical factor for developing acquired resistance of doxorubicin (DOX) in HepG2 tumor spheroids, which could be effectively reversed by MDM2 inhibitor MI-773, thereby improving anticancer effects. Therefore, a pH-sensitive liposomal formulation of DOX and MI-773 (LipD/M@CMCS) were developed for recovering p53-mediated DOX resistance in hepatocellular carcinoma. LipD/M@CMCS were composed of cationic liposomes covered with carboxymethyl chitosan (pI = 6.8), and were stable in the physiological condition (pH 7.4), but rapidly converted to cationic liposomes in tumor acidic microenvironment (pH 6.5), endowing them with tumor specificity and enhanced cellular uptake. We showed that LipD/M@CMCS could not only effectively induce cell apoptosis in HepG2 tumor spheroids, but significantly inhibit tumor growth with minimal adverse effects. In summary, selective regulation of MDM2 in cancer cells is a promising strategy to overcome DOX resistance, and may provide a perspective on the management of malignant tumors.

C-terminal truncated HBx reduces doxorubicin cytotoxicity via ABCB1 upregulation in Huh-7 hepatocellular carcinoma cells

Hepatitis B virus (HBV) encoding the HBV x protein (HBx) is a known causative agent of hepatocellular carcinoma (HCC). Its pathogenic activities in HCC include interference with several signaling pathways associated with cell proliferation and apoptosis. Mutant C-terminal-truncated HBx isoforms are frequently found in human HCC and have been shown to enhance proliferation and invasiveness leading to HCC malignancy. We investigated the molecular mechanism of the reduced doxorubicin cytotoxicity by C-terminal truncated HBx. Cells transfected with C-terminal truncated HBx exhibited reduced cytotoxicity to doxorubicin compared to those transfected with full-length HBx. The doxorubicin resistance of cells expressing C-terminal truncated HBx correlated with upregulation of the ATP binding cassette subfamily B member 1(ABCB1) transporter, resulting in the enhanced efflux of doxorubicin. Inhibiting the activity of ABCB1 and silencing ABCB1 expression by small interfering ribonucleic acid (siRNA) increased the cytotoxicity of doxorubicin. These results indicate that elevated ABCB1 expression induced by C-terminal truncation of HBx was responsible for doxorubicin resistance in HCC. Hence, co-treatment with an ABCB1 inhibitor and an anticancer agent may be effective for the treatment of patients with liver cancer containing the C-terminal truncated HBx.

MicroRNA-383 inhibits doxorubicin resistance in hepatocellular carcinoma by targeting eukaryotic translation initiation factor 5A2.

Drug resistance occurs commonly in cancers, especially in hepatocellular carcinoma (HCC). Accumulating evidence has demonstrated that microRNAs (miRNAs) play a vital role in tumour chemoresistance. However, little is known about the role of miR‐383 in HCC chemoresistance. In the present study, RT‐PCR and western blotting were used to identify the expression profile of miR‐383 and eukaryotic translation initiation factor 5A2 (EIF5A2). The bioinformatics website Targetscan was used to predict the target genes of miR‐383. In vitro and in vivo loss‐ and gain‐of‐function studies were performed to reveal the effects and potential mechanism of the miR‐383/EIF5A2 axis in chemoresistance of HCC cells. The expression level of miR‐383 correlated negatively with doxorubicin (Dox) sensitivity. Overexpression of miR‐383 promoted HCC cells to undergo Dox‐induced cytotoxicity and apoptosis, whereas miR‐383 knockdown had the opposite effects. EIF5A2 was predicted as a target gene of miR‐383. EIF5A2 knockdown sensitized HCC cells to Dox. Moreover, miR‐383 inhibition‐mediated HCC Dox resistance could be reversed by silencing EIF5A2. Finally, we demonstrated that miR‐383 inhibition could enhance Dox sensitivity by targeting EIF5A2 in vivo. The results indicated that miR‐383 inhibited Dox resistance in HCC cells by targeting EIF5A2. Targeting the miR‐383/EIF5A2 axis might help to alleviate the chemoresistance of HCC cells.

