Primary Pituitary Adenoma Cell Culture And The Effect Of Temozolomide On Refractory Pituitary Tumors

Pituitary adenomas are one of the most common types of intracranial tumors, with autopsy studies demonstrating an incidence of 25%. The biology of pituitary adenomas can vary substantially. Some pituitary adenomas are slow growing, incidentally found, and require no treatment whatsoever. On the other extreme, some pituitary adenomas prove refractory to surgery, radiation, and pituitary-suppressive medications (for example, dopamine receptor agonists and somatostatin analogs) and continue to progress. Pituitary carcinomas occur in 0.2% of patients and may metastasize within the CNS. Temozolomide has proven effective in the treatment of patients with high-grade gliomas. Anecdotal case reports have suggested that temozolomide may have efficacy for some patients with aggressive pituitary adenomas, includ-ing pituitary carcinomas. Low MGMT expression has been linked to a favorable clinical response in patients with aggressive pituitary adenomas undergoing treatment with temozolomide. This study evaluates the potential factors that associate with the invitro survival of pituitary adenoma cells and the response of primary pituitary adenoma cell cultures, gonadotroph adenoma cell lines and a patient suffered from refractory pituitary adenoma to temozolomide.Section One Primary Pituitary Adenoma Cell Culture InvitroObjective:To analyze the impact factors for the in vitro survival of pituitary adenoma cells and select the cell culture regimen that maintain the biological properties of pituitary adenoma cells in vitro so as to improve the survival of human pituitary adenoma cells in vitro and guarantee the quality of the cell line that used in the clinical and experimental research.Methods:33 cases of surgical specimens of non-functional pituitary adenoma were isolated inoculated and cultured in vitro. The survial analysis based on the lifetime of these primary cell lines and different separate criteria was performed to evaluate the relevance between them. Cells seeded on Laminin, Matrigel, PPL, Collagen and general culture plate interface were used to observe the disparity of the effect on cell viability.Results:Kaplan Meier survival analysis showed that there was no significant correlation (P> 0.05) between the survival time of pituitary adenoma cells and the age, sex, tumor type, tumor recurrence, tumor invasion, preoperative radiation therapy or the level of Ki-67 LI. Cell viability analysis, based on non-functional pituitary adenoma cell culture on two-dimensional and three-dimensional interface, showed that the effect of Matrigel, Laminin, Collagen group on cell viability was significantly greater than the bare surface of cell culture plates (Matrigel, Laminin group vs. NC, P<0.01; Collagen group vs. NC, P<0.001). There was no significant difference (P>0.05) when Lysine compared with the control group. Cell viability of the cells cultured on Laminin and Matrigel was significantly higher than other cells cultured on other interfaces (P<0.001, vs. Lysine; P <0.01 vs.Collagen). Cell viability collagen group were higher than those Lysine group (P <0.05) while there was no significant difference between Matrigel and Laminin group (P>0.05). Therefore, the sequence of the effect of different cell culture interfaces on pituitary adenoma cell viability was Matrigel, Laminin group> Collagen group> Lysine and general culture plate bare surface group.Conclusion:The in vitro cultured non-functional pituitary adenoma cells growed slowly. There was no significant correlation (P> 0.05) between the survival time of pituitary adenoma cells and the age, sex, tumor type, tumor recurrence, tumor invasion, preoperative radiation therapy or the level of Ki-67 LI. Laminin and Matrigel culture interface can be more powerful than Collagen and PPL to enhance the vitality of the pituitary adenoma cells, and may guarantee a qualified platform for in vitro research.Second Two Effect of Temozolomide on Cell Viability in Gonadotroph Adenoma Cell LinesBackground:Invasive pituitary adenomas are usually refractory to routine neurosurgery, radiosurgery or medications, and alternative therapies are expected. The