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.     

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.     

索拉菲尼联合阿霉素对人肝癌细胞株HepG2的抑制作用

目的：探讨索拉非尼（Sorafenib）和阿霉素（adriamycin）联合用药对肝癌细胞株nepG2的作用及可能的机制。方法：以不同浓度索拉非尼和不同浓度阿霉素分别组成单药组和索拉非尼＋阿霉素联合用药组作用于HepG2细胞,MTT法检测增殖抑制率、流式细胞仪分析细胞周期和凋亡率。结果：索拉非尼、阿霉素单药与联用均能抑制HepG2细胞增殖,呈剂量依赖效应,两药联用有协同效应（P〈0.01）。索拉非尼、阿霉素单药与联用均能诱导HepG2细胞凋亡,并以联合组更为明显,与对照组比较有显著的统计学意义（P〈0.01）。索拉非尼及阿霉素单药作用均可使细胞周期阻滞于G0-G1期,联合用药组G0/Gl期细胞比率低于索拉非尼及阿霉素单药组,S期细胞比率高于单药组;阿霉素能抑制HepG2细胞Survivin mRNA表达诱导细胞的凋亡。结论：索拉非尼与阿霉素联合作用于人肝癌HepG2细胞具有协同作用,其机制可能是通过多途径共同抑制HepG2细胞增殖及诱导细胞凋亡。     

Multikinase inhibitor Sorafenib and HDAC inhibitor suberoylanilide hydroxamic acid suppress confluent resistance of cancer cells to recombinant protein izTRAIL

Suppression of human tumor cell resistance to TRAIL-induced apoptosis in confluent cultures, using molecular target drugs (sorafenib and SAHA) at non-toxic concentrations was studied. Sorafenib, a multikinase inhibitor, and SAHA, an inhibitor of histone deacetylase, effectively suppressed resistance of confluent human cells derived from the skin carcinoma (A431 cell line) and fibrosarcoma (HT-1080 cell line). The effectiveness of suppression of confluent resistance with these inhibitors for human carcinoma A431 cells was significantly higher than that for the human ovarian carcinoma OVCAR-3 cells. For all cell lines studied, suppression of confluent resistance with SAHA was more effective than when sorafenib was used. The possible reason for increasing tumor cell resistance in confluent cultures and the importance of this phenomenon for understanding drug resistance of cells in the tumor tissue are discussed.     

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.     

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic "BH3-domain only" PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-β (TGF-β) through the intrinsic pathway and to tumor necrosis factor-α (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-β or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines.     

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.     

Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells

Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRb and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.     

Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells

Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRb and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.     

microRNA-1274a, a modulator of sorafenib induced a disintegrin and metalloproteinase 9 (ADAM9) down-regulation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with poor prognosis. Sorafenib, a multikinase inhibitor, has been widely used to treat patients with advanced HCC in clinic. We postulated that microRNAs (miRNA) might be involved in HCC target-chemotherapy with sorafenib. MiRNA profile of HepG2 was evaluated after cells were treated with vehicle or sorafenib and alterations in miRNA expression occurred with 14 miRNAs. MiR-1274a, which is up-regulated by sorafenib, could significantly repress expression of ADAM9, a protease that is involved in sorafenib target-therapy of HCC, in HCC cells. Taken together, our data emphasizes a new miRNA-based mechanism of sorafenib antitumor therapy.     

Mcl-1-dependent activation of Beclin 1 mediates autophagic cell death induced by sorafenib and SC-59 in hepatocellular carcinoma cells

We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway.     

Suppression of Natural Killer Cells by Sorafenib Contributes to Prometastatic Effects in Hepatocellular Carcinoma

Sorafenib, a multi-tyrosine kinase inhibitor, is a standard treatment for advanced hepatocellular carcinoma (HCC). The present study was undertaken to determine whether the growth and metastasis of HCC were influenced in mice receiving sorafenib prior to implantation with tumors, and to investigate the in-vivo and in-vitro effect of sorafenib on natural killer (NK) cells. In sorafenib-pretreated BALB/c nu/nu mice and C57BL/6 mice, tumor growth was accelerated, mouse survival was decreased, and lung metastasis was increased. However, the depletion of NK1.1⁺ cells in C57BL/6 mice eliminated sorafenib-mediated pro-metastatic effects. Sorafenib significantly reduced the number of NK cells and inhibited reactivity of NK cells against tumor cells, in both tumor-bearing and tumor-free C57BL/6 mice. Sorafenib down-regulated the stimulatory receptor CD69 in NK cells of tumor-bearing mice, but not in tumor-free mice, and inhibited proliferation of NK92-MI cells, which is associated with the blocking of the PI3K/AKT pathway, and inhibited cytotoxicity of NK cells in response to tumor targets, which was due to impaired ERK phosphorylation. These results suggest immunotherapeutic approaches activating NK cells may enhance the therapeutic efficacy of sorafenib in HCC patients.     

Sorafenib-induced Scrotal Eczema

Sorafenib is an orally active, small-molecule multikinase inhibitor that blocks tumor cell proliferation and angiogenesis. Studies have shown that it is a highly potent, selective inhibitor of vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor-β, RAF, FLT-3, and c-Kit. This drug was recently approved for the treatment of metastatic renal cell carcinoma and hepatocellular carcinoma. We report a case of a patient treated with sorafenib for metastatic renal cell carcinoma who developed scrotal eczema.Key words: Sorafenib, Scrotal eczema, Hand-foot skin reactionSorafenib is approved by the US Food and Drug Administration for use in metastatic renal cell carcinoma.¹ It is a multikinase inhibitor that blocks both tumor cell proliferation and angiogenesis. ² Up to 90% of patients receiving sorafenib have reported dermatologic symptoms, including handfoot skin reaction, facial erythema, subungual hemorrhage, alopecia, pruritus, and xerosis.³ Here we report on a patient on sorafenib who presents with the uncommon adverse effect of scrotal eczema.     

C/EBPβ enhances efficacy of sorafenib in hepatoblastoma

Hepatoblastoma (HB) is the predominant hepatic neoplasm in infants and young children. Sorafenib has been used to treat adult and pediatric hepatocellular carcinoma. However, efficacy of monotherapy of sorafenib in HB is not sustained. In this study, we tested a possible combinatory therapy of sorafenib with the CCAAT/enhancer-binding proteins (C/EBP) overexpression in HB cell line. Firstly, we evaluated the expression level of C/EBPβ in the patients with HB by analyzing The Cancer Genome Atlas data. Lower level of C/EBPβ was observed in tumor tissues in comparison with matched normal tissues. Next, we observed that combination of sorafenib and C/EBPβ overexpression led to dramatic growth and migration inhibition of live tumor cells which implied promising probability for clinical trial. Mechanistically, C/EBPβ which can be downregulated by Ras v12, augmented messenger RNA and protein levels of p53. These data suggested that a combination of sorafenib and C/EBPβ overexpression inhibited tumor growth synergistically and provided a promising approach to treat HB.     

Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death,Upregulation of p53 and Triggers Low Neurotoxicity

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin’s (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin’s effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.     

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory G_i proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-x_L, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.