MicroRNA-92b augments sorafenib resistance in hepatocellular carcinoma via targeting PTEN to activate PI3K/AKT/mTOR signaling

Sorafenib (SOR) resistance is still a significant challenge for the effective treatment of hepatocellular carcinoma (HCC). The mechanism of sorafenib resistance remains unclear. Several microRNAs (miRNAs) have been identified as playing a role in impairing the sensitivity of tumor cells to treatment. We examined the mechanism behind the role of miR-92b in mediating sorafenib resistance in HCC cells. We detected that miR-92b expression was significantly upregulated in SOR-resistant HepG2/SOR cells compared to parental HepG2/WT cells. After transfection with miR-92b inhibitor, the proliferation of HepG2/SOR cells was remarkably weakened and rates of apoptosis significantly increased. PTEN was considered to be a functional target of miR-92b according to a luciferase reporter assay. Knockdown of PTEN significantly impaired the ability of miR-92b inhibitor on increasing sorafenib sensitivity of HepG2/SOR cells. Furthermore, we confirmed by western blotting and immunofluorescence that miR-92b can mediate sorafenib resistance by activating the PI3K/AKT/mTOR pathway in HCC cells by directly targeting PTEN. These findings further validate the mechanism of miR-92b in SOR resistance in HCC treatment.     

Valproic acid overcomes transforming growth factor-β-mediated sorafenib resistance in hepatocellular carcinoma

Sorafenib is a multi-kinase inhibitor approved for hepatocellular carcinoma, but rarely causes tumor regression in patients with chronic liver diseases. To investigate whether growth factor-mediated signaling is involved in sorafenib resistance, HepG2 and PLC/PRF/5 hepatoma cells were exposed to epidermal growth factor (EGF), hepatocyte growth factor (HGF) or transforming growth factor-β (TGF-β) prior to treatment with sorafenib. Furthermore, to identify an effective combination treatment with sorafenib, growth factor-sensitized cells were treated with sorafenib alone or in combination with celecoxib, lovastatin or valproic acid (VPA). Trypan blue staining and Annexin V assays showed that the cytotoxic effect of sorafenib was inhibited by 15-54% in cells sensitized to TGF-β (P<0.05). Western blotting analysis showed that TGF-β significantly activated extracellular signal-regulated kinase (ERK)-mediated AKT signaling, and sorafenib failed to suppress both ERK and AKT in TGF-β-sensitized cells. The decreased anti-tumor effect of sorafenib was rescued by chemical inhibition of ERK and AKT. When TGF-β-sensitized cells were treated with sorafenib plus VPA, the levels of phosphorylated ERK and AKT were considerably suppressed and the numbers of dead cells were increased by 3.7-5.7-fold compared with those exposed to sorafenib alone (P<0.05). Moreover, low dose sorafenib-induced cell migration was effectively suppressed by combination treatment with sorafenib and VPA. Collectively, TGF-β/ERK/AKT signaling might play a critical role in sorafenib resistance in hepatoma cells, and combination treatment with VPA may be effective against this drug resistance.     

Decreased IL-6 induces sensitivity of hepatocellular carcinoma cells to sorafenib

Sorafenib has been recommended as a new palliative therapy for advanced hepatocellular carcinoma (HCC). However, the sensitivity of HCC cells to sorafenib is declined along with the extension of medication time, and the clinical outcome varies with patients receiving sorafenib therapy. Therefore, in the present study, we attempted to investigate the effect and mechanisms of IL-6 on sensitivity of HCC cells to sorafenib regarding the cell proliferation and apoptosis. Tissues from patients with HCC and its paracarcinoma tissues were collected for IL-6 expression determination. SiRNA (si) IL-6 was transfected into SMMC-7721 cells to evaluate the effects of IL-6 on cell sensitivity to sorafenib by RT-PCR, western blot, CCK-8 and flow cytometry assay. Results indicated that IL-6 was significantly upregulated in tumor tissues than that of paracarcinoma tissues. Furthermore, sorafenib significantly inhibited cell proliferation, IL-6 level and activation of p-PI3K/AKT while promoted the cell apoptosis rate and Caspase3 level compared as the control group, which were further promoted by administration of si IL-6. Therefore, downregulating IL-6 could be a potential treatment to increase the cell sensitivity of HCC cells to sorafenib.     

