Effects Of Sorafenib In Combination Research Articles

Ursolic acid enhances the antitumor effects of sorafenib associated with Mcl-1-related apoptosis and SLC7A11-dependent ferroptosis in human cancer

As a broad-spectrum oral small molecule inhibitor targeting multikinase, sorafenib is currently approved for the clinical treatment of several types of cancer as a single agent. A considerable number of clinical trial results have indicated that combination therapies involving sorafenib have been shown to improve treatment efficacy and may lead to novel therapeutic applications. Ursolic acid (UA), a natural pentacyclic triterpene compound extracted from a great variety of traditional medicinal plants and most fruits and vegetables, exhibits a wide range of therapeutic potential, including against cancer, diabetes, brain disease, liver disease, cardiovascular diseases, and sarcopenia. In the present study, we investigated the antitumor effects of sorafenib in combination with ursolic acid and found that the two agents displayed significant synergistic antitumor activity in in vitro and in vivo tumor xenograft models. Sorafenib/UA induced selective apoptotic death and ferroptosis in various cancer cells by evoking a dramatic accumulation of intracellular lipid reactive oxygen species (ROS). Mechanistically, the combination treatment promoted Mcl-1 degradation, which regulates apoptosis. However, decreasing the protein level of SLC7A11 plays a critical role in sorafenib/UA-induced cell ferroptosis. Therefore, these results suggest that the synergistic antitumor effects of sorafenib combined with ursolic acid may involve the induction of Mcl-1-related apoptosis and SLC7A11-dependent ferroptosis. Our findings may offer a novel effective therapeutic strategy for tumor treatment.

Targeting Na+ /K+ -ATPase by berbamine and ouabain synergizes with sorafenib to inhibit hepatocellular carcinoma.

The multikinase inhibitor sorafenib is a first-line drug for advanced hepatocellular carcinoma. The response to sorafenib varies among hepatocellular carcinoma patients and many of the responders suffer from reduced sensitivity after long-term treatment. This study aims to explore a novel strategy to potentiate or maximize the anti-hepatocellular carcinoma effects of sorafenib. We used hepatocellular carcinoma cell lines, western blotting, various antagonists, siRNA and tumour xenografts mouse model to determine the anti- hepatocellular carcinoma effects of sorafenib in combination with berbamine or other Na+ /K+ -ATPase ligands. Berbamine and the cardiotonic steroid, ouabain, synergize with sorafenib to inhibit hepatocellular carcinoma cells growth. Mechanistically, berbamine induces Src phosphorylation in Na+ /K+ -ATPase-dependent manner, leading to the activation of p38MAPK and EGFR-ERK pathways. The Na+ /K+ -ATPase ligand ouabain also induces Src, EGFR, type I insulin-like growth factor receptor, ERK1/2 and p38MAPK phosphorylation in hepatocellular carcinoma cells. Treatment of hepatocellular carcinoma cells with Src or EGFR inhibitor inhibits the induction of ERK1/2 phosphorylation by berbamine. Moreover, sorafenib inhibits the induction of Src, p38MAPK, EGFR and ERK1/2 phosphorylation by berbamine and ouabain. Importantly, combination of sorafenib with berbamine or ouabain synergistically inhibits both sorafenib-naïve and sorafenib-resistant hepatocellular carcinoma cells growth. Co-treatment of hepatocellular carcinoma cells with berbamine and sorafenib significantly induces cell death and significantly inhibits hepatocellular carcinoma xenografts growth in vivo. Berbamine or other Na+ /K+ -ATPase ligands have a potential for improving sorafenib responsiveness in hepatocellular carcinoma. Targeting Na+ /K+ -ATPase represents a novel strategy to potentiate the anti- hepatocellular carcinoma effects of sorafenib.

