The Radiosensitization Effects Of γ-Secretase Inhibitor-Ⅰ On Glioblastoma Cells

IntroductionGlioblastoma, grade IV malignant glioma based on the World Health Organization classification, is the most common malignant brain tumors in adults. Although marked progress has been achieved in diagnosis and treatments of this disease, the prognosis is still poor with a median survival of less than 1 year. Ionizing radiation represents the most effective therapy for gliomas, especially glioblastoma, but remains only palliative because a few radioresistant tumor cells survive the radiation and recur rapidly. Mechanisms underlying radioresistance of tumor cells have been studied for years, effective yet simple therapies are still waiting to be found to enhance the radiosensitivity of glioblastoma. y-secretase inhibitors (GSIs), which block the presenilin-gamma secretase complex, have been actively investigated for their potential to block the generation of Aβpeptide that is associated with Alzheimer's disease. Now they have gained increasingly more attention as novel anti-cancer drugs because of their ability to block Notch signaling pathway. In normal stem cells, Notch signaling pathway plays important roles in promoting self-renewal and repressing differentiation. In multiple tumors, including gliomas,aberrant Notch signaling has been found to be essential for survival and proliferation of tumor cells. Several forms of GSIs have been verified to have striking antineoplastic effects on Notch-expressing tumors. Cell cycle arrest and induction of apoptosis were usually involved in GSI-induced cytotoxicity. Additionally, GSIs were found to sensitize tumor cells to chemotherapies by inhibition of pro-survival pathway regulated by Notch signaling. All these studies strongly suggested a potential clinical application of GSI in cancer therapy. However, unwanted toxicity toward normal cells and tissues is a major concern that cannot be neglected. The combination of GSI with typical therapy, such as radiation, seems to be possible in achieving better curative effects, while reducing side effects. But there is no direct evidence that small concentrations of GSI, which has limited effects on tumor cells alone, compromise tumor cells to other therapies.In current study, we found that a tripeptide GSI (z-Leu-leu-Nle-CHO), called GSI-Ⅰ, enhance the radiosensitivity of human glioblastoma cell lines U87 and U251 at a rather small concentration. This GSI-Ⅰ-induced radiosensitivity was related to depletion of CD133+ subpopulations in the cell lines, which showed greater radioresistance than did CD133- cells. The preferential killing of CD133+ cells by small concentration of GSI-Ⅰsuggests that GSI-Ⅰmay target the CD133+ cells. Therefore, we propose that GSI-Ⅰmay be used at a rather small concentration to serve as radiasensitizer for glioblastoma. Materials and MethodsGlioblastoma cell lines U251 and U87 were used in our experiments. GSI-Ⅰ(Calbiochem, CA, USA) were dissolved in DMSO to 1mg/mL and stored at-20℃. When used, it was diluted in media to final concentration.MTT assays were used to test the cytotoxic effects of GSI-Ⅰon U87 and U251 cells. Cell cycle arrest and apoptosis induced by GSI-Ⅰwere analyzed by flow cytometry. The inhibition of Notch signaling pathway was examined by quantitative real-time RT-PCR analysis of Hes-1, a target gene of Notch signaling pathway. The influence of GSI-Ⅰon expression of some cell cycle-related proteins and apoptosis-related proteins were examined by immunoblotting.The radiosensitization effects of small concentration of GSI-Ⅰon U87/U251 cells were evaluated by colony formation assays. CD133+ cells of U87 and U251 cells were separated by magnetic activated cell sorting (MACS). CD133+U87/U251 cells were cultured in Neural stem cell medium (NSC medium), which was composed of DMEM/F12,20 ng/mL basic fibroblast growth factor (bFGF; peprotech),20 ng/mL epidermal growth factor (EGF; peprotech), and 20μl/mL B27 supplement (Life Technologies). CD133+fractions of U87 and U251 cells were determined by flow cytometry analysis using CD133-PE antibody. Trypan blue staining was used by studied the cytotoxic effects of GSI-Ⅰon CD133+/CD133- U87/U251 cells respectively.U87/U251 cells (with or without treatments) were injected subcutaneously into 4-week-old female athymia nude mice.105 viable cells in 0.2mL PBS were used in each injection. After 3-4 weeks, when the xenografts of control groups reached the longest diameter of 10mm, all mice were killed, and the tumor engrafts were removed and photographed.All experiments were conducted