Ⅰ型γ分泌酶抑制剂在恶性胶质瘤放射增敏中的作用与机制研究
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摘要
前言恶性胶质瘤是最常见的原发性恶性脑肿瘤,其浸润性生长的特点及对放、化疗的不敏感导致临床治疗相当棘手。手术切除配合术后放疗是目前最常用的治疗手段,但疗效差强人意,约70%的恶性胶质瘤在术后1年复发[1-4]。究其原因,肿瘤难以彻底切除,残留瘤细胞对后续放疗的敏感性差是导致恶性胶质瘤复发率高的主要因素。因此,如何提高恶性胶质瘤的放疗敏感性成为攻克这一肿瘤的主要努力方向。
肿瘤细胞放射抗性的产生多与下列因素有关：①肿瘤细胞超强的DNA损伤修复能力。部分肿瘤细胞高表达DNA损伤修复相关蛋白,能迅速修复受损DNA,因此对放射治疗产生抗性[5]。②肿瘤细胞较强的抗凋亡能力[15]。恶性肿瘤细胞中某些促存活、抗凋亡通路及分子,如表皮生长因子受体通路,PI3K-Akt通路,p53, Survivin等往往高表达,致使肿瘤细胞抵抗射线诱导的凋亡。最近有文献报道,Notch通路是促进恶性胶质瘤细胞放疗抗性的一条重要信号通路,而其机制正是通过上调胶质瘤细胞内Akt等抗凋亡通路的表达[8]。因此削弱肿瘤细胞的DNA损伤修复能力,促进其凋亡是恶性肿瘤放射增敏的主要策略之一。③肿瘤干细胞的放疗抗性。近年来研究发现,肿瘤干细胞在恶性肿瘤的放射抵抗中发挥不可忽视的作用[13],其机制正与肿瘤干细胞超强的DNA损伤修复能力及抗凋亡能力有关。存活下来的肿瘤干细胞成为肿瘤复发的根源。这一点,我们在实验中也有类似发现。
目前对肿瘤细胞放射增敏的研究已进展到分子治疗手段,例如针对放射敏感基因的小分子干扰RNA(siRNA).单克隆抗体[14]等。但这些方法都存在一些问题——基因转移效率低成为制约基因治疗应用的瓶颈；针对单一基因的治疗在体内实验中很难得到令人满意的结果,等等[15]。这些都促使我们寻找一种新的更安全有效,且易于操作的放疗增敏方法,而我们的研究工作发现,γ分泌酶抑制剂(gamma secretase inhibitor, GSI)有望成为一类理想的放射增敏剂。
GSI能抑制细胞内γ分泌酶的活性。过去,人们对GSI的研究多集中在阿尔茨海默病的治疗上[16]。近年来人们发现,GSI还具有很强的抗肿瘤效应[17],而这主要是通过抑制Notch信号通路实现的。Notch信号通路已被证实与多种肿瘤的发生进展密切相关,如急性淋巴细胞性白血病[21,21]、卵巢癌[22]、乳腺癌[23]、髓母细胞瘤及恶性胶质瘤等[24,25]。目前已被体外/体内实验证实具有抗肿瘤活性的GSI有多种[26-30],但有关GSI抗恶性胶质瘤的研究还很少,特别是GSI在恶性肿瘤放射增敏中的作用未见报道。因此,我们着手进行该方面研究。
目的以人恶性胶质瘤细胞株U87、U251细胞为主要实验对象,通过体内/体外实验,研究Ⅰ型γ分泌酶抑制剂(GSI-Ⅰ)对胶质瘤细胞的细胞毒作用及放射增敏作用,并以CD133+胶质瘤干细胞为切入点,探讨GSI-Ⅰ放射增敏的作用机制,为GSI的抗肿瘤效应提供新证据,为恶性胶质瘤的临床治疗,特别是辅助放疗提供新思路。
方法第一部分实验中,我们采用MTT法检测梯度浓度的GSI-Ⅰ对U87、U251细胞生长曲线的抑制作用,利用流式细胞术分析GSI-Ⅰ对U87、U251细胞细胞周期及凋亡的作用,利用免疫印迹法及荧光实时定量RT-PCR等技术研究GSI-Ⅰ对U87、U251细胞中Notch信号通路及其下游一些细胞周期、凋亡相关蛋白表达的影响,初步探讨GSI-Ⅰ细胞毒作用的分子机制。