Novel Combination of Mesenchymal Stem Cell-Conditioned Medium with Sorafenib Have Synergistic Antitumor Effect of Hepatocellular Carcinoma Cells

Objective:Hepatocellular carcinoma (HCC) is the most common liver malignancy. Sorafenib is the first-line systemic treatment for advanced HCCs. However, due to safety concerns, researchers are now looking for ways to boost the efficacy of the medication. One approach for reducing toxicity is combining sorafenib with other agents so that a lower dose of sorafenib is required. Mesenchymal stromal cells (MSCs) can have an inhibitory effect on HCC tumor growth. Mesenchymal Stem Cell-Conditioned Medium (MSC-CM) is the substance extracted from MSC culture and contains most of the potential cytokines secreted by MSCs. We, therefore, anticipated a synergistic Antitumor Effect of sorafenib in Combination with MSC-CM. In this study, we used HepG2 as our target cell lines.Methods:HepG2 cells were treated with sorafenib alone and with sorafenib + MSC-CM. CCK-8 assay was used to evaluate and compare the inhibition of cell growth between the two groups with different treatments.Results:The combination treatment of cell lines with sorafenib and MSC-CM had significantly reduced the values of IC50 compared to the use of sorafenib alone (3.4 vs. 2.7 respectively).Conclusion:This study suggests that a combination of sorafenib with MSC-CM can synergistically suppress the growth of HCC cells.

Sequence‑dependent effect of sorafenib in combination with natural phenolic compounds on hepatic cancer cells and the possible mechanism of action.

Sorafenib (Nexavar, BAY43-9006 or Sora) is the first molecular targeted agent that has exhibited significant therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond well to Sora and novel therapeutic strategies to optimize the efficacy of Sora are urgently required. Plant-based drugs have received increasing attention owing to their excellent chemotherapeutic and chemopreventive activities; they are also well tolerated, non-toxic, easily available and inexpensive. It is well known that certain biologically active natural products act synergistically with synthetic drugs used in clinical applications. The present study aimed to investigate whether a combination therapy with natural phenolic compounds (NPCs), including curcumin (Cur), quercetin (Que), kaempherol (Kmf) and resveratrol (Rsv), would allow a dose reduction of Sora without concomitant loss of its effectiveness. Furthermore, the possible molecular mechanisms of this synergy were assessed. The hepatic cancer cell lines Hep3b and HepG2 were treated with Sora alone or in combination with NPCs in concomitant, sequential, and inverted sequential regimens. Cell proliferation, cell cycle, apoptosis and expression of proteins associated with the cell cycle and apoptosis were investigated. NPCs markedly potentiated the therapeutic efficacy of Sora in a sequence-, type-, NPC dose- and cell line-dependent manner. Concomitant treatment with Sora and Cur [sensitization ratio (SR)=28], Kmf (SR=18) or Que (SR=8) was associated with the highest SRs in Hep3b cells. Rsv markedly potentiated the effect of Sora (SR=17) on Hep3b cells when administered in a reverse sequential manner. By contrast, Rsv and Que did not improve the efficacy of Sora against HepG2 cells, while concomitant treatment with Cur (SR=10) or Kmf (SR=4.01) potentiated the cytotoxicity of Sora. Concomitant treatment with Sora and Cur or Kmf caused S-phase and G2/M phase arrest of liver cancer cells and markedly induced apoptosis compared with mono-treatment with Sora, Cur or Kmf. Concomitant treatment with Sora and Cur reduced the protein levels of cyclins A, B2 and D1, phosphorylated retinoblastoma and B-cell lymphoma (Bcl) extra-large protein. By contrast, Sora and Cur co-treatment increased the protein levels of Bcl-2-associated X protein, cleaved caspase-3 and cleaved caspase-9 in a dose-dependent manner. In conclusion, concomitant treatment with Sora and Cur or Kmf appears to be a potent and promising therapeutic approach that may control hepatic cancer by triggering cell cycle arrest and apoptosis. Additional studies are required to examine the potential of combined treatment with Sora and NPCs in human hepatic cancer and other solid tumor types in vivo.

Strong enhancement by IGF1-R antagonists of hepatocellular carcinoma cell migration inhibition by Sorafenib and/or vitamin K1.

