Liver Cancer Drug Resistance Research Articles

TESC associated with poor prognosis enhances cancer stemness and migratory properties in liver cancer

Liver cancer stem cells (LCSCs) are responsible for recurrence, metastasis, and drug resistance in liver cancer. However, the genes responsible for inducing LCSCs have not been fully identified. Based on our previous study, we found that tescalcin (TESC), a calcium-binding EF hand protein that plays a crucial role in chromatin remodeling, transcriptional regulation, and epigenetic modifications, was up-regulated in LCSCs of spheroid cultures. By searching the Cancer Genome Atlas, International Cancer Genome Consortium, Human Protein Atlas, and Kaplan–Meier Plotter databases, we found that TESC expression was significantly elevated in liver cancer compared with that in normal liver tissue and was predictive of a decreased overall survival rate. Multivariate Cox analysis revealed TESC to be an independent prognostic factor for survival. High TESC expression was positively associated with cancer stem cell pathways, cancer stem cell surface markers, stemness transcription factors, epithelial–mesenchymal transition (EMT) factors, immune checkpoint proteins, and various cancer-related biological processes in liver cancer. Furthermore, TESC was implicated as promoting cancer stem cell properties through its influence on EMT. We demonstrated that TESC is a novel stemness-related gene that can serve as an independent prognostic factor for liver cancer.

Research progress on the mechanism and response strategies of molecular targeted drug resistance in liver cancer

Molecular targeted drugs are one of the treatments for hepatocellular carcinoma (HCC), the primary factor influencing their therapeutic efficacy is drug resistance. Diminished drug intake, greater efflux, improved DNA damage repair capacity, aberrant signal pathways, hypoxia, epithelial-mesenchymal cell transition, and the cellular autophagy system are summarized herein as aspects of the drug resistance mechanism. Simultaneously, effective strategies for addressing drug resistance are elaborated, providing ideas for better clinical treatment of HCC.

LncRNA model predicts liver cancer drug resistance and validate in vitro experiments.

Introduction: Hepatocellular carcinoma (HCC) patients may benefit from chemotherapy, but drug resistance is an important obstacle to favorable prognoses. Overcoming drug resistance is an urgent problem to be solved. Methods: Differential expression analysis was used to identify long non-coding RNAs (LncRNAs) that differed in chemotherapy-sensitive and chemotherapy-resistant patients. Machine learning algorithms including random forest (RF), lasso regression (LR), and support vector machines (SVMs) were used to identify important chemotherapy-related LncRNAs. A back propagation (BP) network was then used to validate the predictive capacity of important LncRNAs. The molecular functions of hub LncRNAs were investigated via qRT-PCR and cell proliferation assay. Molecular-docking technique was used to explore candidate drug of targets of hub LncRNA in the model. Results: A total of 125 differentially expressed LncRNAs between sensitive and resistant patients. Seventeen important LncRNAs were identified via RF, and seven factors were identified via LR. With respect to SVM, the top 15 LncRNAs of AvgRank were selected. Five merge chemotherapy-related LncRNAs were used to predict chemotherapy resistance with high accuracy. CAHM was a hub LncRNA of model and expression high in sorafenib resistance cell lines. In addition, the results of CCK8 showed that the sensitivity of HepG2-sorafenib cells to sorafenib was significantly lower than that of HepG2; and the sensitivity of HepG2-sorafenib cells transfected with sh-CAHM was significantly higher than that of Sorafenib. In the non-transfection group, the results of clone formation experiments showed that the number of clones formed by HepG2-sorafenib cells treated with sorafenib was significantly more than that of HepG2; after HepG2-sorafenib cells were transfected with sh-CAHM, the number of clones formed by Sorafenib treatment was significantly higher than that of HepG2 cells. The number was significantly less than that of HepG2-s + sh-NC group. Molecular Docking results indicate that Moschus was candidate drug for target protein of CAHM. Conclusion: Five chemotherapy-related LncRNAs could predict drug resistance in HCC with high accuracy, and the hub LncRNA CAHM has potential as a new biomarker for HCC chemotherapy resistance.