索拉非尼对敏感人肝癌细胞株MAPK信号通路基因表达的影响

 目的：筛选对索拉非尼敏感的人肝癌细胞株,检测索拉非尼作用于敏感细胞株后MAPK信号通路基因表达谱的变化。方法：索拉非尼作用于人肝癌细胞株Huh7、 MHCC97H、 HepG2 、SMCC7721、 HepG2.2.15和PLC,流式细胞术检测肝癌细胞凋亡,CCK-8测定细胞增殖的抑制率,筛选敏感肝癌细胞株;采用实时定量PCR基因芯片,观察索拉非尼作用于敏感细胞株前后MAPK信号通路基因的表达。结果：索拉非尼作用后,流式细胞术的结果显示,凋亡较明显的细胞株为PLC和HepG2.2.15,相对不明显的细胞株为SMCC7721和MHCC97H;CCK-8测定索拉非尼对PLC、HepG2.2.15、Huh7、HepG2、MHCC97H和SMCC7721细胞的IC50值分别为5.25 μmol/L、5.30 μmol/L、6.80 μmol/L、7.01 μmol/L、11.7 μmol/L和15.0 μmol/L。对索拉非尼相对敏感细胞株和不敏感细胞株分别为PLC和SMCC7721。索拉非尼作用敏感细胞株PLC后,MAPK信号通路表达 >2.0倍的有2个,≤0.5倍的有6个。结论：PLC为对索拉非尼相对敏感的人肝癌细胞株;索拉非尼主要导致MAPK信号通路中与调控细胞周期和活化转录因子相关的基因表达发生变化。     

Novel EGFR-TK inhibitor EKB-569 inhibits hepatocellular carcinoma cell proliferation by AKT and MAPK pathways

Epidermal growth factor receptor (EGFR)-targeted therapies have been effective in some cancers, but not in hepatocellular carcinoma (HCC). The aim of this study was to investigate the drug potential to overcome multi-drug resistance in HCC cells. Thirteen drug-sensitive HCC cells were assessed using the CCK-8 assay. G(0)-G(1) arrest was measured by FACS. Western blot analysis was used to detect the key enzymes in both the Ras/Raf and PI3K pathways. When establishing the IC(50) of HCC to several drugs, including EKB-569, sorafenib, erlotinib, gefitinib, pazopanib, and brivanib, SK-Hep1 cells treated with EKB-569 have shown the highest (72.8%-86.4%) G(0)-G(1) arrest and decreased the phosphorylation of AKT and ERK at the protein level. We found that EKB-569 had higher efficacy in HCC, compared to first generation, reversible EGFR-TK inhibitors. Furthermore, the combination of sorafenib and EKB-569 showed a synergistic effect to inhibit proliferation of SNU-475, previously the most resistant cell to EGFR-TKIs. Therefore, novel EKB-569 in combination with sorafenib may be able to overcome HCC resistance to EGFR-TK inhibitors.     

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre‐loxP‐controlled lentiviral vectors expressing CRIPTO in cell line‐derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft‐derived ex vivo tumour slices. CRIPTO‐overexpressing patient‐derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial‐to‐mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2‐CRIPTO cells formed tumours when injected into immune‐compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High‐level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO‐expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

KAT5 and KAT6B are in positive regulation on cell proliferation of prostate cancer through PI3K-AKT signaling

