Drug-resistant Hepatocellular Carcinoma Cells Research Articles

Protein and metabolic profiles of tyrosine kinase inhibitors co-resistant liver cancer cells

Hepatocellular Carcinoma (HCC) patients often develop resistance to tyrosine kinase inhibitors (TKIs) like sorafenib (SR) and lenvatinib (RR). We established HCC cell lines resistant to these drugs and analyzed the correlation between protein and metabolite profiles using bioinformatics. Our analysis revealed overexpression of MISP, CHMP2B, IL-18, TMSB4X, and EFEMP1, and downregulation of IFITM3, CA4, AGR2, and SLC51B in drug-resistant cells. Differential signals are mainly enriched in steroid hormone biosynthesis, cell adhesion, and immune synapses, with metabolic pathways including cytochrome P450 drug metabolism, amino acid metabolism, and glycolysis. Proteomics and metabolomics analysis showed co-enrichment signals in drug metabolism, amino acids, glucose metabolism, ferroptosis, and other biological processes. Knocking down MISP, CHMP2B, IL-18, TMSB4X, and EFEMP1 significantly reduced drug resistance, indicating their potential as therapeutic response biomarkers. This study characterizes protein and metabolic profiles of drug-resistant HCC cells, exploring metabolite-protein relationships to enhance understanding of drug resistance mechanisms and clinical treatment.

Polyphyllin I attenuates the invasion and metastasis via downregulating GRP78 in drug-resistant hepatocellular carcinoma cells.

Drug resistance to chemotherapy agents presents a major obstacle to the effective treatment of hepatocellular carcinoma (HCC), a common type of liver cancer. Increasing evidence indicates a link between drug resistance and the recurrence of HCC. Polyphyllin I (PPI), a promising pharmaceutical candidate, has shown potential therapeutic advantages in the treatment of sorafenib-resistant hepatocellular carcinoma (SR-HCC cells). In this study, we sought to investigate the mechanism underlying the inhibitory effect of PPI on the invasion and metastasis of SR-HCC cells. Our in vitro studies included scratch wound-healing migration assays and transwell assays to examine PPI's effect on HCC cell migration and invasion. Flow cytometry was employed to analyze the accumulation or efflux of chemotherapy drugs. The results of these experiments demonstrated that PPI increased the susceptibility of HCC to sorafenib while inhibiting SR-HCC cell growth, migration, and invasion. Molecular docking analysis revealed that PPI exhibited a higher binding affinity with GRP78. Western blot analysis and immunofluorescence experiments showed that PPI reduced the expression of GRP78, E-cadherin, N-cadherin, Vimentin, and ABCG2 in SR-HCC cells. Interference with and overproduction of GRP78 in vitro impacted the proliferation, migration, invasion, and metastasis of HCC cells. Further examination revealed that PPI hindered the expression of GRP78 protein, resulting in a suppressive effect on SR-HCC cell migration and invasion. Histological examination of tumor tissue substantiated that administering PPI via gavage to HepG2/S xenograft nude mice inhibited tumor growth and significantly reduced tumor size, as evidenced by xenograft experiments involving nude mice. Hematoxylin and eosin (HE) staining of tumor tissue specimens, along with immunohistochemistry (IHC), were conducted to evaluate the expression levels of Ki67, GRP78, N-cadherin, Vimentin, and ABCG2. The results indicated that PPI administration decreased the levels of proteins associated with metastasis and markers of drug resistance in tumor tissues, impeding tumor growth and spread. Overall, our findings demonstrated that PPI effectively suppressed the viability, proliferation, invasion, and metastasis of SR-HCC cells both in vitro and in vivo by modulating GRP78 activity. These findings provide new insights into the mechanism of PPI inhibition of SR-HCC cell invasion and metastasis, highlighting PPI as a potential treatment option for sorafenib-resistant HCC.

Sorafenib Resistance Contributed by IL7 and MAL2 in Hepatocellular Carcinoma Can Be Overcome by Autophagy-Inducing Stapled Peptides.

