Vitamin K1 enhances sorafenib-induced growth inhibition and apoptosis of human malignant glioma cells by blocking the Raf/MEK/ERK pathway
© Du et al.; licensee BioMed Central Ltd. 2012
Received: 30 November 2011
Accepted: 21 April 2012
Published: 21 April 2012
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.
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.
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.
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.
Malignant gliomas are the most prevalent primary brain tumors in adults, exhibiting a high rate of cell proliferation and migration activities . Despite tremendous efforts in the improvement of therapeutics, such as surgery, radiotherapy and chemotherapy, the clinical outcome of gliomas remains dismaying .
Recent publications indicate that the majority of gliomas display upregulated Raf kinase , which is an essential serine/threonine kinase constituent of the mitogen-activated protein kinase (MAPK) pathway. Raf is recruited to the cellular membrane and activated by GTP-bound activated Ras. Activated Raf phosphorylates MAPK kinase 1/2 (MEK 1/2) , which in turn phosphorylates and activates extracellular signal-regulated kinase 1/2 (ERK 1/2). Then, ERK activation leads to phosphorylation of a variety of transcription factors and results in induction of gene expression and proliferation . The upregulation of the Raf/MEK/ERK pathway has been proven to take part in the amplification of mitogenic stimuli and promotion of cellular proliferation of malignant gliomas. Therefore downregulation of the Raf/MEK/ERK pathway may be a valuable therapy for glioma patients .
Sorafenib (Nexavar, BAY 43–9006) is an oral, small molecule multikinase inhibitor that was originally developed as a Raf kinase inhibitor . Subsequent studies have demonstrated that it also inhibits receptor tyrosine kinases, including vascular endothelial growth factor (VEGFR) and platelet-derived growth factor receptor (PDGFR) . Sorafenib has shown a preclinical antitumor effect against a variety of tumor types and has been approved by the US Food and Drug Administration (FDA) for the treatment of advanced renal carcinoma and unresectable hepatocellular carcinoma [9–11]. Whereas sorafenib treatment of glioma cell lines and tumor xenografts results in cell growth inhibition and tumor growth regression [12, 13], its use in the clinical treatment of patients with malignant gliomas has yielded disappointing results . Despite its relative ineffectiveness in patients with malignant gliomas, the ability of sorafenib to inhibit tumor cell proliferation suggests that it may be useful in combination with other therapeutic agents.
Vitamin Ks are fat-soluble vitamins that are involved in bone metabolism and blood coagulation . There are two natural forms of vitamin K, vitamin K1 (VK1, mostly found in green leafy vegetables) and vitamin K2 (VK2, synthesized by the intestinal flora). It has been shown that vitamin Ks could inhibit the growth of various types of cancer cells in vitro as a single agent or in combination with other chemotherapy [16, 17]. Recent studies have shown that VK1 can enhance the effects of sorafenib-mediated hepatocellular carcinoma cell growth inhibition through inhibiting the density-enhanced phosphatase 1 (DEP-1)-regulated c-Met-Akt pathway . However, the combinational effect of sorafenib and VK1 on glioma cells has not been studied so far. In this work, we employed the human malignant glioma cell lines BT325 and U251 to evaluate the induction apoptosis and inhibition of cell proliferation of sorafenib in combination with VK1 through the Raf/MEK/ERK signaling pathway.
Cells and reagents
The BT325 cell line was obtained from Beijing Neurosurgical Institute Collection and the U251 cell line was bought from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Sorafenib was purchased from Bayer Corporation (West Haven, CT, USA) and dissolved in dimethylsulfoxide (DMSO) with cell medium to the given concentration with a final DMSO concentration of 0.1%. VK1 was purchased from Sigma-Aldrich Chemical, and dissolved in 99.5% ethanol at a stock concentration of 100 mmol/l and then diluted to appropriate concentrations with medium. DMSO or ethanol was added to medium at 0.1% (V/V) as a solvent control.
BT325 and U251 cells were plated at a density of 5 × 104 cells/ml in 96-well plates (Corning, USA) for 24 h. Then the medium was replaced with fresh DMEM containing various concentrations of sorafenib, VK1 or combination of the two agents for 72 h. Cells were washed twice with phosphate-buffered saline (PBS), and 20 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/ml) was added to each well. After 4 h incubation at 37°C, the culture containing MTT was carefully removed, 200 μl of DMSO was added to each well, and absorbance at 570 nm was measured using MRX II absorbance reader (DYNEX Technologies, Chantilly, VA, USA). The cell viability was assessed by the percentage of absorbance in cells at least three independent tests.