第二部分实验中,我们采用克隆形成实验分析低浓度GSI-Ⅰ对U87、U251细胞放射敏感性的影响；利用磁式细胞分选技术(Magnetic Activated Cell Sorting, MACS)分离U87、U251细胞中的CD133+/CD133-细胞,CD133+细胞培养于无血清的神经干细胞培养基中,CD133-细胞培养于含血清的MEM培养基中。分别研究低浓度GSI-Ⅰ对CD133+/CD133-胶质瘤细胞的放射增敏作用。利用荧光标记的CD133抗体和流式细胞术,分析GSI-Ⅰ对U87、U251细胞及1例原代培养恶性胶质瘤细胞中CD133+细胞比例的影响。采用体外成球实验及台盼蓝染色实验分析GSI-Ⅰ对CD133+/CD133-胶质瘤细胞的毒性作用。最后,利用裸鼠成瘤实验在体内检测GSI-Ⅰ对胶质瘤细胞的成瘤抑制作用及放射增敏作用。
结果第一部分实验主要研究GSI-Ⅰ对胶质瘤细胞株U87及U251的细胞毒作用。MTT结果显示,GSI-Ⅰ对U87及U251细胞的生长曲线有抑制作用,且随着浓度与时间的增加而加大。进一步研究表明,GSI-Ⅰ对胶质瘤细胞的毒性作用主要为细胞周期阻滞及诱导凋亡：2.5μmol/L GSI一Ⅰ作用48h后,U87细胞的G1期细胞比例从43.8±1.99%增加至52.96±1.36%(P=0.003),而S期细胞比例从24.81±1.5%减少为9.34±0.53%(P=0.000),G2期细胞比例略有增加；U251细胞则观察到在G1期、S期细胞比例下降的同时,G2期细胞比例大幅增加(从10.43±1.39%到46.97±2.24%,P=0.000)；在诱导凋亡方面,1μmol/L GSI-Ⅰ作用24h或48h,U87细胞的凋亡率较低,分别为2.8±0.48%和2.99±0.55%；U251细胞的凋亡率稍高,分别为3.85±0.52%和9.22±0.25%;2.5μmol/L GSI-Ⅰ作用48h后,U87细胞的凋亡率达到10.29±0.94%,U251细胞的凋亡率则高达22.99±0.94%。
为探索GSI-Ⅰ细胞毒作用的可能机制,我们采用RT-PCR、qRT-PCR及Western Blot技术检测GSI-Ⅰ对细胞内Notch信号通路的阻断作用及对一些细胞周期、凋亡相关蛋白的影响。结果显示,Notch-2.Notch-3在U87、U251细胞中表达；GSI-Ⅰ作用12-24小时即可降低Notch靶基因Hes-1的表达,证明GSI-Ⅰ对Notch通路有阻断作用。GSI-Ⅰ作用后两种细胞的抗凋亡蛋白Akt及S6K的磷酸化程度略有降低。U87细胞的Cyclin D1表达下调,U251细胞的CyclinB1表达下调,两种细胞的CDK2都有明显下调。说明GSI-Ⅰ对胶质瘤细胞株的抗凋亡及细胞周期蛋白的活性及表达有影响。这可能是GSI-Ⅰ抗胶质瘤效应的分子机制之一。
第二部分实验主要探讨GSI-Ⅰ的放射增敏作用及体内抗肿瘤作用。克隆形成实验显示,低浓度GSI-Ⅰ对U87、U251细胞有显著的放射增敏作用。为探讨放射增敏机制,我们用Western Blot技术检测与DNA修复有关的细胞周期检测点蛋白CHK1、CHK2的表达,发现无影响；检测抗凋亡蛋白Akt的磷酸化水平,发现放射后Akt磷酸化水平增加,在GSI-Ⅰ的作用下则明显降低,提示抑制Akt的活性可能参与GSI-Ⅰ的放射增敏作用。
我们还发现,CD133+胶质瘤干细胞的放射抗性要高于CD133-胶质瘤细胞,而GSI-Ⅰ则显著降低CD133+胶质瘤干细胞的放射抗性。流式细胞分析显示,放射后胶质瘤细胞中的CD133+细胞比例大幅增加,而1μmmol/L GSI-Ⅰ作用24小时可显著减少CD133+细胞比例,提示GSI-Ⅰ通过消除CD133+胶质瘤细胞达到放射增敏的效应。体外成球实验显示,GSI-Ⅰ可明显抑制CD133+胶质瘤干细胞的体外成球能力。台盼蓝染色实验显示,1μmmol/L GSI-Ⅰ对CD133+胶质瘤细胞的毒性大于CD133-胶质瘤细胞,而随着GSI-Ⅰ浓度的增加,它对CD133+/CD133-胶质瘤细胞的毒性作用无差异。说明低浓度GSI-Ⅰ可靶向抑制CD133+胶质瘤干细胞。qRT-PCR结果显示,CD133+U87细胞中Notch-2、Notch-3受体及靶基因Hes-1的表达水平明显高于CD133-细胞,提示GSI-Ⅰ的靶向抑制CD133+胶质瘤细胞可能与此有关。