Lgr5-mediated p53 Repression through PDCD5 leads to doxorubicin resistance in Hepatocellular Carcinoma

The devastating prognosis of hepatocellular carcinoma (HCC) is partially attributed to chemotherapy resistance. Accumulating evidence suggests that the epithelial-mesenchymal transition (EMT) is a key driving force of carcinoma metastasis and chemoresistance in solid tumors. Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), as an EMT inducer, is involved in the potentiation of Wnt signaling in HCC. This study proposes uncovering the roles of Lgr5 in Doxorubicin (Dox) resistance of HCC to improve treatment efficacy for HCC.Methods: We investigated the expression and significance of Lgr5 in HCC tissue and different cell lines. The effect of Lgr5 in EMT and Dox resistance was analyzed in HCC cells and implanted HCC tumor models. A two-hybrid analysis, using the Lgr5 gene as the bait and a HCC cDNA library, was used to screen targeted proteins that interact with Lgr5. The positive clones were identified by coimmunoprecipitation (Co-IP) and Glutathione-S-transferase (GST) pull-down. The impact of the interaction on Dox resistance was investigated by a series of assays in vitro and in vivo .Result: We found that Lgr5 was upregulated and positively correlated with poor prognosis in HCC. Additionally, it functioned as a tumor promoter to increase cell migration and induce EMT in HCC cells and increase the resistance to Dox. We identified programmed cell death protein 5 (PDCD5) as a target gene of Lgr5 and we found that PDCD5 was responsible for Lgr5-mediated Dox resistance. Further analysis with Co-IP and GST pull-down assays showed that the N-terminal extracellular domain of Lgr5 could directly bind to PDCD5. Lgr5 induced p53 degradation by blocking the nuclear translocation of PDCD5 and leading to the loss of p53 stabilization. Lgr5 showed a protection against the inhibition of Dox on the growth of tumor subcutaneously injected. Moreover, Lgr5 suppressed Dox-induced apoptosis via the p53 pathway and attenuated the cytotoxicity of Dox to HCC.Conclusion: Lgr5 induces the EMT and inhibits apoptosis, thus promoting chemoresistance by regulating the PDCD5/p53 signaling axis. Furthermore, Lgr5 may be a potential target gene for overcoming Dox resistance.

FSCN‑1 increases doxorubicin resistance in hepatocellular carcinoma through promotion of epithelial-mesenchymal transition.

Resistance to chemotherapy drugs remains a significant problem for the treatment of many types of cancer. Fascin-1 (FSCN-1) is an actin-bundling protein involved in the invasion and metastasis of a variety of tumors. However, its involvement in drug resistance in hepatocellular carcinoma (HCC) remains unclear. The present study aimed to investigate the function of FSCN-1 in HCC resistance to doxorubicin (DOX). FSCN-1 expression was increased in DOX-resistant HCC cell lines (SNU449 and SNU387) compared with DOX-sensitive cell lines (Huh7 and Hep3B). The resistance of HCC cells to DOX was decreased following FSCN-1 knockdown with small interfering RNA. FSCN-1 knockdown also significantly altered the expression of key markers of epithelial-mesenchymal transition (EMT). Notably, vimentin expression was reduced and epithelial-cadherin expression was increased. Furthermore, when EMT was suppressed through knockdown of Twist, an essential pathway of DOX-induced EMT, the viability of HCC cells following treatment with DOX was not affected by FSCN-1 expression. Furthermore, FSCN-1 knockdown eliminated hypoxia-induced doxorubicin resistance and EMT. The results of the present study indicated that FSCN-1 expression increased DOX resistance in HCC cells via the promotion of EMT, and this phenomenon was maintained in a hypoxic environment. FSCN-1 potentially represents a novel target to overcome resistance to DOX in HCC.

CDKN2B is critical for verapamil-mediated reversal of doxorubicin resistance in hepatocellular carcinoma.

In this study, we explored the function and mechanism of CDKN2B genes in verapamil (VER)-induced reversal of resistance to doxorubicin (ADM) chemotherapy in hepatocellular carcinoma (HCC). We examined 4 HCC cell lines and found that the expression levels of CDKN2B genes correlated with the level of apoptosis induced by ADM+VER. Overexpression of CDKN2B genes promoted apoptosis in cells treated with VER+ADM. CDKN2B knockdown using siRNA weakened the effect of ADM+VER, indicating that ADM+VER promotes HCC cell apoptosis and that CDKN2B genes participate in VER-mediated promotion in tumor cell apoptosis. Future research will further explore the functional mechanism, and the associated signal transduction pathways via which CDKN2B affects HCC drug resistance.

