Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells

Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1^Thr163 phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells.     

Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

Sorafenib is the standard first-line drug for the treatment of advanced hepatocellular carcinoma (HCC), however, its therapeutic efficacy is not satisfactory due to primary or secondary resistance of HCC cells. In the present study, we identified Metaxin 1 (MTX1) as a new regulator of sorafenib resistance in HCC through genome-scale CRISPR activation (CRISPRa) screening. We found that MTX1 was frequently upregulated in HCC tissues and overexpression of MTX1 promoted HCC cell proliferation in vitro and in vivo. As well, MTX1 overexpression increased cell growth rate and decreased cell apoptosis upon sorafenib treatment. Consistently, the resistance induced by MTX1 was also observed in subcutaneous xenograft tumor model. Clinically, high expression of MTX1 was closely related with poor outcomes in HCC patients who received sorafenib treatment. Mechanistically, overexpression of MTX1 could promote HCC cell autophagy via interacting with and inhibiting CDGSH iron sulfur domain 1 (CISD1), an autophagy negative regulator. Taken together, our findings suggest that MTX1 is upregulated in HCC and contributes to sorafenib resistance via a possible mechanism involving CISD1 mediated autophagy.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

GSTZ1 sensitizes hepatocellular carcinoma cells to sorafenib-induced ferroptosis via inhibition of NRF2/GPX4 axis

Increasing evidence supports that ferroptosis plays an important role in tumor growth inhibition. Sorafenib, originally identified as an inhibitor of multiple oncogenic kinases, has been shown to induce ferroptosis in hepatocellular carcinoma (HCC). However, some hepatoma cell lines are less sensitive to sorafenib-induced ferroptotic cell death. Glutathione S-transferase zeta 1 (GSTZ1), an enzyme in the catabolism of phenylalanine, suppresses the expression of the master regulator of cellular redox homeostasis nuclear factor erythroid 2-related factor 2 (NRF2). This study aimed to investigate the role and underlying molecular mechanisms of GSTZ1 in sorafenib-induced ferroptosis in HCC. GSTZ1 was significantly downregulated in sorafenib-resistant hepatoma cells. Mechanistically, GSTZ1 depletion enhanced the activation of the NRF2 pathway and increased the glutathione peroxidase 4 (GPX4) level, thereby suppressing sorafenib-induced ferroptosis. The combination of sorafenib and RSL3, a GPX4 inhibitor, significantly inhibited GSTZ1-deficient cell viability and promoted ferroptosis and increased ectopic iron and lipid peroxides. In vivo, the combination of sorafenib and RSL3 had a synergic therapeutic effect on HCC progression in Gstz1^−/− mice. In conclusion, this finding demonstrates that GSTZ1 enhanced sorafenib-induced ferroptosis by inhibiting the NRF2/GPX4 axis in HCC cells. Combination therapy of sorafenib and GPX4 inhibitor RSL3 may be a promising strategy in HCC treatment.Subject terms: Cancer therapeutic resistance, Cancer therapeutic resistance     

Differential interactions between Beclin 1 and Bcl-2 family members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x(L), Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x(L) was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x(L). Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x(L). Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x(L) and Beclin 1.     

Oncogene-induced autophagy and the Goldilocks principle

Although several oncogenes enhance autophagic flux, the molecular mechanism and consequences of oncogene-induced autophagy remain to be clarified. We have recently shown that expression of oncogenic H-Ras (V12) promotes autophagy through upregulation of Beclin 1 and the BH3-only protein Noxa. H-Ras-expressing cells undergo autophagic cell death as a result of Noxa-mediated displacement of Mcl-1 and Bcl-xL from Beclin 1. Oncogenic H-Ras-induced death is attenuated through knockdown of BECLIN 1, ATG5, or ATG7, or through overexpression of Mcl-1, Bcl-2, Bcl-xL and their close relatives. These observations suggest that high-intensity oncogene activation may be selected against by promoting excessive autophagy, leading to cell death. Consequently, such oncogenes may select for cells with a reduced capacity for autophagy, either through loss of a BECLIN 1 allele or through upregulation of negative regulators of Beclin 1, such as Bcl-2 family members.     