effects of temozolomide (TMZ) on the inhibiton of gonadotroph adenoma cell viability and hormone secretion were evaluated.Methods:Cell viability and IC50 values were evaluated afterαT3-1 cells were treated with TMZ (31.25-1000μM) or vehicle for 0-72 h. Cell cycle changes and extent of apoptosis were detected using flow cytometry, TUNEL and TEM. Molecular mechanism of TMZ was investigated by Caspase-Glo(?) assay and immunoblotting. Gonadotropin secretion were assessed by immunoassay system.Results:TMZ dose-and time-dependently suppressed cell proliferation (P<0.01 vs. control,250μM,24 h) and induced S-phase accumulation and G2/M-phase arrest (P< 0.05 vs. control,250μM,24 h). Early apoptotic cells increased following a 24-h TMZ incubation (P<0.001 vs. control,250μM), consistent with TEM and TUNEL detection that exhibited morphological features of apoptosis. TMZ (250μM) increased the level of caspase-3/-7 6-fold, caspase-9 7-fold, and caspase-8 3-fold after a 24-h incubation, while attenuated Bcl-2 expression (P<0.001 vs. control) and raised the proteolysis of PARP. Both FSH and LH level were significantly decreased by TMZ (P<0.01 vs. control,250μM,24 h).Conclusions:TMZ inhibited cell proliferation and hormone secretion, and induced cell cycle arrest and apoptotic cell death in gonadotroph adenoma cells via both death receptor and mitochondrial pathway, suggesting that it might represent a useful medical management of invasive gonadotroph adenomas.Section Three Effect of Temozolomide on Human Pituitary Adenoma Cells and Its Relationship with the Expression of MGMTObjective:To observe the effect of temozolomide on human pituitary adenoma cells and test the indicative efficacy of MGMT expression on the sensitivity of TMZ for pituitary tumors.Methods:Surgical specimens of 26 patients with pituitary adenoma underwent isolation, inoculation and cell culture. Drug sensitivity test for TMZ on primary pituitary adenoma cells was performed, and the IC50 values was calculated for each cell line. Immunohistochemical detection for MGMT expression of these tumors was performed to correlate with those TMZ IC50 values by Spearman correlation analysis.Results:Among 26 TMZ-treated primary pituitary adenoma cells,24 cases (92.31%, except for 2 cases of nonfunctioning pituitary adenomas cells) demonstrated dose-dependent inhibitory activity,9 cases (34.62%,3 cases of GH adenomas,6 cases of nonfunctioning pituitary adenomas) demonstrated minor IC50 values that less than the maximum dose of TMZ. MGMT immunohistochemistry results showed that 9 cases (34.62%,4 cases of GH adenomas,1 case of prolactinomas,4 cases of nonfunctioning pituitary adenomas) showed low MGMT expression, less than 10%; 14 cases (53.85%, 12 cases of nonfunctioning pituitary adenomas,2 cases of GH adenomas) showed moderate MGMT expression, greater than 10% but less than 50%; 3 cases (11.54%, non-functional pituitary adenomas) showed high level MGMT expression, MGMT expression ratio greater than 50%. Spearman correlation analysis for TMZ IC50 data and the value of MGMT expression ratio showed that the correlation coefficient was-0.094, P>0.05, namely the TMZ IC50 value and the rate of MGMT expression were not significantly correlated.Conclusion:TMZ was able to steadily inhibit about 30%-40% of the primary pituitary adenoma cell lines. There was no significant correlation between TMZ IC50 value of pituitary adenoma cells and tumor MGMT expression ratio, suggesting that MGMT expression can not accurately indicate the sensitivity of pituitary adenoma to TMZ. Further research was needed to analyze the mechanism of TMZ tolerance.Section Four Effect of Temozolomide on a Refractory Pituitary AdenomaObjective:To explore the effective treatment for refractory pituitary adenomas.Method:Based on MGMT expression and sensitive test of the primary pituitary adenoma cells to Temozolomide, we treated a refractory pituitary adenoma patient.Result:MGMT expression in this case was at low level, and primary pituitary adenoma cells were sensitive to temozolomide in vitro. The tumor volume decreased after the first cycle Temozolomide therapy.Conclusion:Temozolomide may be a salvage therapy for those refractory pituitary adenomas with low MGMT expression.