Emerging evidence indicates that combining Sorafenib with vitamin K1 (VK1) may result in a synergistic inhibition of hepatocellular carcinoma (HCC) cell migration and proliferation. Despite this synergy, its benefits may be limited due to drug resistance resulting from cross-talk with the tumor microenvironment. Insulin-like growth factor-1 (IGF1) signaling acts as an important modulator of HCC cell growth, motility and drug resistance. Therefore, we aimed to explore the effects of Sorafenib in combination with VK1 and/or IGF1-R antagonists on HCC cells. Scratch wound migration assays were performed to assess the motility of HCC-derived PLC/PRF/5, HLF and Hep3B cells. The synergistic, additive or antagonistic effects of Sorafenib, VK1 and IGF1-R antagonists on HCC cell motility were assessed using CompuSyn software. The effects mediated by these various compounds on HCC cytoskeleton organization were evaluated using DyLight 554 Phalloidin staining. Proliferation and migration-associated signaling pathways were analyzed in PLC/PRF/5 cells using Erk1/2 and Akt activation kits and Western blotting (Mek, JNK, Akt, Paxillin and p38), respectively. The effects of the IGF1-R antagonists GSK1838705A and OSI-906 on HCC cell migration inhibition after Sorafenib and/or VK1 administration, individually or in combination, were evaluated. We found a synergistic effect in PLC/PRF/5, HLF and Hep3B cells for combinations of fixed doses of GSK1838705A or OSI-906 together with different doses of Sorafenib and/or VK1. The levels of synergy were found to be stronger at higher Sorafenib and/or VK1 concentrations and lower or absent at lower concentrations, with some variation among the different cell lines tested. In addition, we found that in PLC/PRF/5 and HLF cells IGF1-R blockage strongly enhanced the reduction and redistribution of F-actin induced by Sorafenib and/or VK1 through alterations in the phosphorylation levels of some of the principal proteins involved in the MAPK signaling cascade, which is essential for cell migration. Our results indicate that modulation of the efficacy of Sorafenib through combinations with VK1 and/or IGF1-R antagonists results in synergistic inhibition of HCC cell migration.

Combined delivery of sorafenib and a MEK inhibitor using CXCR4-targeted nanoparticles reduces hepatic fibrosis and prevents tumor development.

Liver damage and fibrosis are precursors of hepatocellular carcinoma (HCC). In HCC patients, sorafenib—a multikinase inhibitor drug—has been reported to exert anti-fibrotic activity. However, incomplete inhibition of RAF activity by sorafenib may also induce paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in malignant cells. The consequence of this effect in non-malignant disease (hepatic fibrosis) remains unknown. This study aimed to examine the effects of sorafenib on activated hepatic stellate cells (HSCs), and develop effective therapeutic approaches to treat liver fibrosis and prevent cancer development.Methods: We first examined the effects of sorafenib in combination with MEK inhibitors on fibrosis pathogenesis in vitro and in vivo. To improve the bioavailability and absorption by activated HSCs, we developed CXCR4-targeted nanoparticles (NPs) to co-deliver sorafenib and a MEK inhibitor to mice with liver damage.Results: We found that sorafenib induced MAPK activation in HSCs, and promoted their myofibroblast differentiation. Combining sorafenib with a MEK inhibitor suppressed both paradoxical MAPK activation and HSC activation in vitro, and alleviated liver fibrosis in a CCl4-induced murine model of liver damage. Furthermore, treatment with sorafenib/MEK inhibitor-loaded CXCR4-targeted NPs significantly suppressed hepatic fibrosis progression and further prevented fibrosis-associated HCC development and liver metastasis.Conclusions: Our results show that combined delivery of sorafenib and a MEK inhibitor via CXCR4-targeted NPs can prevent activation of ERK in activated HSCs and has anti-fibrotic effects in the CCl4-induced murine model. Targeting HSCs represents a promising strategy to prevent the development and progression of fibrosis-associated HCC.

