الفهرس | Only 14 pages are availabe for public view |
Abstract n cancer cell, aerobic glycolysis is high and uncoupled with GO. This leads to rise in each of glucose uptake and lactate production and reduction in OXPHOS known as metabolic respiration. This mechanism is called the Warburg’s effect and it is considered as an important cell metabolic reprogramming in cancer. This reprogramming increases cancer cell survival with low ATP yield. There is a great association between glycolytic pathway and cancer cell survival. Cancer cell needs high glucose uptake and this is achieved by GLUTs. GLUT1 is considered as the superior glucose transporter in several types of cancer. GO in cancer cell leads to cancer cell death. Hence, this process is reduced by high PDK expression. Also, LDHA produces lactate from pyruvate resulting in enhancing continuous glycolysis. P53 is a tumor suppressor gene and its expression in cancer cell is inactivated and converted to mutated one to avoid P53 inhibitory effect on many glycolytic enzymes especially GLUT-1. Mutation of P53 directly enhances the expression of PDK in cancer cell. C-Myc oncogene has an important role in disturbing the mechanism of normal oxygen sensing and in regulation of glycolytic enzymes such as LDHA and GLUT1. It also enhances the Warburg effect. PI3K/AKT signaling enhances GLUT1 mRNA expression increasing the rate of aerobic glycolysis. Therefore, the current study was established to investigate the anticancer efficacy of TMZ to inhibit aerobic glycolysis in Ehlrich mouse model and the underlying effects of TMZ on GLUT- 1, PDK, LDHA, p53 and PI3K/AKT signaling were assessed as new mechanisms for treatment of breast malignancy. Methods and results: 1. MTT-Cell Proliferation Assay was accomplished for cytotoxicity assessment of TMZ on MCF-7/breast adenocarcinoma 2. Forty female Swiss mice weighing: 20 g to 25 g were investigated. Intradermal injection of 100 μl EAC suspension was given for each mouse at the lower ventral side (shaving this area previously). Day zero is the start day of EAC suspension injection. After tumor inoculation, four groups of mice were formed randomly, ten mice each. The first group 55 acted as the Ehrlish control. The second group was given Trimetazidine (10 mg/kg/day P.O). The third group was given Trimetazidine (20 mg/kg/day P.O). The forth group was given (30 mg/kg/day P.O).Treatment started at day 7 and continued daily for 14 days 3. The expression of glycolytic parameters in the tumor tissue was determined by quantitative real time PCR. The expression of GLUT1, LDHA, PDK1 and PDK4 genes in the tumor tissue was assessed. 4. Expression of C-myc and p-AKT (ser473) in tumor tissues for four groups was assessed by western blot analysis. 5. Evaluation of P53 in tumor tissues was determined by Immunohistochemical examination. 6. TMZ showed a cytotoxic action against MCF-7 cells, having IC50 value of 2.95 μM. 7. The results of the antitumor activity of TMZ doses (10, 20, 30 mg) in vivo in SEC female mice model showed a significant gradual reduction in tumor weight with increasing doses of TMZ compared to the untreated SEC control group. Moreover, there was a significant decrease in the tumor volume in a dose dependent way upon using TMZ doses. 8. Inhibition of the glycolytic pathway was detected in the tumor tissue of SEC mice injected with graded doses of TMZ. A significant downregulation of the gene expression of GLUT- 1 and the glycolytic enzymes PDK-1, PDK-4 and LDH-A was observed in the three TMZtreated groups in a dose-dependent manner compared to the SEC control group. 9. In western blot, the expression of c-Myc was significantly downregulated in mice treated with 20 mg/kg and 30 mg/kg of TMZ compared with the SEC control group. Similarly, a gradient decrease of p-AKT expression was observed in the three TMZ-treated groups in a dose-dependent manner. 10. Immunohistochemical evaluation results of p53 expression in the tumor tissue revealed a significant rise upon treatment with TMZ in a dose dependent fashion. 11. Molecular docking and in silico studies results showed that TMZ is an AKT and c-Myc selective inhibitor. 56 Conclusion: In conclusion, TMZ is a β-oxidation inhibitor that suppressed tumor development in a SEC model of breast tumor and MCF-7 cells. The results of the current work revealed a novel mechanism of the proposed cytotoxic effect of TMZ on malignant cells, that is inhibition of glycolytic pathway and AKT signaling leading to enhancement of glucose oxidation. Treatment by TMZ inhibited c-myc and AKT signaling in dual in vivo and in silico experiments. It also stimulated apoptosis of cancer cells through stimulating p53. TMZ may be a promising anticancer therapy to combat breast cancer |