裸鼠实验表明,经1μmmol/L GSI-Ⅰ处理的U87细胞,其体内成瘤能力下降,瘤体积比对照组小。对正常U87细胞成瘤的瘤组织,瘤周注射GSI-Ⅰ,可导致瘤组织大片坏死。免疫组化染色结果显示,GSI-Ⅰ能显著降低移植瘤细胞中Hes-1和Nestin的表达,说明Notch信号通路被阻断且肿瘤细胞的“干细胞特性”(Stem-like character)被削弱。
结论1、GSI-Ⅰ在体外可抑制胶质瘤细胞增殖并诱导凋亡,在体内可抑制成瘤并导致瘤组织坏死,具有明确的抗恶性胶质瘤效应。
2、低浓度GSI-Ⅰ(1μmol/L)单独作用对恶性胶质瘤细胞的抑制作用有限,但能显著增加胶质瘤细胞的放射敏感性,而这种放射增敏作用是通过靶向抑制放射抗性较强的CD133+胶质瘤细胞实现的。
Introduction
Glioblastoma, 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 Methods
Glioblastoma 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.
Results
Small 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.
Discussion
Because 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.
肿瘤细胞放射抗性的产生多与下列因素有关：①肿瘤细胞超强的DNA损伤修复能力。部分肿瘤细胞高表达DNA损伤修复相关蛋白,能迅速修复受损DNA,因此对放射治疗产生抗性[5]。②肿瘤细胞较强的抗凋亡能力[15]。恶性肿瘤细胞中某些促存活、抗凋亡通路及分子,如表皮生长因子受体通路,PI3K-Akt通路,p53, Survivin等往往高表达,致使肿瘤细胞抵抗射线诱导的凋亡。最近有文献报道,Notch通路是促进恶性胶质瘤细胞放疗抗性的一条重要信号通路,而其机制正是通过上调胶质瘤细胞内Akt等抗凋亡通路的表达[8]。因此削弱肿瘤细胞的DNA损伤修复能力,促进其凋亡是恶性肿瘤放射增敏的主要策略之一。③肿瘤干细胞的放疗抗性。近年来研究发现,肿瘤干细胞在恶性肿瘤的放射抵抗中发挥不可忽视的作用[13],其机制正与肿瘤干细胞超强的DNA损伤修复能力及抗凋亡能力有关。存活下来的肿瘤干细胞成为肿瘤复发的根源。这一点,我们在实验中也有类似发现。
目前对肿瘤细胞放射增敏的研究已进展到分子治疗手段,例如针对放射敏感基因的小分子干扰RNA(siRNA).单克隆抗体[14]等。但这些方法都存在一些问题——基因转移效率低成为制约基因治疗应用的瓶颈；针对单一基因的治疗在体内实验中很难得到令人满意的结果,等等[15]。这些都促使我们寻找一种新的更安全有效,且易于操作的放疗增敏方法,而我们的研究工作发现,γ分泌酶抑制剂(gamma secretase inhibitor, GSI)有望成为一类理想的放射增敏剂。
GSI能抑制细胞内γ分泌酶的活性。过去,人们对GSI的研究多集中在阿尔茨海默病的治疗上[16]。近年来人们发现,GSI还具有很强的抗肿瘤效应[17],而这主要是通过抑制Notch信号通路实现的。Notch信号通路已被证实与多种肿瘤的发生进展密切相关,如急性淋巴细胞性白血病[21,21]、卵巢癌[22]、乳腺癌[23]、髓母细胞瘤及恶性胶质瘤等[24,25]。目前已被体外/体内实验证实具有抗肿瘤活性的GSI有多种[26-30],但有关GSI抗恶性胶质瘤的研究还很少,特别是GSI在恶性肿瘤放射增敏中的作用未见报道。因此,我们着手进行该方面研究。