Drug resistance poses a great challenge in systemic therapy for hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms associated with resistance to anti-cancer drugs, such as Sorafenib, remain unclear. In this study, we use transposon insertional mutagenesis to generate Sorafenib-resistant HCC cell lines in order to identify potential drug resistant causative genes. Interleukin 7 (IL7) and mal, T cell differentiation protein 2 (MAL2) were identified as candidate genes that promote survival by activating JAK/STAT and PI3K/AKT signaling pathways. Sorafenib-resistant cells exhibited higher clonogenic survival and lower drug sensitivity due to IL7 and MAL2 upregulation. Higher anti-apoptotic effect, clonogenic survival and increased PI3K/AKT/STAT3 activities were observed in IL7 and MAL2 co-overexpressing cells compared with controls or cells overexpressing IL7 or MAL2 individually. Given the critical role of MAL2 in endocytosis, we propose that MAL2 might facilitate the endocytic trafficking of IL7 and its cognate receptors to the plasma membrane, which leads to upregulated JAK/STAT and PI3K/AKT signaling pathways and Sorafenib resistance. Additionally, our previous studies showed that an autophagy-inducing stapled peptide promoted the endolysosomal degradation of c-MET oncogene and overcame adaptive Sorafenib resistance in c-MET+ HCC cells. In this study, we demonstrate that these stapled peptides readily induced autophagy and inhibited the proliferation of both wild-type and Sorafenib-resistant HCC cells co-overexpressing both IL7 and MAL2. Furthermore, these peptides showed synergistic cytotoxicity with Sorafenib in drug-resistant HCC cells co-overexpressing both IL7 and MAL2. Our studies suggest that targeting autophagy may be a novel strategy to overcome IL7/MAL2-mediated Sorafenib resistance in HCC.

Mitochondria-Targeting-Based of Paclitaxel-Loaded Triphenylphosphine-Pluronic F127-Hyaluronic Acid Nanomicelles in Multi-Drug Resistant Hepatocellular Carcinoma

Background:In this study a new novel nanomicelle (TPH) sco-loaded with triphenylphosphine (TPP)-Pluronic F127-hyaluronic acid (HA) and Paclitaxel (PTX) has been designed to treat multidrug resistant hepatocellular carcinoma (HCC).Methods:TPH was initially synthesized by ester bond formation with mitochondria-targeting TPP agent and TPH nanomicelles loaded with PTX (TPH/PTX) had outstanding physical characteristics in human multi drug-resistant HCC cell line Bel7402/5-FU. Cytotoxicity and hemocompatibility assessments, nanomicelle cellular absorption and mitochondrial targeting, andin vivoxenograft imaging was used to evaluate that the nonemicells delivered into target cell and components.Results:The results of fluorescence test showed that TPP could promote the fusion of nanomicells to human multi drugresistant HCC cell line Bel7402/5-FU, and targeted the mitochondria, and also improved the targeting and retention of drugs in liver tumors. The results of cell efficacy showed that TPH/PTX induced a strong apoptosis effect, which could significantly reduce the mitochondrial membrane Zeta potential, increase the level of intracellular ROS and the release of Caspase-3, significantly enhanced the pro-apoptotic protein (Bcl-2), decrease the expression level of anti-apoptotic protein (Bax).Conclusion:TPH/PTX has a promising mitochondrial targeting function, and can enhance the effect of drugs on promoting apoptosis of drug resistant HCC cells.

CISD2 Promotes Resistance to Sorafenib-Induced Ferroptosis by Regulating Autophagy in Hepatocellular Carcinoma.