Sorafenib in combination with chemotherapy as first-line therapy for FLT3-ITD positive acute myeloid leukemia

目的探讨Fms样酪氨酸激酶3内部串联重复（FLT3-ITD）突变阳性的急性髓系白血病（AML）患者的临床特征及预后，分析索拉非尼联合化疗一线治疗该类患者的疗效。方法回顾性分析2013年1月至2016年8月河南省肿瘤医院确诊的53例初治伴FLT3-ITD突变的AML患者资料，研究该类患者发病时生物学特征，并对比分析化疗联合与不联合索拉非尼治疗的疗效。结果53例FLT3-ITD突变阳性AML患者中，M5型所占比例最高（41.5%），外周血WBC中位数为61.00（0.98~920.00）×109/L，其中高白细胞计数（>10×109/L）患者50例（94.3%）；骨髓原始细胞比例中位数为0.730（0.234~0.966）。53例患者总诱导缓解率为56.6%，索拉非尼联合化疗组与单纯化疗组诱导缓解率分别为86.4%（19/22）和35.5%（11/31），差异有统计学意义（P<0.001）。索拉非尼联合化疗组与单纯化疗组患者1年总生存率分别为78.3%和50.0%（P=0.041），1年无进展生存率分别为75.9%和42.4%（P=0.044）。结论FLT3-ITD突变阳性AML具有初发时外周血WBC及骨髓原始细胞比例高、与M5亚型高度伴随的临床特征，索拉非尼联合化疗可明显改善该类患者的诱导缓解率及短期生存。

Synergistic Antitumor Effect of Sorafenib in Combination with ATM Inhibitor in Hepatocellular Carcinoma Cells.

Background: Currently, sorafenib is the only systemic chemotherapy drug for advanced stage Hepatocellular carcinoma (HCC). However, emerging data from some clinical HCC patients indicate that sorafenib alone has only moderate antitumor efficacy, and could not inhibit disease metastasis and progression. KU-55933 is a specific ATM inhibitor, which has pro-apoptotic effect on tumor cells. In this study, we analyzed the synergistic effect of sorafenib and KU-55933 on the proliferation of HCC cell lines.Methods: Three HCC cell lines were treated with sorafenib and KU-55933 alone or combination in vitro to investigate inhibitory effect by MTT and wound healing assay. Epithelial to mesenchymal transition (EMT) phenotype change was investigated after sorafenib and KU-55933 treatment by microscopy. Akt signaling pathway proteins including p-Akt, p-mTOR and p-p70S6K were examined by western blot. In addition, cleaved PARP and autophage-related proteins LC3A/B were detected by western blot.Results: KU-55933 can enhance the effect of sorafenib in inhibiting cell proliferation and migration, overcoming EMT, inducing cell apoptosis via inactivating Akt signaling pathway and inducing autophage. The combination treatment with sorafenib and KU-55933 resulted in a strong synergistic effect in vitro.Conclusion: Our results demonstrate that sorafenib combined with KU-55933 treatment does effectively inhibit proliferation of HCC cell lines synergistically. These data suggests that KU-55933 may be a promising chemosensitizer to sorafenib in the treatment of HCC.

Combination effects of sorafenib with PI3K inhibitors under hypoxia in colorectal cancer.

AimThis study reports the influence of hypoxia on response of colorectal cancer cells to anticancer effects of sorafenib in combination with PI3K inhibitors GDC-0941 and BEZ-235.Materials and methodsAll hypoxic exposures were carried out at 1% O2/5% CO2. Antiproliferation activity was evaluated by 48 hours propidium iodide and 14 days clonogenic assay. Protein levels were evaluated by fluorescence ELISA. Metabolites lactate and glucose were evaluated biochemically.ResultsIn the 48-hour proliferation assay, sorafenib acted synergistically with GDC-0941 but not with BEZ-235. In long-term colony-forming assays, both GDC-0941 and BEZ-235 were shown to potentiate the antiproliferative activity of sorafenib. At the molecular level, the synergism is mediated through inhibition of pAKT, pS6, p4EBP1, pERK, cyclin D1, and Bcl-2. No change in hypoxia-inducible factor-1α (HIF-1α) levels was observed in cells treated with the combination of compounds under hypoxia. A significant reduction in glucose uptake and lactate release was observed in cells treated with the combination of compounds under normoxia and hypoxia.ConclusionCombinations of sorafenib with PI3K inhibitors BEZ-235 and GDC-0941 are efficacious under hypoxia. Thus, these anticancer combinations have a potential to overcome the hypoxia-mediated resistance mechanisms to antiproliferative agents in cancer therapy.