Inhibition of JNK/c-Jun-ATF2 Overcomes Cisplatin Resistance in Liver Cancer through down-Regulating Galectin-1.

Due to drug resistance, the clinical response to cisplatin (CDDP) from patients with liver cancer is unsatisfactory. The alleviation or overcoming of CDDP resistance is an urgent problem to be solved in clinics. Tumor cells rapidly change signal pathways to mediate drug resistance under drug exposure. Here, multiple phosphor-kinase assays were performed and c-Jun N-terminal kinase (JNK) was activated in liver cancer cells treated with CDDP. The high activity of the JNK promotes poor progression and mediates cisplatin resistance in liver cancer, leading to a poor prognosis of liver cancer. Mechanistically, the highly activated JNK phosphorylated c-Jun and ATF2 formed a heterodimer to upregulate the expression of Galectin-1, leading to promoting cisplatin resistance in liver cancer. Importantly, we simulated the clinical evolution of drug resistance in liver cancer by continuous CDDP administration in vivo. In vivo bioluminescence imaging showed the activity of JNK gradually increased during this process. Moreover, the inhibition of JNK activity by small molecular or genetic inhibitors enhanced DNA damage and overcame CDDP resistance in vitro and in vivo. Collectively, our results underline that the high activity of JNK/c-Jun-ATF2/Galectin-1 mediates cisplatin resistance in liver cancer and provides an optional scheme for dynamic monitoring of molecular activity in vivo.

MicroRNA-6838-5p suppresses the self-renewal and metastasis of human liver cancer stem cells through downregulating CBX4 expression and inactivating ERK signaling.

Hepatocellular carcinoma (HCC) is the most common primary human liver malignancy with high mortality. Liver cancer stem cells (CSCs) have been demonstrated to contribute to the recurrence, metastasis and drug resistance of liver cancer. Human HCC cohort analysis indicated that the epigenetic regulator polycomb chromobox homologue 4 (CBX4) was overexpressed in human HCC. Moreover, we found that CBX4 expression was significantly higher in CD44+ CD133+ Hep3B CSCs. Functionally, we demonstrated that CBX4 regulated cell proliferation, self-renewal, and metastasis ability of Hep3B CSCs. Bioinformatics analysis predicted that CBX4 was a direct target of microRNA-6838-5p (miR-6838-5p), which was further confirmed by luciferase reporter assay. MiR-6838-6p was down-regulated in HCC tumors and overexpression of miR-6838-5p attenuated the malignant traits of human liver CSCs in vitro. In addition, we found that miR-6838-5p/CBX4 axis modulates the biological properties of human liver CSCs via regulating ERK signaling. Overexpression of miR-6838-5p suppressed Hep3B xenograft tumor growth in vivo, while CBX4 overexpression abrogated the suppression effect, restored the angiogenesis, epithelial-to-mesenchymal transition (EMT), and ERK signaling in Hep3B tumor. In summary, our findings suggest that miR-6838-5p/CBX4 axis regulates liver tumor development and metastasis, which could be utilized as potential therapeutic target for HCC treatment.

MCM7 affects the cisplatin resistance of liver cancer cells and the development of liver cancer by regulating the PI3K/Akt signaling pathway

Objective Aberrant DNA replication is regarded as a component of cancer development. Minichromosome maintenance protein 7 (MCM7), which is critical for the initiation of DNA replication, is overexpressed in multiple malignancies. The effect of MCM7 on cell proliferation, apoptosis, and drug resistance of liver cancer and its mechanism were investigated in this study. Methods MCM7 expression in normal liver cells, liver cancer cell lines, and tissues, as well as adjacent tissues, was determined by qRT-PCR. CCK-8 and flow cytometry was performed to detect cell viability, apoptosis, and cell cycle, respectively. The related mRNA and protein expressions were detected by qRT-PCR and western blot. Results High expression of MCM7 was found in liver cancer tissues and cells, which results in notably lower survival time of patients. Cisplatin (DDP) could inhibit cell proliferation and affect MCM7 expression. Silencing of MCM7 inhibited cell viability, promoted cell apoptosis, arrested cell cycle at G1 phase, and enhanced the effect of DDP on cancer cells, while overexpression of MCM7 did the opposite. Moreover, silencing of MCM7 inhibited cyclinD1 and Ki-67 expressions. The overexpression of MCM7 increased phosphorylation levels of PI3K and AKT, activated the PI3K/AKT pathway, and weakened the inhibitory effect of DDP on the PI3K/AKT pathway. Conclusion Silencing of MCM7 may inhibit cell proliferation and promote apoptosis by regulating the PI3K/AKT pathway to affect the cell cycle, thus affecting the development of liver cancer, and improving the sensitivity of liver cancer cells to DDP.