Apoptosis analysis by flow cytometer
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences, Sparks, MD, USA) was used to measure the percentage of apoptosis induced by sorafenib and VK1. Cells were cultured in six-well plates at 3 × 105 cells per well and treated with the agents for 10 h. The cells were harvested, washed with cold PBS, and then resuspended in 500 μl of binding buffer. A total of 5 μl of annexinV-FITC solution and 10 μl PI (1 μg/ml) were added to these cells for 30 minutes away from the light. Using flow cytometer (Becton Dickinson, USA) to detect apoptosis through channels two and three. In all, 10,000 cells were collected for each sample.
4′,6-Diamidino-2-phenylindole (DAPI) assay
Cells were cultured on chamber slides and treated with sorafenib, VK1 or their combination. Then, 24 h later, cells were washed with cold PBS and stained with DAPI for 10 minutes away from the light. Nuclear morphological changes were examined using fluorescence microscopy (DFC480; Leica Microsystems, Germany).
Cells were plated in tissue culture dishes overnight and treated with the agents for 24 h. After harvest, the cells were resuspended in lysis buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 2 mM ethylenediaminetetra-acetic acid (EDTA), 1% NP-40) containing protease inhibitor cocktail (Amresco, Solon, OH, USA). Equal amount of total protein extracts were separated by 10% standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (0.45 mm, Millipore, Bedford, MA, USA). Non-specific antibody binding was blocked with 5% fat-free dry milk/Tris-buffered saline (TBS)-Tween 20 (TBST) at room temperature for 1 h. The membrane was then incubated overnight at 4°C with the following specific antibodies to MEK, ERK, phospho-MEK, phospho-ERK, Bcl-2, Mcl-1, Bcl-xl, Bax and β-actin (Cell Signaling Technology). Horseradish peroxidase-linked anti-mouse or anti-rabbit IgG were then used as secondary antibody, and bands were detected with enhanced chemiluminescence reagent (Amersham Bioscience, Piscataway, NJ, USA).
Data were expressed as means ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) via SPSS 13.0 software (SPSS,Chicago, IL, USA). A value of P<0.05 was considered statistically significant.
Inhibition of glioma cell growth by sorafenib plus VK1
Induction of glioma cell apoptosis by sorafenib plus VK1
Inhibition of the Raf/MEK/ERK pathway by sorafenib plus VK1
Involvement of sorafenib plus VK1-mediated apoptosis of glioma cells
The combination of nutritional factors with anticancer agents might be a promising strategy for improving the efficacy of chemotherapy [19, 20]. In the present study, we reported that VK1 had the potential to enhance the antitumor activity of sorafenib in the glioma cell lines BT325 and U251. The effects of the agents on the inhibition of proliferation of glioma cells were measured by MTT assay. The results indicated that VK1 is safe for use in cells up to 100 μM, and sorafenib exerted a dose-dependent effect on cell growth inhibition. In addition, we found that low dose of sorafenib (2.5 μM) plus VK1 (0 to 100 μM) exerted synergistic efficacy in inhibiting cell growth in a time-dependent and dose-dependent manner. To observe the effect of the combined agents on cell apoptosis, we also performed apoptosis analysis using flow cytometry assays and DAPI staining. Our results showed that a combination of sorafenib (2.5 μM) with VK1 (50 μM) induced apoptosis and caused typical apoptotic morphology changes, including condensed chromatin as well as nuclear fragmentation.
The mechanisms by which combination sorafenib and VK1 synergistically induce apoptosis in glioma cells appear to be complex. The upregulation of the Raf/MEK/ERK cascade is one of the principal Ras-regulated pathways, and has been proven to be associated with glioma cell proliferation, survival and migration . It has been suggested that downregulation of the Raf/MEK/ERK pathway may be of great promise as a target for preventing tumor cell growth [8, 9]. In the present study, western blotting analysis showed that low concentrations of sorafenib (2.5 μM) or VK1 (50 μM) when used as single agents did not inhibit phospho-MEK and phospho-ERK levels, whereas the combination of both agents had a strong synergistic action to inhibit phosphorylation of MEK and ERK. These results suggest that the synergistic induction of cell apoptosis by combination of sorafenib and VK1 is, at least partly, attributed to inhibition of the Raf/MEK/ERK signaling pathway.
