Synergistic Cytotoxicity of Vitamins C and K3 Towards Rat Glioma C6 Cells in Vitro

Abstract

Introduction Malignant glioma is the most common primary CNS tumour [1]. Glioblastoma is an incurable brain tumor that usually causes death within 2 years after conventional therapies consisting of surgery, radiation, and chemotherapy [2]. The development of more potent and less toxic compounds represents one of the major goals to overwhelm the poor outcome of patients with glioblastoma [3]. It has been shown that the combination of vitamins C and K3 at the ratio 100:1 is synergistic towards some tumour cell lines in vitro [4]. The aim of this study was to study the cytotoxic action of vitamins C and K3 in vitro and find out whether the action of these vitamins is synergistic for rat glioma C6 cells. Materials and Methods Experiments were performed in vitro with rat glioma C6 cells. The cells were grown in monolayer culture in 60-ml flasks at 37 oC and 5% CO2 in a water-jacketed incubator IR AutoFlow NU-2500E (NuAire, Plymouth, MN, USA). The culture medium consisted of Dulbecco’s modified Eagle’s medium (Product No. D5546, Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) supplemented with 10% fetal bovine serum (F7524, Sigma-Aldrich, Chemie GmbH, Steinheim, Germany), 1% Lglutamine (G7513, Sigma-Aldrich, Chemie GmbH, Steinheim, Germany). All manipulations that required sterile conditions were performed in vertical laminar flow cabinet Aura Vertical SD4 (BIOAIR Instruments, Siziano, Italy) [5]. The cytotoxicity of vitamins C and K3 alone as well as cell sensitivity to combined treatment with vitamin C and vitamin K3 were estimated from the reduction of the cell viability. Cell viability was determined by means of a colony-forming assay [5]. After six days, the colonies were fixed with 96% ethanol (AB Stumbras, Kaunas, Lithuania), stained with a Gram’s crystal violet solution (Fluka Chemie GmbH, Buchs, Germany) and counted under a binocular light microscope (MBS-9, LOMO PLC, St. Petersburg, Russia). The survival of the cells treated with vitamins was calculated as the percentage of the colonies obtained from the untreated control cells [5]. Results At first, the cytotoxic concentrations of vitamin C (VC) and vitamin K3 (VK3) were determined. The cells were treated with vitamin C or vitamin K3 by growing them in the media containing various concentrations of vitamins for 6 days. Then, cell viabilities were determined by means of a colony-forming assay [5]. The obtained dependences of the cell viability on the concentrations of vitamin C and vitamin K3 are shown in Fig. 1. The concentration of vitamin C required to reduce cell survival by 50 % was 0.3 mM for rat glioma C6 cells. Vitamin K3 killed 50 % of rat glioma C6 at a concentration of 6.8 μM. Treatment of cells by both vitamins at the ratio of 100:1 (VC:VK3) greatly enhanced their cytotoxicity towards rat glioma C6 cell line. The vitamin K3 concentration required to kill 50 % of rat glioma C6 cells was reduced from 6.8 to 1.4 mM (Fig. 1). Conclusion It has been found that the cytotoxic action of the mixture of vitamins C and K3 at the ratio of 100:1 is synergistic towards rat glioma C6 cells. Acknowledgement This work was in part supported by grant T-57/09 from the Lithuanian State Science and Studies Foundation.