Inhibition of Autophagy by Chloroquine Enhances the Antitumor Efficacy of Sorafenib in Glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and common brain tumor in adults. Sorafenib, a multi-kinase inhibitor, has been shown to inhibit cell proliferation and induce apoptosis through inhibition of STAT3 signaling in glioblastoma cells and in intracranial gliomas. However, sorafenib also induces cell autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the therapeutic effect of sorafenib on glioblastoma is uncertain. Here, we combined sorafenib treatment in GBM cells (U373 and LN229) and tumors with the autophagy inhibitor chloroquine. We found that blockage of autophagy further inhibited cell proliferation and migration and induced cell apoptosis in vitro and in vivo. These findings suggest the possibility of combination treatment with sorafenib and autophagy inhibitors for GBM.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Role of RAF/MEK/ERK pathway,p‐STAT‐3 and Mcl‐1 in sorafenib activity in human pancreatic cancer cell lines

Sorafenib is a multikinase inhibitor that has shown promising therapeutic results in different tumor histotypes, both as a single agent or in combination with other treatments. We analyzed the in vitro activity of sorafenib in pancreatic cancer, one of the most lethal and chemo‐radio‐resistant tumors, using four human pancreatic cancer cell lines (t3m4, Capan 1, Capan 2, and MiaPaca 2), characterized by different K‐ras gene status and RAF/MEK/ERK profile. Sorafenib exerted a strong anti‐proliferative effect independently of RAS/RAF/MEK/ERK and induced various degrees of apoptosis in the cell lines. The mechanisms involved were explored in detail in t3m4 and Capan 1, in which sorafenib induced the highest and lowest levels of apoptosis, respectively. In t3m4, the RAF/AKT/STAT‐3 rather than the RAF/MEK/ERK pathway was involved, whereas in Capan 1 cells there was a strong decrease in pMEK and pERK which was not accompanied by an important reduction in RAF, AKT, and STAT‐3 proteins or in their phosphorylation. Moreover, U0126‐induced MEK inhibition did not induce apoptosis in any cell line, reinforcing the hypothesis of a MEK/ERK‐independent mechanism of sorafenib activity. Mcl‐1 appears to play a crucial role in sorafenib‐induced apoptosis. In fact, both protein and mRNA were downregulated in t3m4 and upregulated in Capan 1, in which siRNA‐induced silencing resulted in the same level of apoptosis as observed in t3m4. Our results show that sorafenib exerts anti‐proliferative and pro‐apoptotic activity in pancreatic cancer cells. Used singly or in combination with other drugs, it could therefore represent valid treatment for pancreatic cancer. J. Cell. Physiol. 220: 214–221, 2009. © 2009 Wiley‐Liss, Inc.     

Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC.     

Differential Interactions Between Beclin 1 and Bcl-2 Family Members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x_L, Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x_L was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x_L. Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x_L. Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x_L and Beclin 1.     

Oncogene-induced autophagy and the Goldilocks principle

Although several oncogenes enhance autophagic flux, the molecular mechanism and consequences of oncogene-induced autophagy remain to be clarified. We have recently shown that expression of oncogenic H-Ras^V12 promotes autophagy through upregulation of Beclin 1 and the BH3-only protein Noxa. H-Ras-expressing cells undergo autophagic cell death as a result of Noxa-mediated displacement of Mcl-1 and Bcl-xL from Beclin 1. Oncogenic H-Ras-induced death is attenuated through knockdown of BECLIN 1, ATG5, or ATG7, or through overexpression of Mcl-1, Bcl-2, Bcl-xL and their close relatives. These observations suggest that high-intensity oncogene activation may be selected against by promoting excessive autophagy, leading to cell death. Consequently, such oncogenes may select for cells with a reduced capacity for autophagy, either through loss of a BECLIN 1 allele or through upregulation of negative regulators of Beclin 1, such as Bcl-2 family members.     