Synergistic effects of sorafenib in combination with gemcitabine or pemetrexed in lung cancer cell lines with K-ras mutations.

K-ras is currently accepted as the most frequently mutated oncogene in non-small cell lung cancer (NSCLC, including squamous carcinoma, adenocarcinoma, and large cell carcinoma). NSCLC patients with the K-ras mutation appear to be refractory to the majority of systemic therapies. In the present study, the in vitro antitumor effects and correlated molecular mechanisms of sorafenib combined with gemcitabine or pemetrexed were explored in the K-ras mutation-positive NSCLC A549 cell line. Sorafenib was seen to exhibit dose-dependent growth inhibition in the A549 cells, while sorafenib combined with pemetrexed demonstrated a greater synergism compared with sorafenib combined with gemcitabine. Sorafenib arrested the cell cycle at the G1 phase, while gemcitabine and pemetrexed caused arrest at the S phase. The molecular mechanism of this synergism was due to the downstream signalling pathways, which were efficiently suppressed by sorafenib, therefore increasing the incidence of the entry of the chemotherapeutic drugs into the apoptotic pathways. Moreover, sorafenib and pemetrexed demonstrated stronger synergism, demonstrating that inhibiting the Ras/Raf/Mek/Erk and Ras/PI3K/Akt pathways concurrently may achieve improved antitumor effects.

Anti-proliferation effect of sorafenib in combination with 5-FU for hepatocellular carcinoma in vitro: antagonistic performance and mechanism

To investigate the anti-cancer efficacy and mechanism of sorafenib and 5-fluorouracil (5-FU) therapy in vitro using the HCC cell line MHCCLM3. The effects of sorafenib and 5-FU, alone or in combination, on the proliferation of MHCCLM3 cells were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle established by Chou and Talalay. Effects on cell cycle distributions were tested by flow cytometry and expression of proteins related to the RAF/MEK/ERK and STAT3 signaling pathways and cyclinD1 were tested by western blotting. Sorafenib and 5-FU alone or in combination displayed significant efficacy in inhibiting proliferation of the MHCCLM3 cells, with the following inhibition rates: sorafenib: 46.16% +/- 2.52%, 5-FU: 28.67% +/- 6.16%, and sorafenib + 5-FU: 22.59% +/- 6.89%. The sorafenib + 5-FU combination did not provide better results than treatment with either drug alone. The combination index values of the sorafenib and 5-FU treatments were mainly greater than 1, indicating that the two agents induced antagonistic, instead of synergistic, effects on the MHCCLM3 cells. In addition, the MHCCLM3 cells were less sensitive to 5-FU when administrated in combination with sorafenib, as evidenced by the half inhibitory concentration (IC50) significantly increasing from (102.86 +/- 27.84) mg/L to (178.61 +/- 20.73) mg/L (P = 0.003). Sorafenib alone induced G1 phase arrest (increasing from 44.73% +/- 1.63% to 65.80% +/- 0.56%; P less than 0.001) and significantly decreased the proportion of cells in S phase (decreasing from 46.63% +/- 0.65% to 22.83% +/- 1.75%; P less than 0.01), as well as down-regulated cyclinD1 expression (0.57 +/- 0.03-fold change vs. untreated control group; P less than 0.01). 5-FU alone up-regulated cyclinD1 expression (1.45 +/- 0.12-fold change vs. untreated control group; P less than 0.01). Moreover, sorafenib alone significantly inhibited the RAF/MEK/ERK and STAT3 pathways, with the fold-changes of p-C-RAF, p-ERK1/2 and p-STAT3 being 0.56 +/- 0.05, 0.54 +/- 0.02 and 0.36 +/- 0.02, respectively (all P less than 0.01); 5-FU alone produced no significant effects on these pathways. Administered alone, both sorafenib and 5-FU exert anti-tumoral activity on in vitro cultured HCC cells. The sorafenib + 5-FU combination treatment produces antagonistic, rather than synergistic, effects. Sorafenib-inhibited RAF/MEK/ERK and STAT3 signaling and cyclinD1 expression may have induced the observed G1phase arrest and S phase reduction, thereby reducing the cells' sensitivity to 5-FU.

