Zhuvaka K.S. «The Impact of Novel Non-Nucleoside MGMT Inhibitors on Alkylating Therapy of Cancer Cells in Model Systems» – A qualification scientific work as a manuscript.
The thesіs fоr а scіentіfіc degree оf Dоctоr оf Рhіlоsорhy by sрecіаlіty 091 – Bіоlоgy (09 – Bіоlоgy) – Іnstіtute оf Mоleculаr Bіоlоgy аnd Genetіcs NАS оf Ukrаіne, Kyіv, 2024.
One of the approaches to cancer treatment is alkylating chemotherapy, which damages cellular DNA and may lead to tumor cell death. However, its effectiveness is limited by the activation of the MGMT enzyme, which repairs alkylation damage, reducing the cytotoxic effect of drugs and promoting chemoresistance. MGMT inhibitors, the most well-known of which are O6-benzylguanine (O6-BG) and its derivatives, are used to overcome this phenomenon. However, their cytotoxicity and side effects limit their clinical application, necessitating the search for new compounds.
This study tested a series of non-nucleoside MGMT inhibitors developed and synthesized at the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine. Five compounds were selected that showed promising results in preliminary tests. Cytotoxicity was assessed in laryngeal carcinoma cells (HEp-2), where the inhibitors demonstrated low toxicity at a concentration of 10 μM, except for inhibitor 46, which was excluded from further research.
The efficacy of inhibitors 41, 41B, and 89 was evaluated using the clonogenic assay in combination with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). It was found that these inhibitors surpassed the effectiveness of O6-BG, acted over a broader concentration range, and enhanced the cytotoxic effect of the alkylating agent at low doses. Western blot analysis demonstrated that the new inhibitors reduced MGMT levels in HEp-2 and T98G glioma cells, increasing their sensitivity to MNNG. The inhibitors showed a mild effect lasting up to 24 hours.
To investigate the impact of the new inhibitors on key cellular processes, their effects on autophagy levels and the lethality of human tumor cells in culture were analyzed. The results allowed for an evaluation of the cytotoxicity and efficacy of the tested compounds in tumor cells with varying MGMT gene expression levels. Glioma cell lines were used: T98G, characterized by high MGMT expression, and U251MG, which has a methylated MGMT promoter, implying an absence of MGMT protein. It was established that the new inhibitors (41 and 41B) alone did not affect the viability of glioma cells or autophagy levels in either cell line. However, treatment of T98G cells with these inhibitors increased their sensitivity to alkylating agents, whereas such effects were absent in U251MG cells. These findings suggest that the investigated compounds may have therapeutic potential in combination alkylating chemotherapy for gliomas with high MGMT levels in cancer cells.
In in vivo experiments on ICR mice, combination therapy with the inhibitors slowed tumor growth and, in some cases, caused tumor regression. Inhibitor 89 induced regression in 5 out of 6 tumors, whereas 41 and 41B demonstrated less pronounced effects. Western blot confirmed a decrease in MGMT levels in tumors, correlating with tumor regression. This observation highlights the potential clinical relevance of the new inhibitors for treating tumors with high MGMT levels.
Side effects, such as spleen morphological changes and tissue necrosis, were minimal for inhibitor 89, making it the most promising candidate. Analysis of the mechanisms underlying tumor regression revealed caspase-3 activation, indicating a possible apoptotic pathway for cell death.
In conclusion, the novel non-nucleoside MGMT inhibitors (41, 41B, and 89) demonstrated encouraging results by enhancing the efficacy of alkylating therapy at low agent doses. This opens new prospects for developing personalized therapeutic strategies for cancer treatment.