目的以人恶性胶质瘤细胞株U87、U251细胞为主要实验对象,通过体内/体外实验,研究Ⅰ型γ分泌酶抑制剂(GSI-Ⅰ)对胶质瘤细胞的细胞毒作用及放射增敏作用,并以CD133+胶质瘤干细胞为切入点,探讨GSI-Ⅰ放射增敏的作用机制,为GSI的抗肿瘤效应提供新证据,为恶性胶质瘤的临床治疗,特别是辅助放疗提供新思路。
方法第一部分实验中,我们采用MTT法检测梯度浓度的GSI-Ⅰ对U87、U251细胞生长曲线的抑制作用,利用流式细胞术分析GSI-Ⅰ对U87、U251细胞细胞周期及凋亡的作用,利用免疫印迹法及荧光实时定量RT-PCR等技术研究GSI-Ⅰ对U87、U251细胞中Notch信号通路及其下游一些细胞周期、凋亡相关蛋白表达的影响,初步探讨GSI-Ⅰ细胞毒作用的分子机制。
第二部分实验中,我们采用克隆形成实验分析低浓度GSI-Ⅰ对U87、U251细胞放射敏感性的影响；利用磁式细胞分选技术(Magnetic Activated Cell Sorting, MACS)分离U87、U251细胞中的CD133+/CD133-细胞,CD133+细胞培养于无血清的神经干细胞培养基中,CD133-细胞培养于含血清的MEM培养基中。分别研究低浓度GSI-Ⅰ对CD133+/CD133-胶质瘤细胞的放射增敏作用。利用荧光标记的CD133抗体和流式细胞术,分析GSI-Ⅰ对U87、U251细胞及1例原代培养恶性胶质瘤细胞中CD133+细胞比例的影响。采用体外成球实验及台盼蓝染色实验分析GSI-Ⅰ对CD133+/CD133-胶质瘤细胞的毒性作用。最后,利用裸鼠成瘤实验在体内检测GSI-Ⅰ对胶质瘤细胞的成瘤抑制作用及放射增敏作用。
结果第一部分实验主要研究GSI-Ⅰ对胶质瘤细胞株U87及U251的细胞毒作用。MTT结果显示,GSI-Ⅰ对U87及U251细胞的生长曲线有抑制作用,且随着浓度与时间的增加而加大。进一步研究表明,GSI-Ⅰ对胶质瘤细胞的毒性作用主要为细胞周期阻滞及诱导凋亡：2.5μmol/L GSI一Ⅰ作用48h后,U87细胞的G1期细胞比例从43.8±1.99%增加至52.96±1.36%(P=0.003),而S期细胞比例从24.81±1.5%减少为9.34±0.53%(P=0.000),G2期细胞比例略有增加；U251细胞则观察到在G1期、S期细胞比例下降的同时,G2期细胞比例大幅增加(从10.43±1.39%到46.97±2.24%,P=0.000)；在诱导凋亡方面,1μmol/L GSI-Ⅰ作用24h或48h,U87细胞的凋亡率较低,分别为2.8±0.48%和2.99±0.55%；U251细胞的凋亡率稍高,分别为3.85±0.52%和9.22±0.25%;2.5μmol/L GSI-Ⅰ作用48h后,U87细胞的凋亡率达到10.29±0.94%,U251细胞的凋亡率则高达22.99±0.94%。
为探索GSI-Ⅰ细胞毒作用的可能机制,我们采用RT-PCR、qRT-PCR及Western Blot技术检测GSI-Ⅰ对细胞内Notch信号通路的阻断作用及对一些细胞周期、凋亡相关蛋白的影响。结果显示,Notch-2.Notch-3在U87、U251细胞中表达；GSI-Ⅰ作用12-24小时即可降低Notch靶基因Hes-1的表达,证明GSI-Ⅰ对Notch通路有阻断作用。GSI-Ⅰ作用后两种细胞的抗凋亡蛋白Akt及S6K的磷酸化程度略有降低。U87细胞的Cyclin D1表达下调,U251细胞的CyclinB1表达下调,两种细胞的CDK2都有明显下调。说明GSI-Ⅰ对胶质瘤细胞株的抗凋亡及细胞周期蛋白的活性及表达有影响。这可能是GSI-Ⅰ抗胶质瘤效应的分子机制之一。