Connexin 32 affects doxorubicin resistance in hepatocellular carcinoma cells mediated by Src/FAK signaling pathway

Doxorubicin (DOX) is first-line chemotherapy for hepatocellular carcinoma (HCC), but the effect is not satisfactory. The resistance of HCC cells to DOX is the main reason leading to treatment failure. Therefore, it is necessary to study the mechanism of DOX resistance in HCC. In this study, expression of connexin (Cx)32 was significantly decreased in HCC tissues compared with corresponding paracancerous tissues, and activity of the Src/focal adhesion kinase (FAK) signaling pathway was significantly enhanced. Expression of Cx32 was closely associated with activity of the Src/FAK signaling pathway, Cx32, and the Src/FAK signaling pathway was also correlated with degree of HCC differentiation. In DOX-resistant HepG2 cells, compared with DOX-sensitive HepG2 cells, expression of Cx32 was significantly reduced and activity of the Src/FAK pathway increased. After silencing Cx32 in HepG2 cells, activity of the Src/FAK pathway increased and sensitivity to DOX decreased. In contrast, overexpression of Cx32 in HepG2/DOX cells decreased activity of the Src/FAK pathway and increased sensitivity to DOX. Dasatinib and KX2-391, inhibitors of the Src/FAK pathway, significantly increased the sensitivity of HepG2/DOX cells to DOX. The results suggest that Src/FAK is a downstream regulator of Cx32 and Cx32 regulates the sensitivity of HCC cells to DOX via the Src/FAK signaling pathway. Our study demonstrates a potential mechanism of DOX resistance in HCC cells and supports that Cx32–Src/FAK is an important target for reversing drug resistance of HCC.

Long Noncoding RNA lncARSR Promotes Doxorubicin Resistance in Hepatocellular Carcinoma via Modulating PTEN-PI3K/Akt Pathway.

Hepatocellular carcinoma (HCC) is generally resistant to chemotherapy due to intrinsic or acquired drug resistances. Many molecules and signaling pathways are involved in chemo-resistance of HCC cells. However, the contribution of long noncoding RNA (lncRNA) to chemo-resistance of HCC cells is still largely unknown. In this study, we revealed the critical roles of long noncoding RNA lncARSR in chemo-resistance of HCC cells. lncARSR is upregulated in HCC, associated with large tumor size and advanced BCLC stage, and indicts poor prognosis. Functional assays showed that overexpression of lncARSR enhances doxorubicin resistance of HCC cells in vitro and in vivo. And while knockdown of lncARSR increases sensitivity of HCC cells to doxorubicin in vitro and in vivo. Mechanistically, we found that lncARSR physically associates with PTEN mRNA, promotes PTEN mRNA degradation, decreases PTEN expression, and activates PI3K/Akt pathway. PTEN is downregulated in HCC, and the expression of PTEN is negatively correlated with lncARSR in HCC tissues. Furthermore, the effects of lncARSR overexpression on doxorubicin resistance could be reversed by PI3K/Akt pathway inhibitor, and lncARSR knockdown-induced doxorubicin sensitivity could be reversed by PTEN depletion. Taken together, our results showed that upregulated lncARSR promotes doxorubicin resistance in HCC via modulating PTEN-PI3K/Akt pathway, and implied that lncARSR may serve as a promising prognostic biomarker and therapeutic target for HCC chemo-resistance. J. Cell. Biochem. 118: 4498-4507, 2017. © 2017 Wiley Periodicals, Inc.

Cx32 reverses epithelial-mesenchymal transition in doxorubicin-resistant hepatocellular carcinoma.

