Glibenclamide induces apoptosis by activating reactive oxygen species dependent JNK pathway in hepatocellular carcinoma cells

Glibenclamide (Gli) is a widely employed drug in the treatment of type 2 diabetes and many lines of evidence have described its anti-tumor effects in some neoplasms. The aim of the present study was to investigate the effect of Gli on apoptosis of human hepatocellular carcinoma (HCC) cells and to analyze the underlying pathway involved in this action. Two HCC cell lines, HepG-2 and Huh7 were used as the cell models. We found that Gli treatment significantly inhibited cell viability, induced a significant cell-cycle arrest in G2/M-phase and induced apoptosis in both HepG-2 and Huh7 cells. We further verified that apoptosis induction by Gli was accompanied by increase in ROS levels and activation of the JNK pathway. Scavenging of the intracellular ROS with its blocker N-acetyl-L-cysteine (NAC) could mitigate the Gli-induced apoptosis and JNK activation in the two HCC cell lines. Furthermore, inhibition of JNK pathway by its inhibitor SP100625 effectively reduced Gli-induced apoptosis in HCC cells. In conclusion, Gli treatment significantly induced cell apoptosis by promoting ROS-dependent JNK pathway activation in HCC cells. Gli may be a potential clinical anti-tumor drug for HCC.

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Inhibiting p21-activated kinase (PAK7) enhances radiosensitivity in hepatocellular carcinoma

Human & Experimental Toxicology ◽

10.1177/09603271211027948 ◽

2021 ◽

pp. 096032712110279

Author(s):

Y-F Gu ◽

L-T Kong

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Inhibitory Effect ◽

Sulforhodamine B ◽

Huh7 Cells ◽

M Phase ◽

Poorly Differentiated ◽

Well Differentiated ◽

P21 Activated Kinase ◽

Hcc Cells

Objective: In light of the upregulation of p21-activated kinase (PAK7) in a variety of cancers, including hepatocellular carcinoma (HCC), we aimed to investigate the effect of PAK7 on the sensitivity of HCC cells to radiotherapy. Methods: PAK7 expression was determined in normal adult liver epithelial THLE-2 and human HCC cell lines. The effect of ionizing radiation (IR) on the HCC cell viability was evaluated by Sulforhodamine B (SRB) assay. HCC cell lines Mahlavu and Huh7 were chosen to assess the effect of PAK7 shRNAs on the viability, clone formation, apoptosis, cycle distribution and γ-H2AX expression after exposure to IR. Results: As compared to THLE-2 cells, PAK7 was upregulated in poorly differentiated Mahlavu and SK-Hep-1 cells, but moderately or lowly expressed in well-differentiated Huh7 and HepG2 cells. HCC cells with moderate or low expression of PAK7 presented a decreased viability at 2 Gy IR, which had no significant effect on PAK7high HCC cells. Mahlavu and Huh7 cells transfected with PAK7 shRNAs showed increased inhibitory effect of IR on viability. In addition, PAK7 shRNAs reduced clone formation, facilitated the cell apoptosis, arrested cells at G2/M phase, and increased γ-H2AX expression. Moreover, changes above were more evident in the HCC cells co-treated with IR and PAK7 shRNAs. Conclusion: PAK7 downregulation could inhibit the viability, promote the apoptosis, arrest cells in G2/M phase, and induce the DNA damage in HCC cells, thereby enhancing the radiosensitivity in HCC.

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Melatonin enhances the anti-tumor effect of sorafenib via AKT/p27-mediated cell cycle arrest in hepatocarcinoma cell lines

RSC Advances ◽

10.1039/c7ra02113e ◽

2017 ◽

Vol 7 (34) ◽

pp. 21342-21351 ◽

Cited By ~ 6

Author(s):

Fei Long ◽

Chengyong Dong ◽

Keqiu Jiang ◽

Yakun Xu ◽

Xinming Chi ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Cycle Arrest ◽

Hepatocarcinoma Cell ◽

Proposed Model ◽

Hcc Cells

Proposed model elucidating the role of MT in regulating the proliferation of hepatocellular carcinoma (HCC) cells treated with sorafenib.