Histone modifications play important roles in the tumorigenesis and progression of prostate cancer (PCa) and genes involved in histone modifications are seemed as ideal targets for treatment of PCa patients. However, clinical trials have shown that those existing drugs exert the minimal antitumor activity and excess adverse effects on PCa patients. Therefore, it is of great interest to figure out novel specific biomarkers to guide the development of new drugs. In present study, an RNAi screening with 44 genes involved in histone modifications was applied to a PCa cell line, Du145. The results showed that nine genes were in positive regulation of Du145 cell growth. Then four selected genes (KAT2B, KAT5, KAT6B and HDAC1) were found to exert this effect by a gene-specific manner when silenced. And then KAT5 or KAT6B silenced cells were subjected to DNA microarray analysis. The common differentially expressed genes were analyzed by Ingenuity Pathway Analysis (IPA) and found that PDEF signaling, EIF2 signaling and PI3K signaling was suppressed following by KAT5 or KAT6B silencing. Subsequent immunoblotting assay showed that AKT signaling was inhibited, which suggested that KAT5 or KAT6B regulates cancer cell growth through PI3K-AKT signaling. Together with our published data [31] that AURKA inhibitoin increased drug sensitivity of DU145, our work demonstrated the underlying mechanism that how the acetylation enzyme regulates cancer cells survial and might provide potential therapeutic targets for prostate cancer patients in future epigenetic drug development.     

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells

Background/Aims

Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells.

Methods

Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis.

Results

Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin.

Conclusions

Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.     

LY3214996 relieves acquired resistance to sorafenib in hepatocellular carcinoma cells

Background: Sorafenib, an oral multi-kinase inhibitor of rapidly accelerated fibrosarcoma; vascular endothelial growth factor receptor-2/3, platelet-derived growth factor receptor, c-Kit, and Flt-3 signaling, is approved for treatment of advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib is often diminished because of acquired resistance through the reactivation of ERK signaling in sorafenib-resistant HCC cells. In this work, we investigated whether adding LY3214996, a selective ERK1/2 inhibitor, to sorafenib would increase the anti-tumor effectiveness of sorafenib to HCC cells.Methods: The Huh7 cell line was used as a cell model for treatment with sorafenib, LY3214996, and their combination. Phosphorylation of the key kinases in the Ras/Raf/MAPK and PI3K/Akt pathways, protein expression of the cell cycle, and apoptosis migration were assessed with western blot. MTT and colony-formation assays were used to evaluate cell proliferation. Wound-healing assay was used to assess cell migration. Cell cycle and apoptosis analyses were conducted with flow cytometry.Results: LY3214996 decreased phosphorylation of the Ras/Raf/MAPK and PI3K/Akt pathways, including p-c-Raf, p-P90RSK, p-S6K and p-eIF4EBP1 activated by sorafenib, despite increased p-ERK1/2 levels. LY3214996 increased the anti-proliferation, anti-migration, cell-cycle progression, and pro-apoptotic effects of sorafenib on Huh7^R cells.Conclusions: Reactivation of ERK1/2 appears to be a molecular mechanism of acquired resistance of HCC to sorafenib. LY3214996 combined with sorafenib enhanced the anti-tumor effects of sorafenib in HCC. These findings form a theoretical basis for trial of LY3214996 combined with sorafenib as second-line treatment of sorafenib-resistant in advanced HCC.     

Sorafenib: complexities of Raf-dependent and Raf-independent signaling are now unveiled

Hepatocellular carcinoma (HCC) is the most common primary cancer worldwide. The only current drug available for clinical treatment of HCC is sorafenib, which inhibits multiple signaling kinases including Raf family members, platelet-derived growth factor receptor, vascular endothelial growth factor receptors 1 and 2, c-Kit, and Fms-like tyrosine kinase 3. Many studies have revealed that the mechanism underlying the antitumor effect of sorafenib is complex. Because sorafenib inhibits C-Raf more potently than B-Raf, the therapeutic efficacy of sorafenib is strongly influenced by the relative expression and activity of B-Raf and C-Raf and the complex interactions between these factors. Moreover, Rafindependent signaling mechanisms have recently emerged as important pathways of sorafenib-induced cell death. Basic research studies have suggested that using sorafenib as part of a combination therapy may improve its effect, although this has yet to be confirmed by clinical evidence. Further studies of the functional mechanism of sorafenib are required to advance the development of targeted therapy for HCC. To aid future work on sorafenib, we here review the current literature pertaining to sorafenib signaling and its clinical efficacy in both monotherapy and combination therapy.     

