Cell and Tissue Journal

Abstract Aim: Cancer remains a global health problem, with ovarian cancer ranking fifth among cancers affecting women and the leading cause of cancer-related death in women. There are different types pf ovarian cancer, including Epithelial ovarian cancer, Stromal tumors and Germ cell tumors.  Epithelial ovarian cancer is the most common type that includes several subtypes, including serous carcinoma and mucinous carcinoma. To this end, several factors have been identified to increase your risk of ovarian cancer, including older age, inherited gene changes, family history of ovarian cancer, being overweight or obese, postmenopausal hormone replacement therapy, endometriosis, and never having been pregnant. The current strategies to treat ovarian cancer include surgery, chemotherapy, radiotherapy and targeted therapies, and hormone therapy. Scientists continue to investigate the foundational mechanisms involving cancer development, as well as to find new drugs and metabolites having the capacity to control cancer. Alpha-ketoglutarate (AKG), a critical metabolite in the Krebs cycle involved in cellular energy production and the regulation of gene expression. Recent studies have shown that AKG may have the potential to enhance the efficacy of cancer treatments, by modulating the tumor microenvironment and improving the immune response against cancer cells. This study investigates the effect of AKG on ovarian cancer cells.
Material and methods: SKOV3 cells were obtained from Tehran University and cultured in complete culture medium (RPMI, 10% Fetal bovine serum (FBS), and 1% Penicillin-Streptomycin (Pen/Strep)). To find the proper concentration of AKG, SKOV3 cells were cultures in 96-well plate, and treated with different concentration of AKG (range 20 to 220 µM). After 24 and 48 h, the viability of the cells was determined using MTT assay. Based on the results obtained from viability assay, 200 μM of AKG was selected for the next assessments. To evaluate the effect of AKG on SKOV3 cell proliferation using plotting a growth curve, cells were cultured in the presence (200 μM AKG) and absence of AKG, and counted the number of cells every 24h for one week. To determine the population doubling time (PDT), the cells were cultured in the presence (200 μM AKG) and absence of AKG for 72h, then the cell were collected and the number of living cells was counted using Neubauer Chamber. The PDT was calculated using a related standard method. To assess colony formation potential, the SKOV3 cell were cultured in the presence (200 μM AKG) and absence of AKG. After 7 days, the cells were fixed using 10% formalin solution, then the colonies were stained using crystal violet dye, and the number of colonies were counted using inverted microscope. To investigate the effect of AKG on the migration rate of SKOV3 cells, the cells were cultured in complete medium to reach 85% confluence, then treated with mitomycin (10 μM) for 3h, then Created a scratch in the cell monolayer using a sterile pipette tip. the cells were cultured in the presence (200 μM AKG) and absence of AKG for 3 days. The images of the scratch were taken at regular intervals using a microscope, and the closure of the scratch over time was analyzed. To analyze the cell cycle profile, the SKOV3 cell were cultured in the presence (200 μM AKG) and absence of AKG for 48h. Then, the cells were collected and analyzed using a flow cytometry.
Results: Based on the MTT assay, 200 μM AKG was determined as the proper concentration to investigate other biological behaviors of SKOV3 cells. Colony forming assay showed a decrease in the number and size of colonies in the AKG-treated group (P<0.05). In addition, the cell doubling time increased in the treatment group, indicating slower growth rate (P<0.05). Growth curve analysis confirmed reduced cell growth in treated group. Cell cycle analysis showed a higher percentage of treated cells arrested in S and G1 phases. The scratch assay showed slow cell migration and metastasis in the cells treated with 200 µM AKG.
Conclusion: In conclusion, AKG has an inhibitory effect on the proliferation, viability, migration in SKOV3 ovarian cancer cells, highlighting its potential as an adjuvant treatment with existing therapies. More research is necessary to fully investigate the therapeutic effect of AKG in ovarian cancer.