Synergistic antitumour activity of sorafenib in combination with tetrandrine is mediated by reactive oxygen species (ROS)/Akt signaling

Background:Sorafenib is a potent inhibitor against Raf kinase and several receptor tyrosine kinases that has been approved for the clinical treatment of advanced renal and liver cancer. Combining sorafenib with other agents has been shown to improve its antitumour efficacy by not only reducing the toxic side effects but also preventing primary and acquired resistance to sorafenib. We have previously observed that tetrandrine exhibits potent antitumour effects in human hepatocellular carcinoma. In this study, we investigated the synergistic antitumour activity of sorafenib in combination with tetrandrine.Methods:This was a two-part investigation that included the in vitro effects of sorafenib in combination with tetrandrine on cancer cells and the in vivo antitumour efficacy of this drug combination on tumour xenografts in nude mice.Results:Combined treatment showed a good synergistic antitumour effect yet spared nontumourigenic cells. The potential molecular mechanism may be mainly that it activated mitochondrial death pathway and induced caspase-dependent apoptosis in the cancer cells. Accumulation of intracellular reactive oxygen species (ROS) and subsequent activation of Akt may also be involved in apoptosis induction.Conclusion:The antitumour activity of sorafenib plus tetrandrine may be attributed to the induction of the intrinsic apoptosis pathway through ROS/Akt signaling. This finding provides a novel approach that may broaden the clinical application of sorafenib.

Antiproliferative effects of sorafenib and pegylated IFN-α2b on human liver cancer cells in vitro and in vivo

Novel therapeutic strategies are needed to treat patients with advanced hepatocellular carcinoma (HCC). Combination therapy of sorafenib and type I interferon (IFN) has substantial activity in patients with metastatic renal cell carcinoma. We investigated the antiproliferative effects of sorafenib in combination with pegylated interferon-α2b (PEG-IFN-α2b) on human hepatocellular carcinoma (HCC) cells in vitro and in vivo. A poorly differentiated HCC cell line derived from a patient with hepatitis C virus infection, HAK-1B and the moderately differentiated HCC cell line KIM-1 were used in this study. We demonstrated a synergistic antiproli ferative effect of combination therapy on HAK-1B cells in vitro. In the in vivo study, a significant reduction of tumor volume and weight were observed in the combination group in both HAK-1B and KIM1 tumors, although synergistic effects were not clearly observed. The density of CD34-positive microvessels was significantly lower and cleaved caspase-3-positive apoptotic cell numbers were higher, in the sorafenib group and the combination group compared to the control or PEG-IFN-α2b group in both HAK-1B and KIM-1 tumors. Ki67 labeling index was significantly lower in the combination group compared to the control group in KIM-1 tumors. In conclusion, our results suggest that the combination therapy may be more effective for the treatment of HCC cases with variable sensitivity to antitumor effects of single therapy with either sorafenib or PEG-IFN-α2b.

IS1-4 - Synergistic Anti-Tumor Effect of Sorafenib in Combination with AKT Inhibitors in Hepatocellular Carcinoma

ABSTRACT Background Sorafenib is currently the only approved agent for hepatocellular carcinoma (HCC). However, its clinical efficacy in HCC is limited. Previous studies indicate that compensatory activation of AKT may contribute to the resistance to sorafenib. We hypothesized that combination of sorafenib and AKT inhibitors could enhance the antitumor effect of sorafenib in HCC. Methods We analyzed the cellular and molecular effects of sorafenib in combination with inhibitors of AKT in human HCC cell lines, and evaluated their combinational effects against HCC in vitro and in vivo. Results HCC cells, including Huh7, Hep3B, and SKhep1 cells, expressed high levels of p-AKT in a prompt and sustained manner upon treated with sorafenib. Down-regulation of AKT by siRNA or inhibition of phospho-AKT by AKT inhibitors (MK-2206 or perifosine) enhanced the anti-proliferative effect of sorafenib in cultured HCC cells. The combination indexes of sorafenib and AKT inhibitors were Conclusions Inhibition of AKT by AKT inhibitors improved the antitumor effect of sorafenib against HCC in vitro and in vivo. Combination of AKT inhibitors and sorafenib warrants further consideration for clinical development in HCC. (The study was supported by NSC97-2628-B-002-004-MY3 and NSC100-2325-B-002-043-.)

