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.
 

Abstract Aim: As the second leading cause of death worldwide, cancer has been a long-standing and rapidly evolving focus of biomedical research and practice. Ovarian cancer is one of the deadly malignancies of women, which is known as the "silent killer". Because its symptoms usually appear when the disease has reached advanced stages and is mostly incurable. On the other hand, currently common treatment methods are associated with various limitations, failures and side effects, which have made researchers pay more attention to the compounds extracted from plants as anti-tumor and anti-cancer agents in the last two decades. The studies conducted on the various properties of Ashwagandha (Withanaia somnifera) show that this plant has therapeutic effects and anti-cancer properties that can even be effective on the process of apoptosis mediated by different genes, including P53. Today, P53 is known as a gene It has a key role in all types of cancers and mutations in this gene have been observed in 60% of ovarian cancers. Therefore, the aim of this study is to investigate the cytotoxic effects of the hydroalcoholic extract of Ashwagandha (Withanaia somnifera) and the changes in P53 gene expression in response to this substance in ovarian cancer cells (cell line A2780).
Material and method: For this purpose, extraction of Ashwagandha plant was done using soaking method. Then, the compounds present in the extract were determined using standard phytochemical tests. Then A2780 cells were treated with different concentrations of Ashwagandha extract for 24, 48 and 72 hours. The effects of this extract on cell survival were evaluated using the MTT test, and IC50 was calculated. Then, A2780 cells were exposed to concentrations of 250 and 500 µg/ml for 24 and 48 hours, and the level of P53 gene expression was investigated by Real-Time PCR. Finally, statistical analysis and data interpretation was done using GraphPad Prism software.
Results: MTT results showed that Ashwagandha extract at concentrations of 62.5, 125, 250, 500, and 750 µg/ml significantly decreased cell viability in a time- and concentration-dependent manner. The lowest percentage of survival rate is related to the concentration of 750 μg/ml and the time of 72 hours, while the highest percentage is related to the concentration of 62.5 μg/ml and the time of 24 hours. After 24, 48, and 72 hours, respectively, IC₅₀ was obtained at concentrations of 512.6, 339, and 226.6 μg/ml, and Real-Time PCR results showed that the expression of the P53 gene during 24 hours of treatment with concentrations of 250 and 500 μg/ml of extract had increased significantly. Also, increasing the concentration of the extract had a significant effect on the expression of this gene.
Conclusion: The results of this study indicate the cytotoxic effect of Ashwagandha extract on ovarian cancer cells, and by increasing the expression of the P53 gene, it can induce anti-cancer effects. It is hoped that by knowing the mechanisms of action of this medicinal plant and designing new drugs, it will be possible to stop the progression of ovarian cancer and thus help to increase the life span of these patients against this silent killer.

Abstract Aim: The purpose of this study is to investigate the pro-apoptotic and anti-metastatic potentials of Lippia citriodora alcoholic leaf extract on A2780 ovarian cancer cells and to evaluate the expression of E-cadherin as one of the most important marker in metastasis.
Material and Methods: A2780 ovarian cancer cell line was prepared from Pasteur cell bank and cultured in RPMI1640 medium containing 10% FBS and 1% antibiotic. After cell seeding and treatment with different concentrations of extract (10-400 µg/ml), the cells viability and pro-apoptotic potential were examined by MTT assay and acridine orange/ propodium iodide, respectively. Caspase-3 assay and anti-invasive effect were analyzed by migration assay and assessment of E-cadherin expression, respectively. Then, one way ANOVA test was employed for analysis of quantitative data.
Results: Data indicated that L. citriodora alcoholic extract attenuated ovarian cancer cell viability. Acridine orange/ propodium iodide and caspase-3 assays showed that this extract in IC50 concentration (100 µg/ml) induced apoptosis mainly through caspase dependent pathway in the ovarian cancer cells. Migration assay and RT-PCR exhibited that this extract has anti-invasive capability and by up- regulation of E-cadherin prevents the loss of attachment between cancer cells.
Conclusion: The results revealed that L. citriodora alcoholic extract has apoptosis inducing capacity and ability of restoration E-cadherin expression in A2780 cancer cells, which it able to suppress invasive potential of ovarian cancer cells.