Hypoxia, Metabolic Reprogramming, and Drug Resistance in Liver Cancer.

Hypoxia, low oxygen (O2) level, is a hallmark of solid cancers, especially hepatocellular carcinoma (HCC), one of the most common and fatal cancers worldwide. Hypoxia contributes to drug resistance in cancer through various molecular mechanisms. In this review, we particularly focus on the roles of hypoxia-inducible factor (HIF)-mediated metabolic reprogramming in drug resistance in HCC. Combination therapies targeting hypoxia-induced metabolic enzymes to overcome drug resistance will also be summarized. Acquisition of drug resistance is the major cause of unsatisfactory clinical outcomes of existing HCC treatments. Extra efforts to identify novel mechanisms to combat refractory hypoxic HCC are warranted for the development of more effective treatment regimens for HCC patients.

Phenylethanol glycosides from Herba Cistanche improve thehypoxic tumor microenvironment and enhance theeffects of oxaliplatin via the HIF‑1α signaling pathway.

Liver cancer is one of the most common types of malignant tumor, and is characterized by high malignancy, rapid progression, high morbidity and mortality. Oxaliplatin (OXA) has been reported to have marked efficiency against advanced liver cancer with tolerable toxicity. In solid tumors, the hypoxic microenvironment promotes epithelial-mesenchymal transition (EMT), which can also induce drug resistance of liver cancer to platinum drugs. Herba Cistanche (Cistanche tubulosa) has been frequently used in traditional Chinese medicine and the phenylethanol glycosides from Herba Cistanche (CPhGs) are the major active components. The present study aimed to investigate the effects of CPhGs on viability, apoptosis, migration and invasion of liver cancer cells. HepG2 liver cancer cells were divided into the control, DMSO, CoCl2, OXA, OXA + CoCl2 and CPhGs + OXA + CoCl2 groups. Subsequently, reverse transcription-quantitative PCR and western blot analysis were performed to determine the expression levels of hypoxia-inducible factor 1α (HIF-1α), lysyl oxidase-like 2 (LOXL2) and EMT-related genes and proteins (i.e., E-cadherin and Twist), in order to investigate the effects of CPhGs on liver cancer. The results demonstrated that CPhGs could enhance the effects of OXA on liver cancer, and inhibit the migration, invasion and apoptotic rate of liver cancer cells. Additionally, CPhGs treatment effectively induced downregulation of HIF-1α, LOXL2 and Twist, and upregulation of E-cadherin. The present findings indicated that CPhGs triggered a significant increase in sensitivity to OXA and suppression of hypoxia-induced EMT in liver cancer by inhibiting the HIF-1α signaling pathway. Therefore, CPhGs may be considered an effective platinum drug sensitizer, which could improve chemotherapeutic efficacy in patients with liver cancer.

Effects of FER1L4-miR-106a-5p/miR-372-5p-E2F1 regulatory axis on drug resistance in liver cancer chemotherapy