Vitamin K1 enhances sorafenib-induced growth inhibition and apoptosis of human malignant glioma cells by blocking the Raf/MEK/ERK pathway

ABSTRACT: BACKGROUND: The combined effects of anticancer drugs with nutritional factors against tumor cells have been reported previously. This study characterized the efficacy and possible mechanisms of the combination of sorafenib and vitamin K1 (VK1) on glioma cell lines. METHODS: We examined the effects of sorafenib, VK1 or their combination on the proliferation and apoptosis of human malignant glioma cell lines (BT325 and U251) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) assay. The signaling pathway changes were detected by western blotting. RESULTS: Sorafenib, as a single agent, showed antitumor activity in a dose-dependent manner in glioma cells, but the effects were more pronounced when used in combination with VK1 treatment. Sorafenib in combination with VK1 treatment produced marked potentiation of growth inhibition and apoptosis, and reduced expression of phospho-mitogen-activated protein kinase kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK). Furthermore, the expression levels of antiapoptotic proteins Bcl-2 and Mcl-1 were significantly reduced. CONCLUSIONS: Our findings indicated that VK1 enhanced the cytotoxicity effect of sorafenib through inhibiting the Raf/MEK/ERK signaling pathway in glioma cells, and suggested that sorafenib in combination with VK1 maybe a new therapeutic option for patients with gliomas.     

Alpha-Fetoprotein Promoter-Driven Cre/LoxP-Switched RNA Interference for Hepatocellular Carcinoma Tissue-Specific Target Therapy

Backgroud

RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.

Methods

Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.

Results

The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.

Conclusions

An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.     

Prognostic significance of Beclin 1-dependent apoptotic activity in hepatocellular carcinoma

Autophagy is believed to be important in tumorigenesis and tumor progression. However, the role of autophagy in hepatocellular carcinoma (HCC), and especially the prognostic value of autophagic proteins, has not been investigated. Our studies described here show decreased basal expression of autophagic genes and their corresponding autophagic activity under conditions of starvation in HCC cell lines, and the autophagy defect correlated well with the highly malignant phenotype of HCC. In addition, in a tissue microarray study of HCC patients who underwent resection, the expression of the autophagy-related protein Beclin 1 was extremely low in tumors, where Beclin 1 could predict the prognosis of HCC patients only in a Bcl-X_L-positive expression background. Based on our results, we propose that autophagy defects that synergize with altered apoptotic activity might facilitate tumor progression and poor prognosis of HCC, due to the fact that autophagy may interact with apoptosis in the regulation of HCC.     

Targeting of distinct signaling cascades and cancer-associated fibroblasts define the efficacy of Sorafenib against prostate cancer cells

Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.     

mcl-1特异性siRNA对HepG2细胞增殖及凋亡的影响

目的：肝癌分子靶向治疗是目前研究的热点,肝癌相关基因mcl-1在肝癌增殖及凋亡中的作用尚不明确,本研究拟探讨mcl-1特异性siRNA对体外培养肝癌细胞HepG2增殖及凋亡的影响。方法：设计、合成有效的mcl-1特异性siRNA序列,体外转染HepG2细胞;通过绘制细胞生长曲线和MTT实验检测mcl-1特异性siRNA对HepG2细胞增殖的影响;通过AnnexinV／PI双标记流式细胞仪检测mcl-1特异性siRNA对HepG2细胞凋亡率的影响。结果：通过绘制细胞生长曲线发现,mcl-1特异性siRNA能够抑制HepG2细胞的增殖（P〈0．05）,MTT实验提示转染mcl-1特异性siRNA24h、48h、72h后,HepG2细胞存活率均显著下降（P〈O．05）;流式细胞仪检测分析发现,转染mcl．1特异性siRNA后AnnexinV＋／PI-细胞百分率显著增高（P〈0．01）,提示mcl-1具有促进HepG2细胞凋亡的作用。结论：Mcl-1蛋白具有促进肝癌细胞增殖,抑制肝癌细胞凋亡的作用,这种分子特性符合肿瘤靶向治疗的要求,Mcl-1可能成为肝癌靶向治疗的潜在靶点。