Sorafenib and HDAC inhibitors synergize to kill CNS tumor cells

The present studies were designed to determine whether the multi-kinase inhibitor sorafenib (Nexavar) interacted with histone deacetylase inhibitors to kill glioblastoma and medulloblastoma cells. In a dose-dependent fashion sorafenib lethality was enhanced in multiple genetically disparate primary human glioblastoma isolates by the HDAC inhibitor sodium valproate (Depakote). Drug exposure reduced phosphorylation of p70 S6K and of mTOR. Similar data to that with valproate were also obtained using the HDAC inhibitor vorinostat (Zolinza). Sorafenib and valproate also interacted to kill medulloblastoma and PNET cell lines. Treatment with sorafenib and HDAC inhibitors radio-sensitized both GBM and medulloblastoma cell lines. Knock down of death receptor (CD95) expression protected GBM cells from the drug combination, as did overexpression of c-FLIP-s, BCL-XL and dominant negative caspase 9. Knock down of PDGFRα recapitulated the effect of sorafenib in combination with HDAC inhibitors. Collectively, our data demonstrate that the combination of sorafenib and HDAC inhibitors kills through activation of the extrinsic pathway, and could represent a useful approach to treat CNS-derived tumors.

Abstract 4337: Effect of sorafenib in combination with cytostatic agents on hepatoblastoma in vitro and in vivo

Abstract Background: Hepatoblastoma (HB) represents the most common malignant liver tumour in childhood. For patients with high risk or recurrent HB treatment results remain still poor due to a developing multidrug resistance during chemotherapy. Therefore new therapy strategies have to be evaluated. The primary objective was to investigate the combined therapy of cytotoxic agents with sorafenib. Sorafenib has been shown to reduce tumour progression and angiogenesis. In order to investigate a treatment close to a clinical setting the effect of the combined administration of cytotoxic agents with sorafenib on HB cell lines and xenotransplanted HB tumours was determined. Methods: Cell viability of two HB cell lines (HUH6 and HepT1) was evaluated in using MTT assays after treatment with sorafenib and different cytostatic agents (cisplatin (CDDP), doxorubicin, irinotecan, topotecan). ERK signalling was investigated by Western blot analysis. NMRI mice (nu/nu) bearing subcutaneous HUH6 derived tumours were treated with Sorafenib orally (30 mg/kg, on days 4-13 and 17-24) alone or in combination with CDDP i.p. (3 mg/kg, on days 1-3 and 14-16). Tumor progression and viability were monitored by tumour volume and AFP levels. Apoptosis was assessed by TUNEL assay. Angiogenesis was determined by CD31 staining and mean vascular density (MVD) was calculated. Results: In vitro the combination of sorafenib with CDDP showed a remarkable decrease in cell viability compared to the other agents tested. However with increasing concentrations of CDDP this additive effect disappeared. CDDP alone showed an enhanced phosphorylation of ERK1/2, which is a common pathway for apoptosis induction through NOXA. CDDP in combination with sorafenib led to an overall reduced phosphorylation level of ERK1/2, which also resulted in a low HB cell viability. Since CDDP and sorafenib have an opposing effect on phosphorylation of ERK1/2, we avoid the concomitant administration of both drugs in vivo. In HB xenografts both sorafenib alone or in alternating combination with CDDP reduced tumour growth compared to the control untreated group (p &amp;lt; 0.05). The combination treatment showed a higher and earlier reduction of tumor volume than sorafenib alone. In addition AFP levels did not significantly change in the treatment groups. Apoptotic areas as determined by TUNEL analysis increased significantly after administration of sorafenib or sorafenib and CDDP (p &amp;lt; 0.08). Furthermore CD31 staining showed a significant inhibition of angiogenesis as revealed by determination of the MVD in both treatment groups (p &amp;lt; 0.02). Discussion: Taken together the combination therapy of Sorafenib and CDDP led to an enhanced decrease in cell viability as well as in tumour progression and angiogenesis than sorafenib alone. Therefore a combined therapy might be a promising option in the treatment of high risk or recurrent hepatoblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4337. doi:1538-7445.AM2012-4337