Liver cancer presents a challenge in today’s healthcare system. This study aimed at investigating the effects of Fer-1 like family member 4 (FER1L4) on chemotherapy resistance and liver cancer development by using clinically collected liver cancer tissues and commercially purchased human liver cancer cisplatin-resistant cell line HUH-7/DDP. Bioinformatics analysis, dual luciferase reporter gene assay, and RNA pull-down were applied to predict and verify the possible binding relationships. The expressions of FER1L4, E2F transcription factor 1 (E2F1), microRNA-106a-5p (miR-106a-5p), or miR-372-5p were altered in the cells, followed by flow cytometry, Cell Counting Kit-8 (CCK-8), and Transwell assays to evaluate apoptotic, proliferative, and invasive abilities in vitro and nude mice xenografts to observe tumor growth in vivo. FER1L4 was highly expressed and miR-106-5p and miR-372-5p were poorly expressed in tumor cells and tissues. FER1L4 knockdown or the overexpression of miR-106-5p and miR-372-5p inhibited the cancerous cell proliferation and invasion while promoting apoptosis. FERIL4 silencing increased the miR-106-5p/miR-372-5p expression to inhibit the E2F1-activated nuclear factor κB (NF-κB) pathway. Besides, overexpressing FER1L4 led to an increased tumor growth in nude mice, which was reversed by the NF-κB inhibitor pyrollidine dithiocarbamate (PDTC). In conclusion, the results indicated that FER1L4 could inhibit the expression of miR-106a-5p/miR-372-5p, to activate E2F1-mediated NF-κB pathway, leading to drug resistance in liver cancer.

MicroRNA in HCC: Biomarkers and Therapeutic Targets

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality. At present, diagnostic methods such as imaging observation, serum testing and tissue biopsy, as well as treatment methods such as surgical resection, radiotherapy, and chemotherapy have certain limitations in clinical interventions for HCC due to the complex pathogenesis and drug resistance of liver cancer, which seriously affect the survival and prognosis of patients. As a large-scale cytokine, microRNA (miRNA) plays an important role in regulating various life activities of cells. Extensive evidence proved that certain miRNAs are specifically expressed in the tissues and blood of HCC patients, and some of them have been confirmed as important factors that can participate in the regulation of key signaling pathways in cancer cells. For this reason, these miRNAs have great potential in clinical diagnosis and treatment of HCC, and can improve the limitations of conventional diagnosis and treatment. Our paper reviews the research on miRNA biomarkers and targets in HCC in recent years, and aims to provide new ideas for the diagnosis and treatment of HCC.

Multivalent rubber-like RNA nanoparticles for targeted co-delivery of paclitaxel and MiRNA to silence the drug efflux transporter and liver cancer drug resistance

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Analogous to the border customs, liver mainly functions as a filter to detoxify chemicals and metabolite administered orally or intravenously. Besides, the liver cancer cells overexpress the drug exporters which cause high drug effluxion from liver cancer cells, leading to chemoresistance and a diminished chemotherapeutic effect on liver cancer. Recently, we found that RNA nanoparticles display rubber-like property that can rapidly deliver therapeutics to tumor site efficiently and the rest of the RNA nanoparticle were cleared by renal excretion within half hour after systemic injection. Therefore, we designed a new multivalent RNA nanoparticle harboring three copies of hepatocyte targeting-ligands, one copy of miR122, and 24 copies of Paclitaxel to overcome the drug effluxion and chemoresistance thus, synergistically treating HCC. The hepatocyte targeting ligands introduce tumor specificity to the RNA nanoparticles as they selectively bind and internalize into liver cancer cells. The rubber-like RNA nanoparticles allow for enhanced targeting ability to the HCC tumors. The RNA nanoparticles carrying miR122 and PTX were delivered to the liver cancer cells efficiently due to their rubber-like property to enhance their EPR as well as the receptor-mediated endocytosis by hepatocyte targeting-ligands. The miR122 efficiently silenced the drug exporters and the oncogenic proteins. The synergistic effect between miR122 and PTX was confirmed by HSA (Highest Single Agent) synergy model. IC50 was determined to be 460 nM. In vivo studies on mice xenografts revealed that the RNA nanoparticle predominantly accumulated in HCC tumor sites and efficiently inhibited the tumor growth after multiple IV injection. This demonstrates the potential of the rubber-like multivalent RNA nanoparticles to conquest the liver cancer, a currently incurable lethal disease.