in triplicate.Datas are presented as mean±S.D. one-way ANOVA was used for analyzing mean values of multiple groups. LSD was used for post hoc test. Independent-samples t-test was used for comparing means between two groups. p< 0.05 was considered statistically significant.ResultsSmall concentration of GSI-Ⅰenhanced radiosensitivity of U87/U251 glioblastoma cells.To determine a appropriate concentration of GSI-Ⅰused in the study, we first examined the cytotoxic effects of gradient concentrations of GSI-Ⅰon U87 and U251 cells. The MTT assays showed that the inhibitory effects of GSI-Ⅰon glioblastoma cell lines were significant (U87:F=27.661, P=0.000; U251:F=92.755,P=0.000) and concentration-dependent. With concentrations higher than 2.5μmol/L, GSI-Ⅰtreating for 48h significantly inhibited the cell growth of both cell lines (P＜0.05), while the 1μmol/L of GSI-Ⅰdid not have such effects. Therefore, we used 1μmol/L of GSI-Ⅰin the subsequent experiments, as the GSI-Ⅰalone at this concentration will not cause severe cell death.To test the radiosensitization effects of 1μmol/L of GSI-Ⅰon the two glioblastoma cell lines, cells were pre-treated with 1μmol/L of GSI-Ⅰfor 24 hours or not, and then irradiated with 3Gy of x-rays. After radiation, colony-formation assays were carried out to measure the single cell's viability of each group. Cells co-treated with GSI-Ⅰand radiation formed the least colonies, significantly less than those formed by cells treated with GSI-Ⅰor IR alone(P＜0.01), suggesting that GSI-Ⅰ-treated U87/U251 cells were more sensitive to radiation than the none-treated cells.The radiosensitization effects of GSI-Ⅰon glioblastoma cells were also confirmed in vivo. Cells, treated with DMSO or 2.5μmol/L of GSI-Ⅰ, were injected into both flanks of athymic nude mice at 105 viable cells per injection. After 21 days, tumors formed by GSI-Ⅰ-treated cells (left flanks) were much smaller than the control ones (right flanks),indicating that GSI-I impaired the propagation ability of the tumor cells in vivo. Then we further studied the radiosensitization effects of GSI-Ⅰat a small concentration. Cells were treated with GSI-Ⅰ(1μmol/L for 24 h), radiation (3Gy),or both, or left untreated as control, then were injected subcutaneously into athymic nude mice with equal numbers of viable cells. The control group formed palpable tumors at all 5 injection sites at day 14 after injection. Cells treated with GSI-Ⅰor radiation alone had a extended latency, but finally formed palpable tumors at day 21-30. Combination of GSI-Ⅰwith radiaton significantly decreased tumor incidence (1/5 for U87 cells,0/5 for U251 cells). Taken together, these results indicate that GSI-Ⅰeffectively inhibited tumor growth in vivo, and enhanced the radiosensitivity of tumor cells at a relatively small concentration.GSI-Ⅰdepleted radioresistant CD133+U87/U251 cells.It has been demonstrated that CD133+ glioma stem cells play critical roles in the radioresistance of glioma cells, as CD133+glioma stem cells performed greater resistance to radiation than did CD 133- glioma cells. Here we wonder whether GSI-Ⅰ-induced radiosensitivity of U87/U251 cells were related to depletion of CD133+ subpopulations. To verify this, we investigate the CD 133+proportion of U87/U251 cells before and after radiation, and found that the CD133+cell fraction were strikingly increased after radiation (from 1.6±0.2% to 20.69±1.26%, P＜0.05 in U87 cells; from 4.66±0.15% to 27.94±0.6%, P＜0.05 in U251 cells). However, when pre-treated with 1μmol/L of GSI-Ⅰfor 24 hours, the CD133+ cell fractions did not increase but even decreased after radiation. The reduction of CD 133+ cell fraction after GSI-Ⅰtreatment was also seen in a primary sample of grade IV glioblastoma. CD133+ glioblastoma cells are believed to be enrichment of cancer stem cells. We found that GSI-Ⅰtreatment reduced Nestin expression in U87 cells, also indicating that GSI-Ⅰimpaired the stem-like cells in glioblastoma cells.To confirm that depletion of CD133+ glioblastoma cells by GSI-Ⅰwas related to the enhanced rediosensitivity, we investigate the radioresistance of CD133+/CD133- glioblastoma cells respectively, and evaluated the radiosensitization effects of GSI-Ⅰon the two cell types. We separated CD133+/CD133- cells from U87/U251 cells by MACS. The efficiency of MACS separation was verified by FACS (Suppl.2). CD133+ cells were grown in NSC medium, and formed tumorspheres from single cells approximately 1 week later. The tumorspheres were confirmed to express Nestin, a marker for NSC. It was shown that CD133+ U87/U251 cells formed more