Evaluation of Pyrimethamine as Apoptosis Inducing Agent on Chemically Induced Gastric Cancer

Abstract

Background: Gastric cancer is one of the leading death modalities amongst all type of cancers. The morbidity and mortality associated with gastric carcinogenesis is ranked second highest amongst all malignant neoplasms. Anti-malarial agents are being evaluated for their potential role in various cancers which are proved to have promising therapeutic effects. Pyrimethamine, belonging to the anti-folate and anti-protozoal class of agent has been suggested to exert anti-tumour effects by inducing apoptotic cancer cell death in gastric cancer via acting on several pathway and mechanisms.Materials and Methods: In-silico docking studies: The molecular docking was performed using GOLD suite software to check the activity of various class of anti-malarial agents and the standard chemotherapeutic agent on target protein caspase-3, which is an effector caspase to induce apoptosis. In-vitro study: The in-vitro study was carried out on AGS cell lines. The cell viability assay (MTT assay) was performed to evaluate the IC50 of pyrimethamine. In-vivo: Gastric cancer was induced chemically by 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) in Wistar rats. MNNG was given in a dose of 100mg/kg for 21 days (3 weeks) daily by oral route. The treatment with pyrimethamine was initiated from 4th week by administering different doses of pyrimethamine alone and in combination with doxorubicin (standard drug). Pyrimethamine was given orally for 30 days (4 weeks) daily orally and doxorubicin (1mg/kg) twice a week for 2 weeks via i.p route. At the end of 8 weeks, the animals were sacrificed and the stomach tissues were isolated from different groups for the evaluation of various parameters. Physical parameters (body weight, food and water intake), tumour parameters (tumour volume, tumour burden and % tumour incidence), oxidative stress parameters (MDA, GSH and SOD), oncogenic marker (CEA), inflammatory markers (IL-6, TNF- α, IFN- γ, IL-1 β, NF- ϰB), histopathological studies, immunohistochemistry (BCL-2) and mRNA expression of caspase-3 was carried out via RT-PCR. Results: In-silico studies The docking results depicted the binding of pyrimethamine on caspase-3 with gold score of 42.43 which was found to be lower than chloroquine, sulfadoxine and quinine but the important and reported amino-acids which are necessary to be present to inhibit the caspase-3 were present in pyrimethamine as well as in doxorubicin. In-vitro studies The MTT assay results depicted that the IC50 of pyrimethamine and doxorubicin was found to be 91.1μM and 4.5 μM respectively after 24 hours.