PurposeSorafenib is a multi-kinase inhibitor that is used as a standard treatment for advanced hepatocellular carcinoma (HCC). However, the mechanism of sorafenib resistance in HCC is still unclear. It has been shown that CISD2 expression is related to the progression and poor prognosis of HCC. Here, we show a new role for CISD2 in sorafenib resistance in HCC.MethodsBioinformatic analysis was used to detect the expression of negative regulatory genes of ferroptosis in sorafenib-resistant samples. The concentration gradient method was used to establish sorafenib-resistant HCC cells. Western blot was used to detect the protein expression of CISD2, LC3, ERK, PI3K, AKT, mTOR, and Beclin1 in HCC samples. Quantitative real-time PCR (qPCR) was used to detect gene expression. CISD2 shRNA and Beclin1 shRNA were transfected to knock down the expression of the corresponding genes. Cell viability was detected by a CCK-8 assay. ROS were detected by DCFH-DA staining, and MDA and GSH were detected with a Lipid Peroxidation MDA Assay Kit and Micro Reduced Glutathione (GSH) Assay Kit, respectively. Flow cytometry was used to detect apoptosis and the levels of ROS and iron ions.ResultsCISD2 was highly expressed in HCC cells compared with normal cells and was associated with poor prognosis in patients. Knockdown of CISD2 promoted a decrease in the viability of drug-resistant HCC cells. CISD2 knockdown promoted sorafenib-induced ferroptosis in resistant HCC cells. The levels of ROS, MDA, and iron ions increased, but the change in GSH was not obvious. Knockdown of CISD2 promoted uncontrolled autophagy in resistant HCC cells. Inhibition of autophagy attenuated CISD2 knockdown-induced ferroptosis. The autophagy promoted by CISD2 knockdown was related to Beclin1. When CISD2 and Beclin1 were inhibited, the effect on ferroptosis was correspondingly weakened.ConclusionInhibition of CISD2 promoted sorafenib-induced ferroptosis in resistant cells, and this process promoted excessive iron ion accumulation through autophagy, leading to ferroptosis. The combination of CISD2 inhibition and sorafenib treatment is an effective therapeutic strategy for resistant HCC.

TPGS-Galactose-Modified Polydopamine Co-delivery Nanoparticles of Nitric Oxide Donor and Doxorubicin for Targeted Chemo-Photothermal Therapy against Drug-Resistant Hepatocellular Carcinoma.

The lack of cancer cell specificity and the occurrence of multidrug resistance (MDR) are two major obstacles in the treatment of hepatocellular carcinoma (HCC). To tackle these challenges, a novel nanoparticle (NP)-based drug delivery system (DDS) with a core/shell structure consisted of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-galactose (Gal)/polydopamine (PDA) is fabricated. The NP is loaded with doxorubicin (DOX) and a nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN) sensitive to heat to afford NO-DOX@PDA-TPGS-Gal. The unique binding of Gal to asialoglycoprotein receptor (ASGPR) and the pH-sensitive degradation of NP ensure the targeted transportation of NP into liver cells and the release of DOX in HCC cells. The near-infrared (NIR) light further facilitates DOX release and initiates NO generation from BNN due to the photothermal property of PDA. In addition to the cytotoxicity contributed by DOX, NO, and heat, TPGS and NO act as MDR reversal agents to inhibit P-glycoprotein (P-gp)-related efflux of DOX by HepG2/ADR cells. The combined chemo-photothermal therapy (chemo-PTT) by NO-DOX@PDA-TPGS-Gal thus shows potent anti-cancer activity against drug-resistant HCC cells in vitro and in vivo and significantly prolongs the life span of drug-resistant tumor-bearing mice. The present work provides a useful strategy for highly targeted and MDR reversal treatment of HCC.

A Novel mRNA-Mediated and MicroRNA-Guided Approach to Specifically Eradicate Drug-Resistant Hepatocellular Carcinoma Cell Lines by Se-Methylselenocysteine.

Despite progress in the treatment of non-visceral malignancies, the prognosis remains poor for malignancies of visceral organs and novel therapeutic approaches are urgently required. We evaluated a novel therapeutic regimen based on treatment with Se-methylselenocysteine (MSC) and concomitant tumor-specific induction of Kynurenine aminotransferase 1 (KYAT1) in hepatocellular carcinoma (HCC) cell lines, using either vector-based and/or lipid nanoparticle-mediated delivery of mRNA. Supplementation of MSC in KYAT1 overexpressed cells resulted in significantly increased cytotoxicity, due to ROS formation, as compared to MSC alone. Furthermore, microRNA antisense-targeted sites for miR122, known to be widely expressed in normal hepatocytes while downregulated in hepatocellular carcinoma, were added to specifically limit cytotoxicity in HCC cells, thereby limiting the off-target effects. KYAT1 expression was significantly reduced in cells with high levels of miR122 supporting the concept of miR-guided induction of tumor-specific cytotoxicity. The addition of alpha-ketoacid favored the production of methylselenol, enhancing the cytotoxic efficacy of MSC in HCC cells, with no effects on primary human hepatocytes. Altogether, the proposed regimen offers great potential to safely and specifically target hepatic tumors that are currently untreatable.

PBK/TOPK promotes chemoresistance to oxaliplatin in hepatocellular carcinoma cells by regulating PTEN.