colonies (56.67±6.67%/79.49±6.75%) than did CD133-U87/U251 cells (22.97±5.41%/20.51±4.62%) after radiation (for CD133+/CD133-U87, P=0.002; for CD133+/CD133-U251, P=0.000), consistent with results from others. With co-treatment of GSI-Ⅰand radiation, cell colonies of CD133+ U87/U251 cells dramatically decreased by 79.75%/95.96%, compared with that treated with radiation alone. However the reduction was less intense for CD 133- U87/U251 cells (41.76%/79.18%), implying that the radiosensitization effects of GSI-Ⅰwere more obvious in CD133+ cells than that in CD133- cells, which means the radioresistance of CD133+ glioblastoma cells can be reversed by GSI-Ⅰ. Therefore, we believed that depletion of CD133+ cells was involved in GSI-Ⅰ-induced radiosensitivity of glioblastoma cells.CD133+ U87/U251 cells displayed preferential sensitivity to GSI-Ⅰtreatment.To further confirm the elimination of CD133+U87/U251 cells by GSI-Ⅰ, we examined the cytotoxic effects of GSI-Ⅰon CD133+/CD133-U87/U251 cells respectively. After treating with 1μmol/L of GSI-Ⅰfor 24,48 and 72 h, cell viability was determined by trypan blue staining. In U87 cells, GSI-Ⅰdemonstrated a significant concentration-dependent effect in increasing the number of dead cells in CD133+ cells (P=0.001), while no effects on CD133- cells (P=0.074). In U251 cells, although both CD133+ and CD133- cells were significantly affected by GSI-Ⅰtreatment, dead cells of CD133+ cells were more than that of CD133-cells (P<0.01) across each time point examined. However, when the concentration of GSI-Ⅰreached 5μmol/L, there was no difference between the cytotoxic effects on CD133+ and CD133- cells. These results suggested that CD133+ cells were more sensitive to small concentration of GSI-Ⅰthan CD133-cells.CD133+ glioblastoma cells are able to form tumorspheres when cultured in serum-free NSC medium. This is a character of tumor stem cells, called "self renewal". Here we examined the effects of GSI-Ⅰon tumorsphere formation of CD133+ U87/U251 cells. The ability to generate tumorspheres of CD133+ cells was severely impaired by treatment of 1μmol/L of GSI-Ⅰ. Although there were still tumorspheres formed with GSI-Ⅰtreatment, there was a clear decrease in both size and quantity.CD133+ U87/U251 cells express higher level of Notch-2, Notch-3 and Hes-1.Previous studies have pointed out that GSI exerted anti-tumor effects by blocking the Notch pathway in tumor cells. We wonder whether the different sensitivity to GSI-Ⅰshowed by CD133+/CD133- glioblastoma cells was related to the different activation of Notch signaling in these two cell types. Having confirmed that Notch-2, Notch-3 and Hes-1, the target gene of Notch signaling pathway, were expressed in U87 and U251 cells, and Hes-1 expression was down-regulated by GSI-Ⅰtreatment in vitro as well as in vivo, indicating that GSI-Ⅰblocked the Notch signaling pathway effectively, we compared the expression level of such genes between CD133+ and CD133- U87/U251 cells. Revealed by qRT-PCR analysis, mRNA levels of Notch-2, Notch-3 and Hes-1 were higher in CD133+ cells than that in CD133- cells, which means CD133+ cells depends more on Notch pathway than do CD133- cells. This may explain why CD133+ cells were more sensitive to GSI-Ⅰ.DiscussionBecause of the infiltrative growth of malignant gliomas, it is difficult to remove the tumor mass thoroughly by surgical resection, while the efficacy of chemotherapy is impaired by the blood brain barrier. Therefore, radiotherapy represents the most effective treatment for malignant gliomas. Although gliomas often respond to radiotherapy, subsequent recurrence is still inevitable, suggesting insufficient killing of tumorigenic cells. To enhance the radiosensitivity of the malignant glioma cells is of great importance in overcoming this disease. Reasons underlying radioresistance are mainly related to the super abilities of tumor cells to repair damaged DNA and to resist apoptosis as well. Cancer stem cells of gliomas, which possess these "super abilities", have been reported to be more resistant to radiation than non-stem glioma cells, and become the source of recurrence. Given the high tumorigenic capacity of glioma stem cells, this paradigm suggests that targeting the glioma stem cells may augment the efficacy of radiotherapy. In current study, we proposed that GSI could be a novel strategy to enhance the radiosensitivity of malignant glioma cells by depletion of radioresistant CD133+glioma stem cells. GSIs are potent inhibitors of Notch signaling, which plays instrumental functions in both stem cells and cancer biology. The