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The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

ABSTRACT: Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5-5 ?M) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.

SUBMITTER: Tesori V

PROVIDER: S-EPMC4361992 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

Tesori Valentina V Piscaglia Anna Chiara AC Samengo Daniela D Barba Marta M Bernardini Camilla C Scatena Roberto R Pontoglio Alessandro A Castellini Laura L Spelbrink Johannes N JN Maulucci Giuseppe G Puglisi Maria Ausiliatrice MA Pani Giovambattista G Gasbarrini Antonio A Gasbarrini Antonio A

Scientific reports 20150317

Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expre ...[more]

PMID: 25779766

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Project description:Objective: identify novel and relevant aspects of Sorafenib action on liver cancer cells. We found that in rat hepatocholangiocarcinoma (LCSC-2) cells, exposure to the MEK/multikinase inhibitor sorafenib did not inhibit ERK phosphorylation nor induced appreciable cell death in the low micromolar range; instead, the drug elicited a raise of intracellular reactive oxygen species (ROS) accompanied by a severe decrease of oxygen consumption and intracellular ATP levels, all changes consistent with mitochondrial damage. Moreover, Sorafenib induced depolarization of isolated rat liver mitochondria, indicating a possible direct effect on the organelle. Microarray analysis of gene expression in sorafenib-trated cells revealed a metabolic reprogramming toward aerobic glycolysis, that likely accounts for resitance to drug toxicity in this cell line. Importantly, cytotoxicity was strongly potentiated by glucose withdrawal from the culture medium or by the glycolytic inhibitor 2-deoxy-glucose, a finding also confirmed in the highly malignant melanoma cell line B16F10. Mechanistic studies revealed that ROS are pivotal to cell killing by the Sorafenib + 2DG combination, and that a low content of intracellular oxidants is associated with resistance to the drug; instead, Thr172phosphorylation/activation of the AMP-activated protein kinase (AMPK), induced by Sorafenib, may exert protective effects, since cytotoxicity was enhanced by an AMPK specific inhibitor and prevented by the AMPK activator Metformin. Overall, this study identifies novel and relevant aspects of Sorafenib action on liver cancer cells, including mitochondrial damage, induction of ROS and a metabolic cell reprogramming towards “glucose addiction”, potentially exploitable in therapy. Microarray analysis of gene expression in sorafenib-trated cells. LCSC-2 cells were incubated with sorafenib 2.5 mM under serum deprivation for 12 hours. The total RNA was isolated from LCSC2, 24h after treatment, using the RNeasy mini kit (Qiagen, Hilden, Germany), RNA was quantified using a UV spectrophotometer and RNA quality and integrity were assessed Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The resulting total RNA was then used to create the biotin-labeled library to be hybridized on GeneChip Rat Gene expression 1.0 st Array (Affymetrix, Santa Clara, CA, USA), according to the recommended experimental protocols provided by the suppliers. The CEL files resulting from the hybridization were analyzed using the Partek® Genomic Suite™ (Partek GS). Gene-level calculation was performed by Robust Multichip Average and normalization by quantile sketch.

Project description:BackgroundHepatocellular carcinoma (HCC) is the sixth most common type of cancer and has a high mortality rate worldwide. Sorafenib is the only systemic treatment demonstrating a statistically significant but modest overall survival benefit. We previously have identified the aurora kinases (AURKs) and FMS-like tyrosine kinase 3 (FLT3) multikinase inhibitor DBPR114 exhibiting broad spectrum anti-tumor effects in both leukemia and solid tumors. The purpose of this study was to evaluate the therapeutic potential of DBPR114 in the treatment of advanced HCC.MethodsHuman HCC cell lines with histopathology/genetic background similar to human HCC tumors were used for in vitro and in vivo studies. Human umbilical vein endothelial cells (HUVEC) were used to evaluate the drug effect on endothelial tube formation. Western blotting, immunohistochemical staining, and mRNA sequencing were employed to investigate the mechanisms of drug action. Xenograft models of sorafenib-refractory and sorafenib-acquired resistant HCC were used to evaluate the tumor response to DBPR114.ResultsDBPR114 was active against HCC tumor cell proliferation independent of p53 alteration status and tumor grade in vitro. DBPR114-mediated growth inhibition in HCC cells was associated with apoptosis induction, cell cycle arrest, and polyploidy formation. Further analysis indicated that DBPR114 reduced the phosphorylation levels of AURKs and its substrate histone H3. Moreover, the levels of several active-state receptor tyrosine kinases were downregulated by DBPR114, verifying the mechanisms of DBPR114 action as a multikinase inhibitor in HCC cells. DBPR114 also exhibited anti-angiogenic effect, as demonstrated by inhibiting tumor formation in HUVEC cells. In vivo, DBPR114 induced statistically significant tumor growth inhibition compared with the vehicle control in multiple HCC tumor xenograft models. Histologic analysis revealed that the DBPR114 treatment reduced cell proliferation, and induced apoptotic cell death and multinucleated cell formation. Consistent with the histological findings, gene expression analysis revealed that DBPR114-modulated genes were mostly related to the G2/M checkpoint and mitotic spindle assembly. DBPR114 was efficacious against sorafenib-intrinsic and -acquired resistant HCC tumors. Notably, DBPR114 significantly delayed posttreatment tumor regrowth and prolonged survival compared with the regorafenib group.ConclusionOur results indicated that targeting AURK signaling could be a new effective molecular-targeted agent in the treatment of patients with HCC.

