In-Vitro And In-Vivo Evaluation of Chemopreventive Potential of Methlyglyoxal on Hep 3b Cell Line and Nitrosodiethylamine (Ndea) Induced Hepatocellular Carcinoma In Balb/C Mice

Abstract

Aim and objective: Primary Hepatocellular Carcinoma (HCC) is the fifth most common cancer in the world and third frequent cancer-related cause of death with increasing incidence worldwide. Increasing progression of cancer and multiple changes occurring in the tumor cells has challenged science to develop tumor specific drugs. Several drugs targeting on tumor cell mitochondria (mitocans) are under investigation. Methylglyoxal (MG) is an aldehyde, synthesized by several enzymatic and non-enzymatic pathways in the body with glucose metabolism as a major source. In our study, we have evaluated in-vitro activity of MG using Hep 3B cell line and in-vivo activity using NDEA induced HCC model in Balb/C mice. An attempt has also been made to evaluate the specificity of MG towards tumor cell mitochondria in comparison with normal cell mitochondria. Materials and methods: In-vitro cytotoxicity assay of MG was performed on Hep 3B cell lines and Vero (normal) cell line. MTT assay and DNA fragmentation assay were performed to get the IC50 value and the fragmentation pattern of MG respectively, in comparison with 5-FU. In-vivo anti-cancer activity was evaluated using Balb/c mice. Animals were divided into four groups: Normal control (NC), NDEA control, MG treated and NDEA + MG treated. For the induction of carcinogenesis (NDEA 100mg/kg, i.p. once a week for three weeks) was administered to animals. After 15 weeks of induction period, MG was administered to the animals at the dose of 36mg/kg (i.p.) for 10 days followed by 50mg/kg (i.p.) for next 10 days. Evaluation of its chemopreventive potential was done by estimating serum biochemical parameters like aminotransferases (ALT and AST) levels, lactate dehydrogenase (LDH) level, glucose level, MG level; oxidative stress parameters in liver homogenate and mitochondria like malondialdehye (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH); SDH assay in liver mitochondria and histopathological evaluation of liver, for signs of tumor development. Results: In-vitro cytotoxicity studies of MG had shown that with less concentration (26.60μM), 50% inhibition of cell growth was observed in Hep 3B cell line. There was minimal cytotoxic effect of MG on normal cell lines when compared to 5-FU. Combination of MG with 5-FU in equal ration had shown improved cytotoxic activity.Administration of NDEA in Balb/c caused significant increase in serum levels of ALT, AST and LDH enzymes, while treatment with MG has shown significant decrease in these levels. There was significant increase in glucose level in MG treated group as compared to normal control group. The treatment of MG in diseased animals had showed significant increase in serum MG level when compared to NDEA control group. Administration of NDEA caused significant increase in MDA level while; treatment with MG caused significant decrease MDA level. On contrary, this was not observed in case of mitochondria. However, SOD and GSH levels significantly decreased in liver mitochondria of NDEA control group when compared to normal control group. Treatment of MG in diseased group had shown increase in SOD and GSH levels when compared to NDEA control group. Significant decrease in SDH level was observed in MG treated group when compared to normal control group. Histopathological evaluation of liver tissue of NDEA control animals showed presence of nuclear pleomorphism, binucleation, mitosis, eosinophilic hepatocytes and central vein distortion. Treatment with MG showed lesser signs of damage however signs of central vein distortion and binucleation were observed. Conclusion: These findings indicate the cytotoxic activity of MG in hepatocellular carcinoma through in-vitro and in-vivo studies. The in-vitro studies indicate the potential of MG to cause cytotoxicity on Hep3B cell line. It was found that MG had shown chemopreventive potential with more specificity towards Succinate dehydrogease (SDH) enzyme on the mitochondria. Inhibition of SDH can cause accumulation of substrates in mitochondria and subsequent activation of HIF-1 and ROS production. Chronic administration of MG in HCC could reveal its better specificity towards mitochondrial ROS production. On contrary, increase in serum glucose levels and MG levels after treatment with MG reveals its ability to cause diabetic complication and formation of advanced glycated end products (AGE’s), respectively. Histopathological evaluation also revealed damage to the liver caused by MG. Owing to the reported toxicity of MG; further studies to establish risk-benefit ratio need to be carried out.