Abstract 5024: Sorafenib in combination with modifiers of epigenetic mechanism induce synergistic cytotoxicity in human liver cancer cell lines

Abstract Background: Hepatocellular carcinoma (HCC) is the third most important cause of cancer death worldwide. Sorafenib, a multikinase inhibitor improved outcomes in patients with hepatocellular carcinoma (HCC) by inhibiting angiogenesis. Epigenetic research suggests that many cancers may not be caused by mutations in genes but by chemical modifications that change how the genes function. DNA methylation and histone deacetylation are two of the more widely studied epigenetic mechanisms. Inhibitors of DNA methylation (Vidaza) and histone deacetylation (SAHA) have shown inhibited cell growth and induced apoptosis in liver cancer cells. We investigated the cytotoxicity of combination of Sorafenib with SAHA or Vidaza in two liver cancer cell lines HepG2 and Hep3B. METHODS: HepG2 and Hep3B cells in log phase growth were treated simultaneously with sorafenib (0.5-10 µM) and/or vidaza (1.25-10 µM) or SAHA (1.25-10 µM) for 72 hours. Following drug treatment, MTS assay was used as the endpoint for cell proliferation and the IC50 values and combination indices (CI) were derived using Calcusyn software (Biosoft, Cambridge, UK). Each combination was repeated at least three times. RESULTS: The mean single agent IC50's for Sorafenib, Vidaza and SAHA were 6.83±0.44 µM, 10.33±2.91µM, and 11.5±4.25 µM, respectively in HepG2 cells and 6.25±0.20 µM, 11.50±0.41 µM, and 4.25±0.20 µM, respectively in Hep3B cells. The combination indices were:CombinationsHepG2Hep3BSorafenib and Vidaza0.72+0.130.73+0.12Sorafenib and SAHA0.77+0.130.68+0.05 CONCLUSIONS: The CI's for both combinations demonstrated synergistic effects in each of the cell lines. The molecular mechanism behind the synergistic effect of sorafenib in combination with vidaza or SAHA in liver cancer in underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5024.

Schedule-dependent effects of sorafenib in combination with paclitaxel on human hepatocellular carcinoma cell line BEL-7402

Sorafenib is a multi-targeted antitumor drug. The monotherapy efficacy of sorafenib is relatively low. This study was to evaluate the schedule-dependent effect of sorafenib in combination with paclitaxel (TAX) on human hepatocellular carcinoma cell BEL-7402, and explore the underlying mechanism. BEL-7402 cells were treated with sorafenib or paclitaxel alone or in three different schedules: sorafenib was give prior to, after, or simultaneously with paclitaxel. The half maximal inhibitory concentration (IC50) of sorafenib and paclitaxel was estimated by MTT. Alteration of cell cycle and apoptosis were analyzed by flow cytometry. The protein level of Bcl-2 in BEL-7402 cells was measured by western blot. At 48 h, the IC50 of sorafenib and paclitaxel for BEL-7402 cells was (2.43+/-0.32) microg/mL and (1.89+/-0.72) microg/mL, respectively. Sorafenib caused cell cycle arrest at S phase, while paclitaxel blocked cells at G2/M phase. S and G2/M phases were extended and a higher apoptotic rate (36.43+/-2.29)% was induced when sorafenib was given after paclitaxel in comparision with other groups (P<0.01). The protein level of Bcl-2 was the lowest in BEL-7402 cells treated with sorafenib after paclitaxel. Administration of sorafenib after paclitaxel induces a higher apoptotic rate in BEL-7402 cells than administration before or simutanously with paclitaxel. This is probably due to that the two drugs act on different cell cycle phases and the expression of Bcl-2 might be involved.

Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo

The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.