| Glioblastomas are the most common primary brain tumors and carry a dismal prognosis, characterized by highly proliferative and invasive nature and recurrent tendency. The current standard therapy for glioblastoma includes maximal safe surgical resection followed by a combination of a concomitant fractioned radiation and low-dose temozolomide (TMZ) and 6 cycles of TMZ therapy (150-200mg/m2 po qd dl-5 every 28 days) after completion of radiotherapy. The outcome for the patients with glioblastoma has been improved significantly after receiving this optimal therapy, but the median survival still ranges from 12 to 15 months.Although advances in chemotherapeutic drugs, alkylating agents remain the most common and effective administration in glioblastoma, based on their lipophilic nature, which can appreciably permeate blood-brain barrier (BBB), and their intrinsic cytotoxic activities. Temozolomide (TMZ), an orally administered cytotoxic alkylating agent with broad-spectrum antitumor activity, is a bioavailable imidazotetrazine derivative of the alkylating agent dacarbazine. It penetrates BBB into the brain easily and undergoes rapid chemical conversion in the systemic circulation at physiological pH to its active compound, MTIC (5-(3-methyltriazen-1-yl)imidazole-4-carboximide), which degrades to a DNA-alkylating species, subsequently, is cytotoxic via the formation of O6-and N7-alkylguanine lesions in DNA, this procedure does not require hepatic metabolism for activation. TMZ becomes the most perspective chemotherapeutic drug against gliomas, on base that it is more effective, easier to administer and has fewer side-effects than its competitors. However, it has also failed to improve prognosis in some patients with glioblastoma. The unsatisfactory outcome with chemotherapy has chiefly been originated from intrinsic or acquired chemoresistance of glioblastoma cells. Tumor cell resistance to chemotherapeutic drugs is one of the main causes of treatment failure, Chemotherapy resistance of tumor cells is mediated by many factors. Among these factors, the association between O6-methylguanine-DNA methyltransferase (MGMT) and chemo-resistance is more concerned. MGMT is a key enzyme in the DNA repair network, which catalyzes the transfer of the methyl group from O6-methylguanine to a cysteine residue of its active site. In this single step reaction, DNA-lesions caused by alkylating substances are repaired. MGMT subsequently is irreversibly inactivated and degraded.The radiation effect on tumors is mainly mediated by free radicals production and DNA damage which could induce apoptotic tumor cell death. This mechanism has been demonstrated by a number of studies on radiotherapy for tumor cells. It has been shown that temozolomide could enhance sensitivity of tumor cells to radiation. The improved therapeutic response in a combination of a concomitant radiation and temozolomide would result from not only the TMZ cytotoxicity synergistic to radiation therapy but also the radiosensiting effect of TMZ.Malignant glioma often accompanied by severe peritumor edema and reactive brain edema after surgery and radiation. Glucocorticoid such as methylprednisolone (MP) is a very effective medication in the management of cerebral edema. However, some recent studies reported that glucccorticoid could inhibit tumor cell death induced by radiotherapy or chemotherapy. In our previous study, we found that the cell growth was not affected after methyiprednisolone was added alone to cultured malignant glioma cells. But the inhibition of tumor cell growth was reduced significantly if methyiprednisolone was added following radiation or TMZ administration to cultured malignant glioma cells, compared to radiation or TMZ administration only without methylprednisolone application. Therefore, this study was aimed to find out whether the methylprednisolone caused tumor cell death resistance in the management of synchronous radiotherapy with TMZ chemotherapy and the possible mechanism related to this induced resistance. No study was report on this topic till now globally.In the first part of this study was aimed to elucidate whether the methylprednisolone affected MGMT expression in human glioblastoma cells treated with TMZ. Sulforhodamine B assay was used to calculate the cell viability percentage. Western Blot and RT-PCR were used to detect MGMT expression in U251 cells in different treated group. Compared to control group, significant cell growth inhibition was shown in TMZ treated group and TMZ+methylprednisolone treated group after 24h treatment (P<0.05) and this inhibition decreased with time. Cell growth inhibition gradually decreased after 96h in TMZ treated group and after 48h in TMZ+ methylprednisolone treated group(P<0.05). MGMT expression in U251 cells was significantly higher in methyiprednisolone treated group after 24h treatment compared to other treated groups (P<0.05). MGMT expression in TMZ+ methylprednisolone treated group was lower than methylprednisolone treated group but higher than the TMZ treated group and control group (P<0.05). After 120h, the highest level of MGMT expression was appeared in the TMZ + methylprednisolone treated group (P<0.05). In the TMZ treated group, MGMT expression was changed with time and gradually reached the highest level at 120th hour after the exposure to TMZ (P<0.05); MGMT expression began to decreased 72h later after TMZ withdrawal (P<0.05). In the TMZ+ methylprednisolone treated group, MGMT level had the same changing trend as in the TMZ treated group but with higher expression (P<0.05).The second part was focused on the effeet of tcmozolomidc and methylprednisolone on radiosensitivity in glioblastoma cells. Sulforhodamine B (SRB) assay was used to calculate the cell viability percentage. The apoptosis rate of U251 cells were determined by flow cytometry analysis. The expression of Bax and Bcl-2 protein in the treated cells was detected by Western blot. The cell survival rate in the radiation+methylprednisolone treated group was significantly higher than other treated groups (P<0.05). At 24th and 48th hour after the treatment, the cell survival rate in the radiation+ TMZ+ methylprednisolone treated group was higher compared to the radiation+ TMZ treated group (P<0.05), but no difference was found statistically at 72th and 96th hour between above treated group (P> 0.05). In all treated group except control group, the cell apoptosis was significantly increased (P <0.05). In the apoptosis elevated groups, the lowest level of apoptosis in was found in the radiation+methylprednisolone treated group (P<0.05), and no difference of apoptosis rate was found in other groups (P>0.05). The expression of Bax protein was increased significantly in the radiation+TMZ treated group and radiation+ TMZ+ methylprednisolone treated group, compared to radiation treated group, radiation+methylprednisolone treated group and control group (P<0.05). The expression of Bcl-2 protein was increased significantly in the radiation+ methylprednisolone treated group and radiation+ TMZ+ methylprednisolone treated group, compared to radiation treated group, radiation + TMZ treated group and control group (P<0.05). Bax/Bcl-2 expression ratio was the highest in the radiation+ TMZ treated group, while the lowest in the radiation + methylprednisolone treated group (P<0.05). Bax/Bcl-2 expression ratio was the same basically in other three groups.Conclusion:1. Methylprednisolone could induce MGMT expression. The glioma cells express more MGMT and increase resistance to temozolomide cytotoxicity with time when treated by temozolomide combined with methylprednisolone. After temozolomide and methylprednisolone withdrawed, MGMT expression decrease gradually.2. Methylprednisolone could induce radiation resistance in human glioma cells. Temozolomide could increase the radiosensitivity in human glioma cells even if methylprednisolone-induced radiation resistanca happened un those cells.this study suggests the radioresistance induced by methylprednisolone, which is applied to treat brain edema in patients with malignant glioma during the course of radiation, could be overcome by the concomitant radiotherapy and temozolomide for patients. |
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