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Silibinin sensitizes CD133+ hepatocellular carcinoma cells to cisplatin treatment through suppression of OPA1

10.21203/rs.3.rs-42671/v1 ◽

2020 ◽

Author(s):

Jikang Yang ◽

Zhiyuan Xing

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Cytochrome C ◽

Carcinoma Cells ◽

Flow Cytometry Analysis ◽

Western Blot Assay ◽

Huh7 Cells ◽

Mechanism Research ◽

Induced Apoptosis ◽

Hcc Cells

Abstract Background: Drug resistance is still a major obstacle during the cisplatin-based chemotherapy of hepatocellular carcinoma (HCC). Recently, studies have indicated that the population of CD133+ cancer cells is partially responsible for the failure of cancer treatment. However, the potential mechanisms are still unclear.Methods: CD133+ HepG2 and Huh7 cells were sorted via flow cytometry. CCK-8 assay was used to detect the cytotoxicity of cisplatin and silibinin against HCC cells. Western blot assay was performed to detect the protein expression, cleavage of caspases and release of cytochrome c from mitochondria into cytosol. Flow cytometry analysis was used to measure the apoptotic rate of CD133+ HepG2 and Huh7 cells.Results: CD133+ HepG2 and Huh7 cells were observed to exhibit obvious resistance against cisplatin. However, co-treatment with silibinin significantly reduced the cisplatin resistance of CD133+ HepG2 and Huh7 cells. Furthermore, although CD133+ HepG2 and Huh7 cells were resistant to cisplatin-induced apoptosis, co-treatment with silibinin enhanced the cisplatin-induced apoptosis through promoting the release of cytochrome c from mitochondria into cytosol. In the mechanism research, we proved that silibinin inhibited the expression of OPA1 in CD133+ HepG2 and Huh7 cells. Under the stress of cisplatin, silibinin promoted the collapse of mitochondria and increased the release of cytochrome c. As a result, caspases-dependent apoptosis was induced in CD133+ HepG2 and Huh7 cells which were co-treated with cisplatin and silibinin.Conclusion: Silibinin sensitizes CD133+ HCC cells to cisplatin-induced apoptosis through suppression of OPA1.

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Targeting PARP and autophagy evoked synergistic lethality in hepatocellular carcinoma

Carcinogenesis ◽

10.1093/carcin/bgz104 ◽

2019 ◽

Vol 41 (3) ◽

pp. 345-357 ◽

Cited By ~ 7

Author(s):

Wenjing Zai ◽

Wei Chen ◽

Yuxuan Han ◽

Zimei Wu ◽

Jiajun Fan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Parp Inhibitor ◽

Strand Break ◽

Parp Inhibitors ◽

Autophagic Flux ◽

Cycle Arrest ◽

Recombination Repair ◽

Induced Apoptosis ◽

Hcc Cells

Abstract Hepatocellular carcinoma (HCC), one of the most lethal malignancies worldwide, has limited efficient therapeutic options. Here, we first demonstrated that simultaneously targeting poly (ADP-ribose) polymerase (PARP) and autophagy could evoke striking synergistic lethality in HCC cells. Specifically, we found that the PARP inhibitor Niraparib induced cytotoxicity accompanied by significant autophagy formation and autophagic flux in HCC cells. Further experiments showed that Niraparib induced suppression of the Akt/mTOR pathway and activation of the Erk1/2 cascade, two typical signaling pathways related to autophagy. In addition, the accumulation of reactive oxygen species was triggered, which was involved in Niraparib-induced autophagy. Blocking autophagy by chloroquine (CQ) in combination with Niraparib further enhanced cytotoxicity, induced apoptosis and inhibited colony formation in HCC cells. Synergistic inhibition was also observed in Huh7 xenografts in vivo. Mechanistically, we showed that autophagy inhibition abrogated Niraparib-induced cell-cycle arrest and checkpoint activation. Cotreatment with CQ and Niraparib promoted the formation of γ-H2AX foci while inhibiting the recruitment of the homologous recombination repair protein RAD51 to double-strand break sites. Thus, the present study developed a novel promising strategy for the management of HCC in the clinic and highlighted a potential approach to expand the application of PARP inhibitors.