Recently, epithelial-mesenchymal transition (EMT) has been reported to be an important mechanism of drug resistance in numerous types of cancer cells, including hepatocellular carcinoma (HCC). However, the underlying mechanisms remain to be fully elucidated. Connexin (Cx)32 plays a crucial role in hepatocarcinogenesis. The present study investigated the role of Cx32 in the regulation of chemotherapy-induced EMT in HCC. We found that the expression levels of Cx32 and E-cadherin were clearly decreased in HCC tissues compared with the corresponding paracancerous tissues, while the expression level of vimentin was significantly enhanced in HCC tissues. The expression of Cx32 had a strong correlation with the expression of E-cadherin and vimentin. In an invitro study, a doxorubicin (DOX)-resistant liver cell line HepG2/DOX was established from parental HepG2 cells. The results showed that HepG2/DOX cells acquired EMT characteristics, with a decreased expression level of E-cadherin and an enhanced expression level of vimentin, and possessed high migratory abilities and invasiveness. Meanwhile, Cx32 was significantly decreased in the HepG2/DOX cells. Knockdown of Cx32 by shRNA in HepG2 cells induced EMT, while overexpression of Cx32 converted EMT to mesenchymal-epithelial transition (MET) in the HepG2/DOX cells. These results suggest that Cx32 is an important regulator of DOX-induced EMT in HCC. Cx32 could be considered as a novel target to reverse DOX resistance in HCC.

MiR-122 Reverses the Doxorubicin-Resistance in Hepatocellular Carcinoma Cells through Regulating the Tumor Metabolism.

Doxorubicin (DOX) is one of the most commonly used anticancer drugs in the treatment of hepatoma. However, acquired drug resistance is one of the major challenges for the chemotherapy. In this study, a down-regulation of miR-122 was observed in doxorubicin-resistant Huh7 (Huh7/R) cells compared with its parental Huh7 cells, suggesting miR-122 is associated with the chemoresistance. Meanwhile, luciferase reporter assay proved that the PKM2 is the target of miR-122, and we reported that the glucose metabolism is significantly up-regulated in Huh7/R cells. Importantly, overexpression of miR-122 in Huh7/R cells reversed the doxorubicin-resistance through the inhibition of PKM2, inducing the apoptosis in doxorubicin-resistant cancer cells. Thus, this study revealed that the dysregulated glucose metabolism contributes to doxorubicin resistance, and the inhibition of glycolysis induced by miR-122 might be a promising therapeutic strategy to overcome doxorubicin resistance in hepatocellular carcinoma.

ARK5 promotes doxorubicin resistance in hepatocellular carcinoma via epithelial–mesenchymal transition

AMP-activated protein kinase family member 5 (ARK5) overexpression has been reported in many human cancers, and ARK5 is associated with poor prognosis in hepatocellular carcinoma (HCC). However, whether ARK5 is involved in HCC chemoresistance is unclear. The present study aimed to investigate the role of ARK5 in HCC chemoresistance and the underlying mechanism. In this study, we found that SNU387 and SNU449 HCC cell lines overexpressing ARK5 displayed low doxorubicin sensitivity compared to Huh7 and Hep3B HCC cell lines with ARK5 low-expression. And knockdown of ARK5 increased the doxorubicin sensitivity in all HCC cell lines in the manner of inhibiting cell proliferation. Western blotting and immunofluorescence both showed that ARK5 knockdown upregulated E-cadherin and downregulated vimentin, which was consistent with the knockdown of TWIST, indicating ARK5 was involved in epithelial–mesenchymal transition (EMT). Moreover, suppressing ARK5 by siRNA restored E-cadherin and vimentin expression induced by doxorubicin treatment or hypoxia culture. Our results indicated ARK5 confers doxorubicin resistance in HCC via inducing EMT.

Genome-Wide Analysis of miRNA Signature Differentially Expressed in Doxorubicin-Resistant and Parental Human Hepatocellular Carcinoma Cell Lines