One important target for chemotherapy is programmed cell death, and this is determined by the balance of proapoptotic and antiapoptotic proteins. We also detected the Bcl-2 protein family, which regulates the mitochondrial pathway of apoptosis [21, 22]. The Bcl-2 family is composed of antiapoptotic (for example, Bcl-2, Mcl-1 and Bcl-xl) and proapoptotic (for example, Bax) proteins. The antiapoptotic Bcl-2 protein targets intracellular organelles such as the endoplasmic reticulum, outer mitochondrial and nuclear membranes, and then modulates tumor cells responses to apoptosis . Mcl-1 is another highly expressed antiapoptotic protein in malignant tumors and has been implicated in resistance to chemotherapy through a number of signaling pathways . It has also been reported that ERK-mediated phosphorylation is an important regulator of Mcl-1 stability, and downregulation of Mcl-1 is one of the main antitumor effect of sorafenib, either alone or in combination with other agents, on several kinds of tumor cells [25, 26]. In our study, the combination of low concentration sorafenib (2.5 μM) and VK1 (50 μM) exhibited strong synergistic action by inhibiting protein expression of Bcl-2 and Mcl-1, leading to induction of cell apoptosis. However, there were no obvious changes in the expression of Bcl-xl and Bax proteins. These results suggested that the combination of sorafenib with VK1 induced apoptosis in BT325 and U251 cells through downregulating proapoptotic proteins Bcl-2 and Mcl-1.
The present study indicated that VK1 could enhance sorafenib-induced growth inhibition and apoptosis of glioma cells, and this synergistic effectiveness was associated with the downregulation of the Raf/MEK/ERK signaling pathway. These results make the combination of sorafenib and VK1 a promising therapeutic strategy against human malignant gliomas.
This work was supported by the National Natural Science Foundation of China (No. 81001009).
- Sathornsumetee S, Reardon DA, Desjardins A, Quinn JA, Vredenburgh JJ, Rich JN: Molecularly targeted therapy for malignant glioma. Cancer. 2007, 110: 13-24. 10.1002/cncr.22741.View ArticlePubMedGoogle Scholar
- Hadziahmetovic M, Shirai K, Chakravarti A: Recent advancements in multimodality treatment of gliomas. Future Oncol. 2011, 7: 1169-1183. 10.2217/fon.11.102.PubMed CentralView ArticlePubMedGoogle Scholar
- Das G, Shiras A, Shanmuganandam K, Shastry P: Rictor regulates MMP-9 activity and invasion through Raf-1-MEK-ERK signalingpathway in glioma cells. Mol Carcinog. 2011, 50: 412-423. 10.1002/mc.20723.View ArticlePubMedGoogle Scholar
- Goillot E, Raingeaud J, Ranger A, Tepper RI, Davis RJ, Harlow E, Sanchez I: Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway. Proc Natl Acad Sci USA. 1997, 94: 3302-3307. 10.1073/pnas.94.7.3302.PubMed CentralView ArticlePubMedGoogle Scholar
- Du K, Zheng Q, Zhou M, Zhu L, Ai B, Zhou L: Chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway. Curr Microbiol. 2011, 63: 341-346. 10.1007/s00284-011-9985-2.View ArticlePubMedGoogle Scholar
- Lo HW: Targeting Ras-RAF-ERK and its interactive pathways as a novel therapy for malignant gliomas. Curr Cancer Drug Targets. 2010, 10: 840-848. 10.2174/156800910793357970.PubMed CentralView ArticlePubMedGoogle Scholar
- Hahn O, Stadler W: Sorafenib. Curr Opin Oncol. 2006, 18: 615-621. 10.1097/01.cco.0000245316.82391.52.View ArticlePubMedGoogle Scholar
- Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, Cao Y, Shujath J, Gawlak S, Eveleigh D, Rowley B, Liu L, Adnane L, Lynch M, Auclair D, Taylor I, Gedrich R, Voznesensky A, Riedl B, Post LE, Bollag G, Trail PA: BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004, 64: 7099-7109. 10.1158/0008-5472.CAN-04-1443.View ArticlePubMedGoogle Scholar
- Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A, Evers BM: PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res. 2010, 30: 4951-4958.PubMed CentralPubMedGoogle Scholar