Project description:Pulmonary hypertension (PH) and cancer pathology share growth factor- and MAPK stress-mediated signaling pathways resulting in endothelial and smooth muscle cell dysfunction and angioproliferative vasculopathy. In this study, we assessed sorafenib, an antineoplastic agent and inhibitor of multiple kinases important in angiogenesis [VEGF receptor (VEGFR)-1-3, PDGF receptor (PDGFR)-beta, Raf-1 kinase] as a potential PH therapy. Two PH rat models were used: a conventional hypoxia-induced PH model and an augmented PH model combining dual VEGFR-1 and -2 inhibition (SU-5416, single 20 mg/kg injection) with hypoxia. In addition to normoxia-exposed control animals, four groups were maintained at 10% inspired O(2) fraction for 3.5 wk (hypoxia/vehicle, hypoxia/SU-5416, hypoxia/sorafenib, and hypoxia/SU-5416/sorafenib). Compared with normoxic control animals, rats exposed to hypoxia/SU-5416 developed hemodynamic and histological evidence of severe PH while rats exposed to hypoxia alone displayed only mild elevations in hemodynamic values (pulmonary vascular and right ventricular pressures). Sorafenib treatment (daily gavage, 2.5 mg/kg) prevented hemodynamic changes and demonstrated dramatic attenuation of PH-associated vascular remodeling. Compared with normoxic control rats, expression profiling (Affymetrix platform) of lung RNA obtained from hypoxia [false discovery rate (FDR) 6.5%]- and hypoxia/SU-5416 (FDR 1.6%)-challenged rats yielded 1,019 and 465 differentially regulated genes (fold change >1.4), respectively. A novel molecular signature consisting of 38 differentially expressed genes between hypoxia/SU-5416 and hypoxia/SU-5416/sorafenib (FDR 6.7%) was validated by either real-time RT-PCR or immunoblotting. Finally, immunoblotting studies confirmed the upregulation of the MAPK cascade in both PH models, which was abolished by sorafenib. In summary, sorafenib represents a novel potential treatment for severe PH with the MAPK cascade a potential canonical target.

Project description:BackgroundAcute myeloid leukemia (AML) is a genetically heterogeneous cancer that frequently exhibits aberrant kinase signaling. We investigated a treatment strategy combining sorafenib, a multikinase inhibitor with limited single-agent activity in AML, and cytarabine, a key component of AML chemotherapy.MethodsUsing 10 human AML cell lines, we determined the effects of sorafenib (10 μM) on antileukemic activity by measuring cell viability, proliferation, ERK1/2 signaling, and apoptosis. We also investigated the effects of sorafenib treatment on the accumulation of cytarabine and phosphorylated metabolites in vitro. A human equivalent dose of sorafenib in nontumor-bearing NOD-SCID-IL2Rγ(null) mice was determined by pharmacokinetic studies using high performance liquid chromatography with tandem mass spectrometric detection, and steady-state concentrations were estimated by the fit of a one-compartment pharmacokinetic model to concentration-time data. The antitumor activity of sorafenib alone (60 mg/kg) twice daily, cytarabine alone (6.25 mg/kg administered intraperitoneally), or sorafenib once or twice daily plus cytarabine was evaluated in NOD-SCID-IL2Rγ(null) mice bearing AML xenografts.ResultsSorafenib at 10 μM inhibited cell viability, proliferation and ERK1/2 signaling, and induced apoptosis in all cell lines studied. Sorafenib also increased the cellular accumulation of cytarabine and metabolites resulting in additive to synergistic antileukemic activity. A dose of 60 mg/kg in mice produced a human equivalent sorafenib steady-state plasma exposure of 10 μM. The more dose-intensive twice-daily sorafenib plus cytarabine (n = 15) statistically significantly prolonged median survival in an AML xenograft model compared with sorafenib once daily plus cytarabine (n = 12), cytarabine alone (n = 26), or controls (n = 27) (sorafenib twice daily plus cytarabine, median survival = 46 days; sorafenib once daily plus cytarabine, median survival = 40 days; cytarabine alone, median survival = 36 days; control, median survival = 19 days; P < .001 for combination twice daily vs all other treatments listed).ConclusionsSorafenib in combination with cytarabine resulted in strong anti-AML activity in vitro and in vivo. These results warrant clinical evaluation of sorafenib with cytarabine-based regimens in molecularly heterogeneous AML.

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The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

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The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

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