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Restoration of Epigenetically Silenced Sulfatase 1 Expression by 5-Aza-2′-Deoxycytidine Sensitizes Hepatocellular Carcinoma Cells to Chemotherapy-Induced Apoptosis

Medical Epigenetics ◽

10.1159/000375461 ◽

2015 ◽

Vol 3 (1) ◽

pp. 1-18 ◽

Cited By ~ 4

Author(s):

Abdirashid Shire ◽

Gwen Lomberk ◽

Jin-Ping Lai ◽

Hongzhi Zou ◽

Norihiko Tsuchiya ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Methylation ◽

Protein Expression ◽

Mrna Expression ◽

Cell Lines ◽

Gene Copy ◽

Drug Induced ◽

Sulfatase Activity ◽

Induced Apoptosis ◽

Hcc Cells

Background: Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer death worldwide. Sulfatase 1 (SULF1) functions as a tumor suppressor in HCC cell lines in vitro but also has an oncogenic effect in some HCCs in vivo. Aim: The purpose of this study was to examine the mechanisms regulating SULF1 and its function in HCC. Methods: First, SULF1 mRNA and protein expression were examined. Second, we examined SULF1 gene copy numbers in HCC cells. Third, we assessed whether DNA methylation or methylation and/or acetylation of histone marks on the promoter regulate SULF1 expression. Finally, we examined the effect of 5-aza-2′-deoxycytidine (5-Aza-dC) on sulfatase activity and drug-induced apoptosis. Results: SULF1 mRNA was downregulated in nine of eleven HCC cell lines, but only in six of ten primary tumors. SULF1 mRNA correlated with protein expression. Gene copy number assessment by fluorescence in situ hybridization showed intact SULF1 alleles in low-SULF1-expressing cell lines. CpG island methylation in the SULF1 promoter and two downstream CpG islands did not show an inverse correlation between DNA methylation and SULF1 expression. However, chromatin immunoprecipitation showed that the SULF1 promoter acquires a silenced chromatin state in low-SULF1-expressing cells through an increase in di/trimethyl-K9H3 and trimethyl-K27H3 and a concomitant loss of activating acetyl K9, K14H3 marks. 5-Aza-dC restored SULF1 mRNA expression in SULF1-negative cell lines, with an associated increase in sulfatase activity and sensitization of HCC cells to cisplatin-induced apoptosis. Conclusion: SULF1 gene silencing in HCC occurs through histone modifications on the SULF1 promoter. Restoration of SULF1 mRNA expression by 5-Aza-dC sensitized HCC cells to drug-induced apoptosis.

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GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.278.5.e958 ◽

2000 ◽

Vol 278 (5) ◽

pp. E958-E966 ◽

Cited By ~ 48

Author(s):

Zhiwu Lin ◽

Joel M. Weinberg ◽

Ricky Malhotra ◽

Steven E. Merritt ◽

Lawrence B. Holzman ◽

...

Keyword(s):

Cell Death ◽

Glucose Uptake ◽

Cell Lines ◽

Glucose Transporter ◽

Morphological Changes ◽

Control Cell ◽

Jnk Pathway ◽

Glut 1 ◽

Pathway Activation ◽

Induced Apoptosis

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 ± 2.6% in control cells and only 6.0 ± 1.1 and 3.9 ± 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH2-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.

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Protein Phosphatase 4 Promotes Hepatocyte Lipoapoptosis by Regulating RAC1/MLK3/JNK Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/5550498 ◽

2021 ◽

Vol 2021 ◽

pp. 1-16

Author(s):

Xiuqing Huang ◽

Guang Yang ◽

Li Zhao ◽

Huiping Yuan ◽

Hao Chen ◽

...