MicroRNAs Modulate Drug Resistance-Related Mechanisms in Hepatocellular Carcinoma

Primary liver cancer [hepatocellular carcinoma (HCC)] is one of the most common malignant tumors worldwide, causing serious health threats because of its high morbidity and mortality, rapid growth, and strong invasiveness. Patients with HCC frequently develop resistance to the current chemotherapeutic drugs, and this is largely attributed to the high-level heterogeneity of the tumor tissue. MicroRNAs (miRNAs) are a group of master regulators for multiple physiological and pathological processes and play important roles in the tumorigenesis. More recent studies have indicated that miRNAs also play a non-negligible role in the development of drug resistance in liver cancer. In this review, we summarize the data from the latest studies on the mechanisms of drug resistance in liver cancer, including autophagy, membrane transporters, epithelial–mesenchymal transitions (EMTs), tumor microenvironment, and genes and proteins that are associated with apoptosis. The data herein will provide valuable information for the development of novel approaches to tackle drug resistance in the management of liver cancer.

LncRNA MALAT1 modulates cancer stem cell properties of liver cancer cells by regulating YAP1 expression via miR‑375 sponging.

Liver cancer stem cells (CSCs) are functionally defined by their ability to undergo self-renewal, and may contribute to metastasis, recurrence and drug resistance in liver cancer. The long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been implicated in tumor formation and metastasis of liver cancer. However, the exact mechanism by which MALAT1 modulates liver CSC features remains largely unknown. In the present study, the expression level of MALAT1 was elevated in cancer spheroids compared with the corresponding levels noted in parental liver cancer cells, whereas the suppression of MALAT1 resulted in markedly reduced sphere formation and decreased expression of stemness factors in liver cancer cells. Dual-luciferase assay and RNA pull-down assays further indicated an interaction between MALAT1 and microRNA (miR)-375, and identified Yes-associated protein 1 (YAP1) as a direct target of miR-375 in liver cancer cells. In addition, YAP1 expression was correlated with MALAT1 in liver cancer. The reduced expression of YAP1 caused by knockdown of MALAT1 with MALAT1 small interfering RNA (si-MALAT1) could be partially abolished by miR-375 inhibition, suggesting that MALAT1 may regulate YAP1 expression by sponging miR-375. Furthermore, YAP1 overexpression rescued the decrease in CSC features of liver cancer cells caused by si-MALAT1, further supporting that MALAT1-mediated YAP1 signaling was required for the stem-like characteristics of liver CSCs. The present study revealed that MALAT1 may promote CSC properties of liver cancer cells by upregulating YAP1 expression via sponging miR-375. The MALAT1/miR-375/YAP1 axis may serve as a novel target for liver cancer therapy, particularly for the eradication of liver CSCs.

Autophagy-Induced Drug Resistance in Liver Cancer.

Autophagy is a self-destructive process that occurs in the cells during abnormal conditions like protein aggregation due to misfolding, nutrient deprivation, damage to vital cell organelles, pathogenic infections, and during cancer. Typically, autophagy plays a key role in the renovation of new cells by balancing the equilibrium between cell death and cell renewal. Dysregulation of autophagy has a profound effect on protein turnover, mitochondrial homeostasis, clearance of damaged organelles, and cellular metabolism, which lead to neurodegenerative, metabolic, and proliferative diseases. Despite its antitumorigenic role, autophagy can promote cell proliferation by enhancing chemotherapeutic resistance in liver cancer. In the present review, we provide a comprehensive overview and discussion on the role of autophagy in the drug-resistant mechanisms of liver cancer.

Association between NF-κB expression and drug resistance of liver cancer.