Oxaliplatin (OXA) resistance limits the efficiency of treatment for hepatocellular carcinoma (HCC). Studies have shown that the PDZ-binding kinase (PBK) plays important roles in tumors. However, the role of PBK in HCC is still a problem. In this study, we explored whether PBK is involved in the chemoresistance to OXA in HCC. Expressions of PBK in six HCC cell lines and one human hepatocytes line were determined by real-time quantitative PCR and western blot analysis. SNU-182 and HepG2 cells were chosen to induce OXA resistance. PBK was silenced or overexpressed in OXA-resistant and sensitive cell lines. Then, cell proliferation, migration, and invasion were measured by cholecystokinin-8 assay and Transwell assay, respectively. The Cancer Genome Atlas dataset showed that PBK is highly expressed in HCC and signifies poor prognosis to patient with HCC. Results showed that expression of PBK in HCC cells was significantly higher than that in THLE2 cells, and it was further increased in OXA-resistant HCC cells. Silencing of PBK promoted the sensitivity of drug-resistant HCC cells to OXA. Overexpression of PBK relieved the apoptosis induced by OXA and promoted the migration and invasion of OXA-sensitive HCC cells. Thus, this study revealed that high PBK expression is correlated with OXA resistance in HCC cells, which may provide a promising therapeutic target for treating HCC.

MiR-196b-5p regulates the proliferation of drug-resistant hepatocellular carcinoma cell lines by activating NFκB/ABCB1 signaling pathway

Purpose: To explore the molecular function of miR-196b-5p in hepatocellular carcinoma (HCC).Methods: MiR-196b-5p expression levels in HCC tissue samples were assessed by qRT-PCR. MiR-196b-5p was knocked-down or over-expressed in HepG2 cells by transfecting the cells with plasmids expressing either a miR-196b-5p inhibitor or mimic, respectively, while cell proliferation was assessed by MTT assay. The interaction of miR-196b-5p with target molecules was confirmed using luciferase reporter assay. Cell cycle was investigated by flow cytometry, while NFκBIA expression was assessed by western blotting.Results: MiR-196b-5p was over-expressed in HCC, and miR-196b-5p expression levels in patients with HCC were related to tumor grade. MiR-196b-5p over-expression promoted cell proliferation and colony formation and suppressed cell cycle arrest and apoptosis. The results of luciferase reporter assay showed that miR-196b-5p reduced NFκBIA expression in HepG2 cells by binding to a response element in the 3′ UTR of NFκBIA. Further investigation showed that NFκBIA interacts with NFκB1 and reduces the concentration of NFκB1 in HepG2 cells. The promoter of ATP-binding cassette sub-family B member 1 (ABCB1) was also targeted and bound by NFκB1, which altered the expression of ABCB1 in HepG2 cells.Conclusion: MiR-196b-5p regulates cell proliferation in drug-resistant HCC cell lines via activation of the NFκB/ABCB1 signaling pathway.
 Keywords: Hepatocellular carcinoma, miR-196b-5p, NFκBIA, NFκB1, ABCB1

Phosphorylation of cofilin-1 by ERK confers HDAC inhibitor resistance in hepatocellular carcinoma cells via decreased ROS-mediated mitochondria injury

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Despite the availability of several treatment strategies, resistance to chemotherapeutic agents, which limits the effectiveness of anticancer drugs, is a major problem in cancer therapy. In this study, we used a histone deacetylases inhibitor (HDACi) to establish drug-resistant HCC cells and further analyzed the molecular mechanisms underlying the development of resistance in HCC cells. Compared with the parental cells, HDACi-resistant cells showed high metastatic and pro-survival abilities. Two-dimensional electrophoresis data showed that the cofilin-1 (CFL-1) protein was altered in HDACi-resistant cells and was highly expressed in resistant cells compared with parental cells. The molecular function of CFL-1 is actin depolymerization, and it is involved in tumor metastasis. In this study, we showed that CFL-1 inhibition decreased cell migration and increased cell apoptosis in HDACi-resistant cells. We observed that HDACi induced ROS accumulation in cells and apoptosis via promotion of the CFL-1 interaction with Bax and CFL-1 translocation to the mitochondria, resulting in cytochrome C release. Importantly, phosphorylation of CFL-1 by activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) confers strong protection against HDAC inhibitor-induced cell injury. p-CFL-1 shows a loss of affinity with Bax and will not translocate to mitochondria, stably remaining in the cytoplasm. These results indicate that phosphorylation to inactivate CFL-1 decreased the chemosensitivity to HDAC inhibitors and resulting in drug resistance of HCC cells.