- Lu X, Tang X, Guo W, Ren T, Zhao H: Sorafenib induces growth inhibition and apoptosis of human chondrosarcoma cells by blocking the RAF/ERK/MEK pathway. J Surg Oncol. 2010, 102: 821-826. 10.1002/jso.21661.View ArticlePubMedGoogle Scholar
- Hasskarl J: Sorafenib. Recent Results Cancer Res. 2010, 184: 61-70. 10.1007/978-3-642-01222-8_5.View ArticlePubMedGoogle Scholar
- Siegelin MD, Raskett CM, Gilbert CA, Ross AH, Altieri DC: Sorafenib exerts anti-glioma activityin vitroandin vivo. Neurosci Lett. 2010, 478: 165-170. 10.1016/j.neulet.2010.05.009.PubMed CentralView ArticlePubMedGoogle Scholar
- Jane EP, Premkumar DR, Pollack IF: Coadministration of sorafenib with rottlerin potently inhibits cell proliferation and migration in human malignant glioma cells. J Pharmacol Exp Ther. 2006, 319: 1070-1080. 10.1124/jpet.106.108621.View ArticlePubMedGoogle Scholar
- Nabors LB, Supko JG, Rosenfeld M, Chamberlain M, Phuphanich S, Batchelor T, Desideri S, Ye X, Wright J, Gujar S, Grossman SA, New Approaches to Brain Tumor Therapy (NABTT) CNS Consortium: Phase I trial of sorafenib in patients with recurrent or progressive malignant glioma. Neuro Oncol. 2011, 13: 1324-1330. 10.1093/neuonc/nor145.PubMed CentralView ArticlePubMedGoogle Scholar
- Krueger T, Westenfeld R, Schurgers L, Brandenburg V: Coagulation meets calcification: the vitamin K system. Int J Artif Organs. 2009, 32: 67-74.PubMedGoogle Scholar
- Okayasu H, Ishihara M, Satoh K, Sakagami H: Cytotoxic activity of vitamins K1, K2 and K3 against human oral tumor cell lines. Anticancer Res. 2001, 21: 2387-2392.PubMedGoogle Scholar
- Showalter SL, Wang Z, Costantino CL, Witkiewicz AK, Yeo CJ, Brody JR, Carr BI: Naturally occurring K vitamins inhibit pancreatic cancer cell survival through a caspase-dependent pathway. J Gastroenterol Hepatol. 2010, 25: 738-744. 10.1111/j.1440-1746.2009.06085.x.View ArticlePubMedGoogle Scholar
- Carr BI, Wang Z, Wang M, Cavallini A, D’Alessandro R, Refolo MG: c-Met-Akt pathway-mediated enhancement of inhibitory c-Raf phosphorylation is involved in vitamin K1 and sorafenib synergy on HCC growth inhibition. Cancer Biol Ther. 2011, 12: 531-538. 10.4161/cbt.12.6.16053.View ArticlePubMedGoogle Scholar
- Huang CS, Lyu SC, Hu ML: Synergistic effects of the combination of beta-ionone and sorafenib on metastasisof human hepatoma SK-Hep-1 cells. Invest New Drugs. in pressGoogle Scholar
- Roy CS, Karmakar S, Banik NL, Ray SK: Synergistic efficacy of sorafenib and genistein in growth inhibition by downregulating angiogenic and survival factors and increasing apoptosis throughupregulation of p53 and p21 in malignant neuroblastoma cells having N-Mycamplification or non-amplification. Invest New Drugs. 2010, 28: 812-824. 10.1007/s10637-009-9324-7.View ArticleGoogle Scholar
- Yip KW, Reed JC: Bcl-2 family proteins and cancer. Oncogene. 2008, 27: 6398-6406. 10.1038/onc.2008.307.View ArticlePubMedGoogle Scholar
- Fulda S, Galluzzi L, Kroemer G: Targeting mitochondria for cancer therapy. Nat Rev Drug Discov. 2010, 9: 447-464. 10.1038/nrd3137.View ArticlePubMedGoogle Scholar
- Heath-Engel HM, Chang NC, Shore GC: The endoplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family. Oncogene. 2008, 27: 6419-6433. 10.1038/onc.2008.309.View ArticlePubMedGoogle Scholar
- Akgul C: Mcl-1 is a potential therapeutic target in multiple types of cancer. Cell Mol Life Sci. 2009, 66: 1326-1336. 10.1007/s00018-008-8637-6.View ArticlePubMedGoogle Scholar
- Yu C, Bruzek LM, Meng XW, Gores GJ, Carter CA, Kaufmann SH, Adjei AA: The role of Mcl-1 downregulation in the proapoptotic activity of the multikinase inhibitor BAY 43–9006. Oncogene. 2005, 24: 6861-6869. 10.1038/sj.onc.1208841.View ArticlePubMedGoogle Scholar
- Domina AM, Vrana JA, Gregory MA, Hann SR, Craig RW: MCL1 is phosphorylated in the PEST region and stabilized upon ERK activation in viable cells, and at additional sites with cytotoxic okadaic acid or taxol. Oncogene. 2004, 23: 5301-5315. 10.1038/sj.onc.1207692.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.