Association between the expression of nuclear factor κB (NF-κB) and the drug resistance of hepatoma cells was investigated. HepG-2 cells and HepG2/ADM cells were cultured, respectively. The morphology and status of the two groups of cells were observed by cell white light. The immunofluorescence by NF-κB and MDR1 staining on HepG-2 cells and HepG2/ADM cells, respectively, was applied and the fluorescence expression in the two groups of cells was observed. RT-qPCR was used to detect the expression of NF-κB and MDR1 mRNA, the NF-κB and MDR1 protein expression was detected by western blot analysis. The results of cell white illumination showed that the structure of HepG-2 and HepG2/ADM cells was complete and the cell morphology was normal, and there was no significant difference, and could be used for comparative study. Immunofluorescence staining showed that the expression of NF-κB and MDR1 in HepG-2 cells was very low, while the expression of NF-κB and MDR1 in HepG2/ADM cells was increased significantly. The RT-qPCR results showed that NF-κB and MDR1 mRNA expression in HepG-2 cells was very low, while NF-κB and MDR1 mRNA expression in HepG-2/ADM cells was significantly increased, and western blot results showed that NF-κB and MDR1 protein expression in HepG-2 cells was very low, while NF-κB and MDR1 protein expression in HepG-2/ADM cells was increased significantly. The results of variance analysis showed that there was significant difference in the expression of the control group and paeonol group (P<0.01). In conclusion, the expression of NF-κB in the drug-resistant cells of liver cancer is closely related to the resistance-related gene MDR1. This result may provide a new solution for the drug resistance of liver cancer.

Metformin reverses stem cell‑like HepG2 sphere formation and resistance to sorafenib by attenuating epithelial‑mesenchymal transformation

Cancer stem cells (CSCs) have been reported to be associated with the recurrence and drug resistance of liver cancer. In the present study, stem cell-like HepG2 cell spheres were enriched using stem cell conditioned culture medium. As expected, stem-like HepG2 cell spheres exhibited increased resistance to sorafenib. Metformin, a common drug used to treat type 2 diabetes mellitus, reduced the diameters and numbers of stem-like HepG2 spheres, and increased their sensitivity to sorafenib. Western blotting confirmed that low doses of metformin reversed the epithelial-mesenchymal transformation (EMT) process of HepG2 spheres. These results suggested that metformin enhanced sensitivity to sorafenib, which was probably through reversal of the EMT process of sphere-forming cells and by reducing the formation of CSCs.

Knockdown of TWIST enhances the cytotoxicity of chemotherapeutic drugs in doxorubicin-resistant HepG2 cells by suppressing MDR1 and EMT.

The transcription factor twist family bHLH transcription factor1 (TWIST), which is a member of the basic helix-loop-helix class of proteins, is known to induce epithelial-mesenchymal transition (EMT) and promote cancer metastasis. TWIST has previously been reported to be associated with multidrug resistance (MDR), since its depletion increases drug sensitivity. Although these previous studies have established a strong association between EMT and MDR, the molecular mechanism remains obscure. The present study demonstrated that TWIST protein expression was elevated in liver cancer, and was positively correlated with multidrug resistance protein1 (MDR1) expression. Conversely, MDR1 was negatively correlated with E‑cadherin expression in liver cancer samples. In addition, the present study indicated that doxorubicin-resistant HepG2 (R‑HepG2) cells acquired an EMT phenotype. TWIST was also more highly expressed in R‑HepG2 cells compared with in parental HepG2 cells. Knockdown of TWIST increased the sensitivity of R‑HepG2 cells to 5-fluroracil, cisplatin and doxorubicin through a reduction in MDR1 expression and drug efflux ability. Furthermore, knockdown of TWIST in R‑HepG2 cells inhibited the migratory ability of cells and suppressed the EMT phenotype. These findings demonstrated that targeting TWIST may be considered a novel strategy to overcome drug resistance in liver cancer.

CXCR4-targeted lipid-coated PLGA nanoparticles deliver sorafenib and overcome acquired drug resistance in liver cancer

Sorafenib, a multikinase inhibitor, has been used as an anti-angiogenic agent against highly vascular hepatocellular carcinoma (HCC) – yet associated with only moderate therapeutic effect and the high incidence of HCC recurrence. We have shown intratumoral hypoxia induced by sorafenib activated C-X-C receptor type 4 (CXCR4)/stromal-derived factor 1α (SDF1α) axis, resulting in polarization toward a tumor-promoting microenvironment and resistance to anti-angiogenic therapy in HCC. Herein, we formulated sorafenib in CXCR4-targeted lipid-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) modified with a CXCR4 antagonist, AMD3100 to systemically deliver sorafenib into HCC and sensitize HCC to sorafenib treatment. We demonstrated that CXCR4-targeted NPs efficiently delivered sorafenib into HCCs and human umbilical vein endothelial cells (HUVECs) to achieve cytotoxicity and anti-angiogenic effect in vitro and in vivo. Despite the increased expression of SDF1α upon the persistent hypoxia induced by sorafenib-loaded CXCR4-targeted NPs, AMD3100 attached to the NPs can block CXCR4/SDF1α, leading to the reduced infiltration of tumor-associated macrophages, enhanced anti-angiogenic effect, a delay in tumor progression and increased overall survival in the orthotopic HCC model compared with other control groups. In conclusion, our results highlight the clinical potential of CXCR4-targeted NPs for delivering sorafenib and overcoming acquired drug resistance in liver cancer.