anti-tumor effects of GSIs have been widely reported in the recent years. Although accumulating data strongly suggest a potential clinical application of GSIs in cancer theapeutics, one of the major challenges on the way toward this goal is the untoward side effects associated with the inhibitors, especially the cytotoxicity in normal tissues. For the reasons that Notch signaling pathway widely participated in cellular physiology in normal tissues and GSIs do not exclusively target the Notch pathways, the side effects caused by GSIs are almost inevitable. The only way to balance the efficacy and toxicity of GSIs seems to be identifying a therapeutic window, in which the cytotoxic effects caused by GSIs alone is limited, whereas it can be exacerbated by conventional therapies, such as radiation. In current study, we describe a possibility that GSI-Ⅰcould be used at a small concentration to enhance the radiosensitivity of glioblastoma cells. Our results demonstrated that GSI-Ⅰinhibited glioblastoma cells growth concentration-dependently. At 1μmol/L, the inhibitory effects of GSI-Ⅰon U87/U251 cells are insignificant. However, pre-treated with 1μmol/L of GSI-Ⅰ, cells became more sensitive to radiation than before. Therefore, we believe that 1μmol/L of GSI-Ⅰsensitize the glioblastoma cells to radiation.The cytotoxic effects of GSI-Ⅰat 1μmol/L were rather limitied, then how it affects the radioresistance of glioblastoma cells? Our results suggest that it might be related to depletion of CD133+ glioblastoma cells. CD133 has been widely accepted as a selection marker for enrichment of brain tumor stem cells in both primary tumor specimens and cell lines. According to previous studies as well as ours, CD133+glioma cells showed greater radioresistance than did CD133- glioma cells. FACS revealed that CD133+ subpopulation of glioblastoma cells strikingly increased after radiation, indicating that it was CD133- cells that were killed mainly after radiation. However, pre-treatment of 1μmol/L of GSI-Ⅰprevented the increase of CD133+ cells after radiation, suggesting that GSI-Ⅰinhibited the survival of CD133+ cells. Further investigation indicated that radiosensitization effects of GSI-Ⅰwere more apparent in CD133+ cells than in CD133-cells. Collectively, these results suggest that depletion of CD133+ cells is involved in GSI-Ⅰ-induced radiosensitization of glioblastoma cells. To further confirm the preferential sensitivity of CD133+ glioblastoma cells to GSI-Ⅰtreatment, we examined the cytotoxic effects of GSI-Ⅰon CD133+/CD133- U87/U251 cells respectively. Results showed that with treatment of 1μmol/L of GSI-Ⅰ,cytotoxicity was more severe in CD133+ cells than in CD 133- cells. However, when the concentration of GSI-Ⅰincreased to 2.5μmol/L, there was no difference of cytotoxicity between the two cell types. These results indicated that CD133+ glioblastoma cells were more vulnerable to small concentrations of GSI-Ⅰ. Why would this happen? Taking into account that Notch signaling plays a critical role in maintenance of not only normal stem cells, but also cancer stem cells, it is plausible that inhibition of Notch signaling would impair the stem-like cells in tumors, leading to depletion of cancer stem cells. Similar results were found in others'reports. Notably, our results demonstrate that the preferential inhibition of CD133+ glioblastoma cells by GSI-Ⅰwas only obvious in a small concentration, which imply that the dependence on Notch signaling to maintain cell survival and proliferation is different between CD133+ and CD133- cells. The qRT-PCR results revealed that the mRNA level of Notch-2, Notch-3 and the target gene Hes-1 were higher in CD133+ than in CD133- cells, which means Notch signaling is more important to CD133+ cells. Cells more dependent on a particular signaling pathway are more easily affected by inhibition of such pathway. This explains why CD133+ cells displayed preferential sensitivity to small concentrations of GSI-Ⅰ.In summary, our data support that GSI,at a rather small concentration, sensitize glioblastoma cells to radiation by depletion of CD133+ cells. There is a promising future for the clinical application of GSI, as side effects of GSI could possibly be avoided by small doses and locally drug delivery. Furthermore, application of GSI is not limited in glioblastomas, because Notch signaling pathway is critical in regulation of cancer stem cells not only in neural tumors, but also in cancers from breast, lung, pancreas and prostate. It is plausible that GSI become a universal radiosensitizer in the near future.