第二部分实验主要探讨GSI-Ⅰ的放射增敏作用及体内抗肿瘤作用。克隆形成实验显示,低浓度GSI-Ⅰ对U87、U251细胞有显著的放射增敏作用。为探讨放射增敏机制,我们用Western Blot技术检测与DNA修复有关的细胞周期检测点蛋白CHK1、CHK2的表达,发现无影响；检测抗凋亡蛋白Akt的磷酸化水平,发现放射后Akt磷酸化水平增加,在GSI-Ⅰ的作用下则明显降低,提示抑制Akt的活性可能参与GSI-Ⅰ的放射增敏作用。
我们还发现,CD133+胶质瘤干细胞的放射抗性要高于CD133-胶质瘤细胞,而GSI-Ⅰ则显著降低CD133+胶质瘤干细胞的放射抗性。流式细胞分析显示,放射后胶质瘤细胞中的CD133+细胞比例大幅增加,而1μmmol/L GSI-Ⅰ作用24小时可显著减少CD133+细胞比例,提示GSI-Ⅰ通过消除CD133+胶质瘤细胞达到放射增敏的效应。体外成球实验显示,GSI-Ⅰ可明显抑制CD133+胶质瘤干细胞的体外成球能力。台盼蓝染色实验显示,1μmmol/L GSI-Ⅰ对CD133+胶质瘤细胞的毒性大于CD133-胶质瘤细胞,而随着GSI-Ⅰ浓度的增加,它对CD133+/CD133-胶质瘤细胞的毒性作用无差异。说明低浓度GSI-Ⅰ可靶向抑制CD133+胶质瘤干细胞。qRT-PCR结果显示,CD133+U87细胞中Notch-2、Notch-3受体及靶基因Hes-1的表达水平明显高于CD133-细胞,提示GSI-Ⅰ的靶向抑制CD133+胶质瘤细胞可能与此有关。
裸鼠实验表明,经1μmmol/L GSI-Ⅰ处理的U87细胞,其体内成瘤能力下降,瘤体积比对照组小。对正常U87细胞成瘤的瘤组织,瘤周注射GSI-Ⅰ,可导致瘤组织大片坏死。免疫组化染色结果显示,GSI-Ⅰ能显著降低移植瘤细胞中Hes-1和Nestin的表达,说明Notch信号通路被阻断且肿瘤细胞的“干细胞特性”(Stem-like character)被削弱。
结论1、GSI-Ⅰ在体外可抑制胶质瘤细胞增殖并诱导凋亡,在体内可抑制成瘤并导致瘤组织坏死,具有明确的抗恶性胶质瘤效应。
2、低浓度GSI-Ⅰ(1μmol/L)单独作用对恶性胶质瘤细胞的抑制作用有限,但能显著增加胶质瘤细胞的放射敏感性,而这种放射增敏作用是通过靶向抑制放射抗性较强的CD133+胶质瘤细胞实现的。
Introduction
Glioblastoma, 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 Methods
Glioblastoma 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.
Results
Small 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.
Discussion
Because 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.
引文
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2. Kleihues P, Cavenee WK. Pathology and genetics of tumours of the nervous system. In:World Health Organization Classification of Tumours of the Nervous System, Editorial and Consensus Conference Working Group. IARC Press, Lyon, France.