JNK signaling in hepatocarcinoma cells is associated with the side population upon treatment with anticancer drugs.

Liver cancer is one of the most drug-resistant cancer types, and cancer stem cells are related to drug resistance. c-Jun-N-terminal kinase (JNK) signaling is involved in drug resistance, and the side population of cells (SP cells) can be used as a model to study liver cancer stem cells. We sought to evaluate the relationship between SP cells and JNK signaling in hepatocarcinoma cells. For this purpose, we examined cell proliferation and the SP cell ratio following treatment of Huh7 cells with the anticancer drugs 5-fluorouracil (5-FU) and paclitaxel. The expression of phospho-stress-activated protein kinase (SAPK)/JNK in the treated cells was evaluated using immunoblotting. 5-FU and paclitaxel treatment increased the number of SP cells and JNK phosphorylation, and decreased cell survival. Huh7 and HepG2 cells were also treated with SP600125, a JNK inhibitor, to study the relationship between SP cells and JNK signaling. The increase in the number of SP cells and the SAPK/JNK and c-Jun phosphorylation was reverted by SP600125 treatment in these cells. We also used immunohistochemistry and showed that SAPK/JNK and c-Jun phosphorylation are increased in hepatocarcinoma tissues. In conclusion, our results demonstrate that the number of SP cells and SAPK/JNK phosphorylation are increased upon treatment with anticancer drugs, and that this increase is blocked by inhibition of JNK signaling. These findings suggest that drug resistance in liver cancer may involve an increase in the number of SP cells following JNK activation.

Pharmacological Isolation of Experimental Models of Drug-resistant Hepatocellular Carcinoma Cell Line

Drug resistance is one of the major challenges facing the success of chemotherapy against human hepatocarcinoma (HCC) as well as other types of cancer. Studies with cell lines can serve as initial screening for agents that could modulate drug resistance. Development of a good experimental model of drug-resistant cells is a prerequisite for the success of such cellular studies; but could be laborious and generally time-consuming. Additionally, the high mortality rate associated with advanced HCC calls for a probe into the mechanism of resistance by developing experimental model that mimics clinical method of its treatment. Consequently, we have reported a simplified method of selection of drug-resistant hepatocarcinoma cells from human hepatocellular carcinoma (HEPG2) cell line using pharmacologic agents, cisplatin (CDDP) and 5-fluorouracil (5-FU). HEPG2 cell line was incubated for 24 hours with different concentrations of CDDP (0 - 20 μM) or 5-FU (0 - 100 μM). Cell viability was assayed by CCK-8 (Cell Counting Kit) analysis, and the inhibitory concentrations (IC50) for CDDP and 5-FU were established by dose-dependent cytotoxicity curves. The IC50(s) were confirmed by flow cytometric analysis of cell death due to CDDP or 5-FU. Clinical method of treatment was imitated by treating the parental HEPG2 cell line in pulse, at the optimal concentration of either CDDP or 5-FU for 4 to 6 hours. Induction was repeated 6 times, whilst allowing the cells to attain at least 70% confluence between intervals of induction. The resultant drug-resistant sublines, (HEPG2CR) and (HEPG2FR) were found to be stable after over 3 months of drug withdrawal and maintenance in drug-free medium. This was done with the views of establishing a simple, efficient and direct protocol for the development of good cellular models for the study of drug resistance in liver cancer, with possible application in other cancer types.