Keyword(s):

Protein Phosphatase ◽

Hepatic Insulin Resistance ◽

Jnk Pathway ◽

Pathway Activation ◽

Jnk Signaling Pathway ◽

Induced Apoptosis ◽

Jnk Activation ◽

First Time

Lipotoxicity-induced apoptosis, also referred to as lipoapoptosis, is one of the important initial factors promoting the progression from hepatosteatosis to nonalcoholic steatohepatitis (NASH). Saturated free fatty acids (SFAs), which are increased significantly in NASH, are directly hepatotoxic which induce hepatocyte lipoapoptosis. Previously, we reported that protein phosphatase 4 (PP4) was a novel regulator of hepatic insulin resistance and lipid metabolism, but its role in hepatic lipoapoptosis remains unexplored. In this study, we found out that PP4 was upregulated in the livers of western diet-fed-induced NASH mice and SFA-treated murine primary hepatocytes and HepG2 cells. In addition, we found for the first time that suppression of PP4 decreased SFA-induced JNK activation and expression of key modulators of hepatocyte lipoapoptosis including p53-upregulated modulator of apoptosis (PUMA) and Bcl-2-interacting mediator (Bim) and reduced hepatocyte lipoapoptosis level as well both in vitro and in vivo. Further study revealed that PP4 induced JNK activation and lipoapoptosis-related protein expression by regulating the RAC1/MLK3 pathway instead of the PERK/CHOP pathway. The effects of palmitate-treated and PP4-induced lipoapoptosis pathway activation were largely abolished by RAC1 inhibition. Moreover, we identified that PP4 interacted with RAC1 and regulated GTPase activity of RAC1. In conclusion, these results demonstrated that PP4 was a novel regulator of hepatocyte lipoapoptosis and mediated hepatocyte lipoapoptosis by regulating the RAC1/MLK3/JNK signaling pathway. Our finding provided new insights into the mechanisms of this process.

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AEG-1/miR-221 Axis Cooperatively Regulates the Progression of Hepatocellular Carcinoma by Targeting PTEN/PI3K/AKT Signaling Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms20225526 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5526 ◽

Cited By ~ 5

Author(s):

Maheshkumar Kannan ◽

Sridharan Jayamohan ◽

Rajesh Kannan Moorthy ◽

Siva Chander Chabattula ◽

Mathan Ganeshan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathway ◽

Akt Signaling ◽

Akt Signaling Pathway ◽

Migration Assay ◽

Cycle Arrest ◽

Significant Expression ◽

Induced Apoptosis ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is the third leading malignancy worldwide, causing mortality in children and adults. AEG-1 is functioned as a scaffold protein for the proper assembly of RNA-induced silencing complex (RISC) to optimize or increase its activity. The increased activity of oncogenic miRNAs leads to the degradation of target tumor suppressor genes. miR-221 is an oncogenic miRNA, that plays a seminal role in carcinogenesis regulation of HCC. However, the molecular mechanism and biological functions of the miR-221/AEG-1 axis have not been investigated extensively in HCC. Here, the expression of miR-221/AEG-1 and their target/associate genes was analyzed by qRT-PCR and Western blot. The role of the miR-221/AEG-1 axis in HCC was evaluated by proliferation assay, migration assay, invasion assay, and flow cytometry analysis. The expression level of miR-221 decreased in AEG-1 siRNA transfected HCC cells. On the other hand, there were no significant expression changes of AEG-1 in miR-221 mimic and miR-221 inhibitor transfected HCC cells and inhibition of miR-221/AEG-1 axis decreased cell proliferation, invasion, migration, and angiogenesis and induced apoptosis, cell cycle arrest by upregulating p57, p53, PTEN, and RB and downregulating LSF, MMP9, OPN, Bcl-2, PI3K, AKT, and LC3A in HCC cells. Furthermore, these findings suggest that the miR-221/AEG-1 axis plays a seminal oncogenic role by modulating PTEN/PI3K/AKT signaling pathway in HCC. In conclusion, the miR-221/AEG-1 axis may serve as a potential target for therapeutics, diagnostics, and prognostics of HCC.