Abstract 1047: The multikinase inhibitor K252a suppresses hepatocellular carcinoma tumor growth in a mouse xenograft model

Abstract There are several ongoing clinical trials of molecular-targeted therapy for hepatocellular carcinoma (HCC), but no clearly effective therapy has yet emerged. Under such conditions, we have been focusing on potentially powerful anticancer effects of the novel multikinase inhibitor K252a, since it targets unique kinases, including PKC, CaMKII, c-Met, TrkB, MLCK, and phosphorylase kinase. Indeed, the former five targets are expressed in HCC tissues and involved in cellular proliferation, invasion, and metastasis (Yoshiji et al., Cancer Res 1999, etc.). The broad spectrum of the targets may block escape pathways in HCC cells, that are resistant to conventional anticancer drugs and even sorafenib. The AIM of this study was to assess whether K252a suppressed cellular proliferation and altered malignant phenotypes such as epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Materials and Methods: The human HCC cell lines HAK-1A, HAK-1B (Yano et al., Hepatology 1993), KYN-2 (Yano et al., Pathol Int 1988), and Huh7 were used in this study. Effects of K252a on the proliferation and the cell cycle were assessed by a commercialized kit (Promega) and flow cytometry, respectively. Protein expression and localization was analyzed by Western blot and immunocytochemistry, respectively. Cellular mRNA level was evaluated by real-time PCR using TaqMan probes. HAK-1B-based xenograft model in nude mice was used to evaluate in-vivo efficacy of K252a. Results: K252a suppressed proliferation and clearly caused G2 cell-cycle arrest in all the cell lines studied. Additionally, the agent induced both polygonal transformation in cell shape and increase in cell size in several HCC cell lines having endogenous mesenchymal features. In Western blot analysis and immunocytochemistry, the expression levels of E2A, an EMT-regulating transcriptional factor, were universally decreased in the cells treated with K252a, in concert with increased expression levels of E-cadherin in some cell lines. In real-time PCR analyses, the decrease in E2A mRNA levels and the increase in E-cadherin mRNA levels were ubiquitously found. Of note, xenografted tumor tissues obtained from mice treated with K252a exhibited significant increase in E-cadherin protein expression, in comparison with those from control mice. Conclusions: Our findings suggest that K252a has robust antitumor effects on HCC through, at least in part, reversing EMT and inducing G2 cell-cycle arrest. This kind of multikinase inhibitor may help us establish the proof-of-concept for closing escape routes in drug-resistant HCC cells. Citation Format: Mitsuhiko Abe, Hironori Koga, Takafumi Yoshida, Hiroshi Masuda, Masahiro Sakata, Yu Ikezono, Toru Nakamura, Eitaro Taniguchi, Takumi Kawaguchi, Hirohisa Yano, Takuji Torimura, Michio Sata. The multikinase inhibitor K252a suppresses hepatocellular carcinoma tumor growth in a mouse xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1047. doi:10.1158/1538-7445.AM2014-1047

MiRNA-195 sensitizes human hepatocellular carcinoma cells to 5-FU by targeting BCL-w

The role of microRNA-195 in developing acquired drug resistance in hepatocellular carcinoma cells was investigated. Expression profiling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miRNA-195 in BEL-7402/5-FU cells. Overexpression of miRNA-195 sensitized BEL-7402/5-FU cells to anticancer drugs. Consistent with these findings, miR-195 antisense oligonucleotide induced drug resistance in BEL-7402/5-FU cells. Also, the basal levels of the anti-apoptotic protein Bcl-w were high in BEL-7402/5-FU cells and miR-195 overexpression repressed Bcl-w protein level and inhibited the luciferase activity of a Bcl-w 3' untranslated region-based reporter construct in both BEL-7402/5-FU and BEL-7402 cells. These results indicate that miR-195 could improve the drug sensitivity at least in part by targeting Bcl-w to increase cell apoptosis in hepatocellular carcinoma cells.

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from −15 to −80 °C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.