3. Black PM. Brain tumor, part I [J]. N Engl J Med,1991,324:1471-1476.
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6. Lammering G, Hewit TH, Holmes M, Valerie K, Hawkins W, Lin PS, Mikkelsen RB, Schmidt-Ullrich RK. Inhibition of the type Ⅲ epidermal growth factor recep tor variant mutant receptor by dominant2negative EGFR CD533 enhances malignant glioma cell radiosensitivity[J]. Clin Cancer Res,2004,10 (19): 6732-6743.
7. A Diaz Miqueli, J Rolff, M Lemm, I Fichtner, R Perez and E Montero. Radiosensitisation of U87MG brain tumours by anti-epidermalgrowth factor receptor monoclonal antibodies[J]. British Journal of Cancer,2009,100:950-958.
8. Nakamura JL, Karlsson A, Arvold ND, Gottschalk AR, Pieper RO, Stokoe D, Haas-Kogan DA. PKB/Akt mediates radiosensitization by the signaling inhibitor LY294002 in human malignant gliomas[J]. J Neurooncol,2005,71 (3): 215-222.
9. Shono T, Tofilon PJ, Schaefer TS, Parikh D, Liu TJ, Lang FF. Apoptosis induced by adenovirus mediated P53 gene transfer in human glioma correlates with site specificphorylation[J]. Cancer Res,2002,62 (4):1069-1076.
10. Chakravarti A, Zhai GG, Zhang M, Malhotra R, Latham DE, Delaney MA, Robe P, Nestler U, Song Q, Loeffler J. Survivin enhances radiation resistance in primary human glioblastoma cells via caspase independen mechanisms[J]. Oncogene,2004,23 (45):7494-7506.
11. Jialiang Wang, Timothy P. Wakemanc, Justin D. Lathiad, Anita B. Hjelmeland, Xiao-Fan Wang, Rebekah R. White, Jeremy N. Richd, Bruce A. Sullengera. Notch Promotes Radioresistance of Glioma Stem Cells[J]. Stem Cells.2010 Jan;28(1):17-28.
12. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. Identification of human brain tumour initiating cells. Nature,2004; 432(7015):396-401.
13. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells p romote radioresistance by p referential activation of the DNA damage response[J]. Nature,2006,444 (7120):756-760.
14. A Diaz Miqueli, J Rolff, M Lemm, I Fichtner, R Perez and E Montero. Radiosensitisation of U87MG brain tumours by anti-epidermalgrowth factor receptor monoclonal antibodies[J]. British Journal of Cancer,2009,100:950-958
15.邹慧超,赵世光.胶质瘤放射增敏治疗分子生物学研究进展[J].现代肿瘤医学2008,16(5)：853-854.
16. Selkoe D, Kopan R. Notch and presenilin:regulated intramembrane proteolysis links development and degeneration[J]. Annu Rev Neurosci 2003;26:565-97.
17. Shih I.M., and Wang T.L., Notch Signaling, γ-Secretase Inhibitors, and Cancer Therapy[J]. Cancer Res.2007.67:1879-1882.
18. Hitoshi S, Alexson T, Tropepe V, Donoviel D, Elia AJ, Nye JS, Conlon RA, Mak TW, Bernstein A, and van der Kooy D. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells[J]. Genes Dev.2002.16:846-858.
19. Morrison SJ, Perez SE, Qiao Z, Verdi JM, Hicks C, Weinmaster G, and Anderson DJ. Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells[J]. Cell.2000,101:499-510.
20. Grabher C, von Boehmer H, Look AT. Notch 1 activation in the molecular pathogenesis of T-cell acute lymphoblastic leukaemia[J]. Nat Rev Cancer 2006;6:347-59.
21. Aster JC. Deregulated NOTCH signaling in acute T-cell lymphoblastic leukemia/lymphoma:new insights, questions, and opportunities[J]. Int J Hematol 2005;82:295-301.
22. Park JT, Li M, Nakayama K, Mao TL, Davidson B, Zhang Z, Kurman RJ, Eberhart CG, Shih IeM, Wang TL. Notch-3 gene amplification in ovarian cancer[J]. Cancer Res 2006;66:6312-8.
23. Gallahan D, Callahan R. The mouse mammary tumor associated gene INT3 is a unique member of the NOTCH gene family (NOTCH4) [J]. Oncogene 1997; 14:1883-90.
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