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Sempervirine Inhibits Proliferation and Promotes Apoptosis by Regulating Wnt/β-Catenin Pathway in Human Hepatocellular Carcinoma

Frontiers in Pharmacology ◽

10.3389/fphar.2021.806091 ◽

2021 ◽

Vol 12 ◽

Author(s):

Rongcai Yue ◽

Haiping Liu ◽

Yaxin Huang ◽

Jing Wang ◽

Dongmei Shi ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cyclin B1 ◽

Potential Mechanism ◽

Active Components ◽

Cycle Arrest ◽

Induced Apoptosis ◽

Toxic Plant ◽

Anti Tumor Activity ◽

Hcc Cells

Gelsemium elegans (G. elegans) Benth., recognized as a toxic plant, has been used as traditional Chinese medicine for the treatment of neuropathic pain and cancer for many years. In the present study, we aim to obtain the anti-tumor effects of alkaloids of G. elegans and their active components in hepatocellular carcinoma (HCC) and the potential mechanism was also further investigated. We demonstrated that sempervirine induced HCC cells apoptosis and the apoptosis was associated with cell cycle arrest during the G1 phase, up-regulation of p53 and down-regulation of cyclin D1, cyclin B1 and CDK2. Furthermore, sempervirine inhibited HCC tumor growth and enhances the anti-tumor effect of sorafenib in vivo. In addition, inactivation of Wnt/β-catenin pathway was found to be involved in sempervirine-induced HCC proliferation. The present study demonstrated that alkaloids of G. elegans were a valuable source of active compounds with anti-tumor activity. Our findings justified that the active compound sempervirine inhibited proliferation and induced apoptosis in HCC by regulating Wnt/β-catenin pathway.

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All-trans Retinoic Acid combined with oxaliplatin suppressed proliferation of chemo-resistant hepatocellular carcinoma cells by inducing G2/M cell cycle arrest

Archives of Medical Science ◽

10.5114/aoms/141321 ◽

2021 ◽

Author(s):

Juxian Sun ◽

Hua Liu ◽

Jie Shi ◽

Chang Liu ◽

Zongtao Chai ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Retinoic Acid ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Resistant Cell ◽

Cycle Arrest ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

M Phase

IntroductionTo investigate the effects and mechanisms of all-trans retinoic acid (ATRA) with and without oxaliplatin (OXA) on chemotherapy-resistant hepatocellular carcinoma cell lines.Material and methodsOXA-resistant cell lines (CSQT-2-R and Hep3b-R) and subcutaneous xenograft model were used in this study. MTT assay, flow cytometry, crystal violet assay, transwell assay and western-blotting were conducted to evaluate the effects of co-treatment with ATRA and OXA on OXA-resistant HCC in vivo and in vitro. The differences between two groups were analyzed using ANOVA. All statistical tests in the study were two-sided, and statistical significance was set at P<0.05.ResultsWe established two oxaliplatin-Resistant HCC cell lines (CSQT-2-R and Hep3b-R). The drug resistance ability can be increased up to 100% than their parental cells(CSQT-2 and Hep3b) in certain concentration of OXA. ATRA alone could not inhibited the viability of CSQT-2-R and Hep3b-R, but it can enhance the ability of OXA on apoptosis than OXA alone (75% vs 35%, p<0.05), which may be related to decreased p-AKT expression. Moreover, the co-treatment of two drugs arrest the cell cycle of OXA-resistant cell at G2/M phase by up-regulating CylinB1 protein.ConclusionsATRA combined with OXA can elicit cell cycle arrest of CSQT-2-R and Hep3b-R at G2/M phase, thereby inhibiting the proliferation of resistant HCC cell, which provides a new treatment for chemotherapy-resistant HCC.

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