Chemotherapy regiments have been widely used in the treatment of a variety of human malignancies including hepatocellular carcinoma (HCC). A major cause of failure in chemotherapy is drug resistance of cancer cells. Resistance to doxorubicin (DOX) is a common and representative obstacle to treat cancer effectively. Individual microRNA (miRNA) has been introduced in the evolution of DOX resistance in HCC in recent studies. However, a global and systematic assessment of the miRNA expression profiles contributing to DOX resistance is still lacking. In the present study, we applied high-throughput Illumina sequencing to comprehensively characterize miRNA expression profiles in both human HCC cell line (HepG2) and its DOX-resistant counterpart (HepG2/DOX). A total of 269 known miRNAs were significantly differentially expressed, of which 23 were up-regulated and 246 were down-regulated in HepG2/DOX cells, indicating that part of them might be involved in the development of DOX resistance. In addition, we have identified 9 and 13 novel miRNAs up- and down-expressed significantly in HepG2/DOX cells, respectively. miRNA profiling was then validated by quantitative real-time PCR for selected miRNAs, including 22 known miRNAs and 6 novel miRNAs. Furthermore, we predicted the putative target genes for the deregulated miRNAs in the samples. Function annotation implied that these selected miRNAs affected many target genes mainly involved in MAPK signaling pathway. This study provides us a general description of miRNA expression profiling, which is helpful to find potential miRNAs for adjunct treatment to overcome DOX resistance in future HCC chemotherapy.

Overexpression of Yes-associated protein confers doxorubicin resistance in hepatocellullar carcinoma

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide and is highly resistant to chemotherapy. Yes-associated protein (YAP) is the downstream effector of the Hippo signaling pathway, which is frequently overexpressed in many types of cancers. Amplification of the YAP gene and overexpression of YAP in HCC have previously been reported to contribute to hepatocyte malignant transformation and tumor progression. In this study, we aimed to investigate the potential role of YAP in HCC chemoresistance. Overexpression of YAP resulted in resistance against doxorubicin-induced apoptosis in HCC cell lines, whereas suppression of the endogenous YAP expression by RNA interference demonstrated the reverse effect. Western blotting revealed that, following exposure to doxorubicin, YAP-overexpressing cells exhibited decreased cleaved PARP, increased phosphorylation of Akt and ERK1/2, and elevated Bcl-xL expression in comparison to the vector control. Inhibition of YAP expression sensitized HCC cells to doxorubicin, by exhibiting increased cleaved PARP, decreased levels of phosphorylated Akt, phosphorylated ERK1/2 and Bcl-xL expression. In addition, pretreatment with the MEK1/2 inhibitor U0126 but not the PI3-K inhibitor LY294002 significantly enhanced doxorubicin-induced apoptosis and decreased Bcl-xL expression in YAP-overexpressing HCC cells. Our data provide evidence that overexpression of YAP plays an important role in conferring doxorubicin resistance to HCC, which is at least partially mediated by YAP-induced activation of the MAP kinase pathway. Targeting YAP may be a promising adjunct for overcoming doxorubicin resistance in HCC.

MDR1 gene expression: its effect on drug resistance to doxorubicin in human hepatocellular carcinoma cell lines.

Hepatic tumors are resistant to many chemotherapeutic agents. Although elevated MDR1 (also known as PGY1) gene expression has been shown in such tumors, no direct association has been established between the gene expression and multidrug resistance. To evaluate the role of the MDR1 gene in the drug resistance of hepatoma, we tested nine human hepatoma cell lines for their expression of the MDR1 gene. We measured the MDR1 messenger RNA (mRNA) expression by RNA slot-blot analysis and by immunocytochemical staining with a P-glycoprotein-specific monoclonal antibody, MRK16. The in vitro chemosensitivity of these cell lines to fluorouracil, doxorubicin, mitomycin C, cisplatin, and etoposide (VP-16) was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. For doxorubicin cytotoxicity, we also tested the potentiating effect of several multidrug resistance-reversing agents. Slot-blot analysis and immunocytochemistry showed that two cell lines expressed high levels of MDR1 mRNA, one expressed an intermediate level, and all others were low expressors. The MTT assay results showed that all cell lines tested were generally resistant to chemotherapeutic agents. The assay area under the curve (AUC) was within a clinically achievable range only for VP-16 in one of nine cell lines. When the IC50 values were compared among the cell lines, the results revealed a close association with the MDR1 gene expression only for doxorubicin resistance. Verapamil and quinidine lowered the IC50 values of doxorubicin for MDR1-positive cell lines. The lowered assay AUC levels for both reversing agents, however, were still higher than the clinically achievable range. These results indicate that the MDR1 gene probably has a role in doxorubicin resistance in hepatocellular carcinoma and that the resistance can be overcome by some multidrug resistance-reversing agents. Some widely used anticancer agents might be ineffective for treating hepatocellular carcinoma in clinical situations even when combined with reversing agents.