The exogenous factors and the endogenous metabolic products can generate reactive species for DNA alkylation [1], O6-guanine in particular. The MGMT enzyme protects cells from such disorders. It functions in cells to restore the native DNA structure in a one-step irreversible and suicidal reaction by transfering the alkyl group from the Oxygen in the DNA to a cysteine residue in the catalytic pocket of MGMT. This protects cells from cytotoxic, carcinogenic and oncogenic DNA alkylation lesions, allowing preservation and transmission of correct and unaltered genetic information during cell division. An unrepaired O6-methylguanine causes point mutations due to mis-pair with thymine during replication, leading to G: C → A: T transition [2]. Significant number of such errors could affect the cell viability and lead to cell death [3]. Therefore, the expression of this protein in cells is crucial.
On the other hand, the MGMT expressed in tumor cells limits the effectiveness of alkylating chemotherapy. Thus there is a demand for MGMT expression regulators or for the modulators of its protein product activity, and many articles are devoted to this issue every year. It is known that the MGMT expression varies considerably, but the reasons for this variation are not well established. It has been shown that the synthetic glucocorticoid dexamethasone, which is used to relieve inflammation and edema in the postoperative period, activates MGMT expression [4]. Thus the effectiveness of the concomitant or subsequent alkylating chemotherapy is significantly reduced. It is unknown how the other drugs used in the cancer treatment influence on the MGMT expression.
Combination of the chemotherapy and the hormone- and immunotherapy is widely-used for the treatment of many types of cancer. However, a little is known about effects of these drugs on the MGMT expression. Therefore, we analysed the MGMT promoter region for the cis-regulatory elements. We predicted novel potential hormone response elements, including such for steroid hormones and thyroid hormone receptor-like factors. The first to be tested are estrogen response elements and progesterone response elements, as these hormones are important in the treatment of hormone-sensitive tumors. Therefore, the knowledge about the effect of these hormones on the MGMT as one of the alkylating chemotherapy limiting factor could help to plan the correct and effective treatment for the patient.
We found that both β-estradiol and progesterone positively regulate MGMT expression in vitro on mRNA and protein level. However, we cannot provide a clear answer as to whether this regulation occurs through the cis-regulatory element in the promoter or through the membrane receptor. This question needs further study.
We investigated the influence of the recombinant interferon α2β synthesized in transgenic plants N. benthamiana on MGMT quantity in both cancer (HЕp-2) and non-cancer (E8) originated human cells. We found that the interferon α2β tended to reduce the amount of MGMT protein in all the cells studied. However, compared to tumor HЕp-2 cells, this effect was weaker in E8 cells and was observed only at the two highest interferon concentrations. Thus, we first identified inhibitory effect of the recombinant interferon α2β (synthesized in plants N. benthamiana) on the expression of the DNA repair enzyme MGMT in human cells. We revealed that the effect of inhibition by interferon α2β was stronger in cancer cells than in non-cancer cells.
We proposed a mechanism of this effect. In particular, interferon α2β inhibits MGMT through NF-κB transcription factor, which positively regulates the MGMT expression through the response element in the promoter [5].
To limit the tumor cell resistance to chemotherapy it is possible not only affect the MGMT protein or mRNA amount, but also the activity of the enzyme itself. So, by using bioinformatics we investigated the possibility to alter the MGMT protein activity by certain post-translational modifications. We found numerous potential sites of acetylation, ubiquitination, SUMOylation, and phosphorylation within the protein molecule.
Also another promising method for reducing the MGMT amount in tumor cells during chemotherapy is the use of enzyme inhibitors. O6-benzylguanine and O6 (4-bromothienyl) guanine (Lomeguatrib) in combination with various alkylating agents are undergoing stage II and III clinical trials to treat temozolomide- or carmustine-resistant tumors [6,7,8]. However, these inhibitors have toxic side effects, so the development and testing of new less toxic inhibitors are relevant. Therefore, we investigated a number of low molecular weight non-nucleoside organic compaunds (designed and synthesized in the Department of Biomedicinal Chemistry of the Institute of Molecular Biology and Genetics of NASU) for their ability to reduce the amount of MGMT enzyme in cells in vitro and identified some of the most promising potential MGMT inhibitors