Shc3高表达抑制人肝癌细胞系凋亡并参与肝癌耐药

目的探讨Shc3对人肝细胞癌HCC细胞凋亡及耐药机制。方法选取稳定下调Shc3的人肝细胞癌系HCCLM3和HepG2细胞及其对照组scramble细胞,稳定上调Huh7和HepG2细胞及其对照组pCDH细胞。Western blot检测Shc3、MEK、p-MEK、ERK和p-ERK蛋白表达;real-time PCR检测Shc3 mRNA表达;凋亡实验检测细胞凋亡;CCK-8法检测细胞增殖。结果成功验证Shc3降表达及过表达细胞系;与scramble组比较,Shc3降表达组细胞MEK/ERK通路激活程度明显降低(P<0.05),细胞凋亡比例增加(P<0.05);与pCDH组比较,Shc3表达上调增加细胞对索拉菲尼耐受性(P<0.05),并且MEK/ERK通路激活程度明显增强(P<0.05)。结论Shc3可通过干扰HCC细胞凋亡,激活MEK/ERK通路,增加HCC细胞对索拉菲尼的耐药性,本研究结果为靶向Shc3治疗肝细胞癌提供了理论依据和实验室基础。     

HBV X siRNA与5-氮-2'-脱氧胞苷对裸鼠肝癌皮下移植瘤作用的研究

目的 目的 探讨靶向HBV X的siRNA(X-siRNA)和5-氮-2′-脱氧胞苷(5-aza-dC)对HBV相关肝细胞癌生长的影响及可能机制.方法 设计合成X-siRNA及对照siRNA,用siRNA处理HepG2/GFP-HBx细胞,RT-PCR法测定处理细胞的HBV X基因表达;将HepG2/GFP、HepG2/GFP-HBx细胞接种于裸鼠皮下建立裸鼠肝癌皮下移植瘤,分别用X-siRNA、5-aza-dC单独或联合处理裸鼠并观察移植瘤生长;甲基化PCR测定移植瘤组织p16基因甲基化.结果 RT-PCR检测示X-siRNA处理的细胞HBV X mRNA水平明显降低;裸鼠体内实验显示HepG2/GFP-HBx组的皮下移植瘤体积明显大于HepG2/GFP组(P＜0.05);X-siRNA与5-aza-dC处理组移植瘤体积明显小于未处理组(P＜0.05);甲基化PCR检测示HepG2/GFP-HBx组移植瘤组织存在p16甲基化而HepG2/GFP组未检出p16甲基化;X-siRNA、5-aza-dC处理的移植瘤组织中p16基因甲基化减低.结论 X-siRNA及甲基化抑制剂可能通过逆转p16甲基化而能有效抑制肝细胞癌生长,具有潜在应用价值.     

Retracted: Physcion Synergistically Enhances the Cytotoxicity of Sorafenib in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common human malignancy. Physcion is a naturally occurring anthraquinone derivative found in plant and marine sources. Our previous studies have indicated that physcion could suppress tumor growth and induce apoptosis in HCC. This study was aimed to investigate the effect of a combination of physcion and sorafenib on HCC. Our findings indicated that physcion could significantly augment the antiproliferative and proapoptotic activities of sorafenib in vitro and in vivo. Mechanistically, the synergistic effect correlates with physcion-induced suppression of Notch3/AKT signaling. This preclinical evidence highlights the potential application of physcion in the treatment of HCC. Anat Rec, 302:2171–2177, 2019. © 2019 American Association for Anatomy     

Quantitative methylation analysis of multiple genes using methylation-sensitive restriction enzyme-based quantitative PCR for the detection of hepatocellular carcinoma

DNA methylation is a promising biomarker for cancer. This study was aimed at investigating the methylation levels of multiple genes in hepatocellular carcinoma (HCC) and to identify a combination of methylation markers that would be useful for the diagnosis of HCC. The methylation status of a panel of nine tumor-associated genes (APC, GSTP1, RASSF1A, CDKN2A, SFRP1, RUNX3, SOCS1, Hint1, and HIC-1) in 8 normal liver tissues and 47 paired HCCs and non-tumorous tissues (NTs) was determined using a modified methylation-sensitive, restriction enzyme-based quantitative PCR (MSRE-qPCR) method. The methylation levels of six genes (APC, CDKN2A, GSTP1, RASSF1A, SFRP1 and RUNX3) were significantly higher in HCCs than in adjacent NTs (P < 0.05). Although the AUC (area under the curve) for each individual gene was low to moderate (range: 0.576 to 0.835) according to receiver operator characteristic (ROC) curve analysis, the combination analysis of these six genes resulted in an increase of AUC of 0.954 with 85.1% sensitivity, 89.4% specificity, 88.9% positive predictive value, and 85.7% negative predictive value in discriminating HCC tissues from NT tissues. These results indicate that the analysis of a combination of these six methylated genes may be a promising method for the risk assessment and diagnosis of HCC.     

Interaction of key pathways in sorafenib-treated hepatocellular carcinoma based on a PCR-array

This study aimed to identify the key pathways and to explore the mechanism of sorafenib in inhibiting hepatocellular carcinoma (HCC). The gene expression profile of {"type":"entrez-geo","attrs":{"text":"GSE33621","term_id":"33621"}}GSE33621, including 6 sorafenib treated group and 6 control samples, was downloaded from the GEO (Gene Expression Omnibus) database. The differentially expressed genes (DEGs) in HCC samples were screened using the ΔΔCt method with the homogenized internal GAPDH. Also, the functions and pathways of DEGs were analyzed using the DAVID. Moreover, the significant pathways of DEGs that involved in HCC were analyzed based on the Latent pathway identification analysis (LPIA). A total of 44 down-regulated DEGs were selected in HCC samples. Also, there were 84 biological pathways that these 44 DEGs involved in. Also, LPIA showed that Osteoclast differentiation and hsa04664-Fc epsilon RI signaling pathway was the most significant interaction pathways. Moreover, Apoptosis, Toll-like receptor signaling pathway, Chagas disease, and T cell receptor signaling pathway were the significant pathways that interacted with hsa04664. In addition, DEGs such as AKT1 (v-akt murine thymoma viral oncogene homolog 1), TNF (tumor necrosis factor), SYK (spleen tyrosine kinase), and PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1 (alpha)) were the common genes that involved in the significant pathways. Several pathway interaction pairs that caused by several downregulated genes such as SYK, PI3K, AKT1, and TNF, were identified play curial role in sorafenib treated HCC. Sorafenib played important inhibition roles in HCC by affecting a complicate pathway interaction network.     

Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most fatal human malignancies, but the molecular mechanisms of hepatocarcinogenesis remain unclear. Although p53 mutations are frequently observed in Asian HCC, it is not a common event in Western HCC. Recent studies suggest that tumor suppressor genes (TSGs) can also be silenced through epigenetic disruption, such as promoter CpG island methylation, during carcinogenesis. To further understand the molecular mechanism of hepatocarcinogenesis, we have investigated the promoter methylation status of nine TSGs (SOCS-1, GSTP, APC, E-cadherin, RAR-beta, p14, p15, p16, and p73) in 51 cases of HCC using methylation-specific polymerase chain reaction. We found that 82% of HCCs had methylation of at least one TSG promoter. The most frequently methylated TSGs in HCC were: SOCS-1 (65%), GSTP (54%), APC (53%), E-cadherin (49%), and p15 (49%). Methylation of SOCS-1, GSTP, APC, E-cadherin, and p15 was more frequent in HCC than in nontumor liver (P < 0.05). Methylation of SOCS-1, GSTP, and p15 was also significantly more frequent in HCC than cirrhotic liver (P < 0.05). Although methylation of one or two genes could be seen in both nontumor and cirrhotic livers, 53% of the HCC cases had three or more TSG promoters methylated, in comparison to 0% in nontumor liver and 13% in cirrhosis (P = 0.001). Methylation of SOCS-1, APC, and p15 was more frequently seen in hepatitis C virus-positive HCC than hepatitis C virus/hepatitis B virus-negative HCC. Our data suggest that promoter hypermethylation of TSGs is a common event in HCC and may play an important role in hepatocarcinogenesis.