RhoB controls the 24 kDa FGF-2-induced radioresistance in HeLa cells by preventing post-mitotic cell death

Farnesylated Ras oncoprotein induces a cellular resistance to ionizing radiation that can be reversed by farnesyltransferase inhibitors (FTI). We previously demonstrated that, expression of the 24 kDa FGF2 isoform in wild type ras bearing HeLa cells, induced radioresistance which was also reversed by FTI. We tested the hypothesis that wild type Ras or RhoB, which has been proposed as a potential FTI target, could control the FGF-2-induced radioresistance mechanisms. For this, we expressed inducible dominant negative forms of Ras (RasN17) and Rho (RhoBN19) in 24 kDa FGF2 transfected HeLa cells and analysed their survival after irradiation. While no cell survival modification was observed after RasN17 induction, the expression of RhoBN19 induced a radiosensitization of FGF2 radioresistant HeLa cells in the same range as the one observed after a 48 h treatment with the specific FTI, R115777. Moreover, we showed that activated RhoB but not RhoA induced radioresistance in NIH3T3 cells. The radiosensitizer effect of RhoBN19 expression was due to the induction of the radiation induced post-mitotic cell death. Taken together, these data demonstrate that 24 kDa FGF-2-induced radioresistance is controlled by Rho pathways and suggest that RhoB should be a major determinant in cellular resistance to ionizing radiation.     

Inhibition of Rho pathways induces radiosensitization and oxygenation in human glioblastoma xenografts

We previously demonstrated in vitro that inhibiting the biological pathways of the small GTPase Rho radiosensitizes the human glioma U87 cell line. The aim of this study was to determine if Rho might be involved in the control of in vivo radiosensitivity altogether by controlling cellular radioresistance and by modifying tumor microenvironment. We demonstrate here that the in vivo induction of the dominant negative of Rho, RhoBN19, in U87 xenografts induces a significant decrease of tumor cell survival after irradiation more important than the one we previously observed in vitro. This in vivo increased effect of RhoBN19 expression is due to the improvement of the tumor oxygenation associated with a significant decrease of the vessel density and of the metalloproteinase 2 (MMP2) expression. Moreover, in vitro RhoBN19 expression in U87 cells leads to the inhibition of MMP2 activity. Our results demonstrate for the first time that inhibiting Rho pathways modifies the in vivo radiosensitivity of human glioma cells by controlling intrinsic radioresistance, hypoxia and angiogenesis. These data strongly suggest that Rho should be a major determinant of cellular resistance to ionizing radiation.     

Alphavbeta3 and alphavbeta5 integrins control glioma cell response to ionising radiation through ILK and RhoB

Integrins are extracellular matrix receptors involved in tumour invasion and angiogenesis. Although there is evidence that inhibiting integrins might enhance the efficiency of radiotherapy, little is known about the exact mechanisms involved in the integrin-dependent modulation of tumor radiosensitivity. The purpose of this study was to investigate the role of alphavbeta3 and alphavbeta5 integrins in glioblastoma cell radioresistance and overall to decipher the downstream biological pathways. We first demonstrated that silencing alphavbeta3 and alphavbeta5 integrins with specific siRNAs significantly reduced the survival after irradiation of 2 glioblastoma cell lines: U87 and SF763. We then showed that integrin activity and integrin signalling pathways controlled the glioma cell radiosensitivity. This regulation of glioma cell response to ionising radiation was mediated through the integrin-linked kinase, ILK, and the small GTPase, RhoB, by two mechanisms. The first one, independent of ILK, consists in the regulation of the intracellular level of RhoB by alphavbeta3 or alphavbeta5 integrin. The second pathway involved in cell radiosensitivity consists in RhoB activation by ionising radiation through ILK. Furthermore, we demonstrated that the alphavbeta3/alphavbeta5 integrins/ILK/RhoB pathway controlled the glioma cells radiosensitivity by regulating radiation-induced mitotic cell death. This work identifies a new biological pathway controlling glioblastoma cells radioresistance, activated from the membrane through alphavbeta3 and/or alphavbeta5 integrins via ILK and RhoB. Our results are clues that downstream effectors of alphavbeta3 and alphavbeta5 integrins as ILK and RhoB might also be promising candidate targets for improving the efficiency of radiotherapy and thus the clinical outcome of patients with glioblastoma.     

Preclinical evidence that SSR128129E – A novel small-molecule multi-fibroblast growth factor receptor blocker – Radiosensitises human glioblastoma

Resistance of glioblastoma to radiotherapy is mainly due to tumour cell radioresistance, which is partially controlled by growth factors such as fibroblast growth factor (FGF). Because we have previously demonstrated the role of FGF-2 in tumour cell radioresistance, we investigate here whether inhibiting FGF-2 pathways by targeting fibroblast growth factor receptor (FGFR) may represent a new strategy to optimise the efficiency of radiotherapy in glioblastoma.Treating radioresistant U87 and SF763 glioblastoma cells with the FGFR inhibitor, SSR12819E, radiosensitises these cells while the survival after irradiation of the more radiosensitive U251 and SF767 cells was not affected. SSR128129E administration to U87 cells increases the radiation-induced mitotic cell death. It also decreased cell membrane availability of the FGFR-1 mainly expressed in these cells, increased this receptor’s ubiquitylation, inhibited radiation-induced RhoB activation and modulated the level of hypoxia inducible factor, HIF-1α, a master regulator of hypoxia, thus suggesting a role of FGFR in the regulation of hypoxia pathways. Moreover, treating orthotopically U87 xenografted mice with SSR128129E before two subsequent local 2.5 Gy irradiations significantly increased the animals neurological sign free survival (NSFS) compared to the other groups of treatment. These results strongly suggest that targeting FGFR with the FGFR blocker SSR128129E might represent an interesting strategy to improve the efficiency of radiotherapy in glioblastoma.     

HDJ-2 as a target for radiosensitization of glioblastoma multiforme cells by the farnesyltransferase inhibitor R115777 and the role of the p53/p21 pathway

Resistance of glioblastoma multiforme to radiotherapy poses a major clinical challenge. Farnesyltransferase inhibitors (FTI), such as R115777, have potential to increase radiotherapeutic benefit in this disease, although their mechanism of action is unclear. In our study with eight glioblastoma multiforme cell lines, the most sensitive ones underwent cell cycle arrest in response to FTI treatment. Radiosensitization by FTIs, however, seemed to involve other pathways. If R115777 treatment was initiated < 6 hours before irradiation, all eight glioblastoma multiforme lines were radiosensitized. However, if the time between drug and radiation was extended to 24 hours, cells harboring wild type but not mutated p53 were able to counteract drug-induced radiosensitization. The involvement of the p53/p21 pathway in the development of resistance was confirmed by showing that U87 cells transfected with human papillomavirus E6 to block p53 or interfering RNA to inhibit p21 stayed radiosensitive for 24 hours after drug treatment. The time dependency of R115777-induced radiosensitization suggested that the initial FTI target for early radiosensitization was short-lived, and that a p21-directed pathway restored resistance. Consideration of prenylated molecules that could potentially be involved led us to consider HDJ-2, a co-chaperone of heat shock protein 70. This hypothesis was strengthened by finding that cellular radiosensitivity was increased by genetic inhibition of HDJ-2, whereas overexpression conferred radioresistance. Importantly, irradiation of cells caused HDJ-2 to migrate from the cytoplasm to the nucleus, and this migration was inhibited by prior FTI treatment. These results have clinical relevance in that they help explain the variability in responses to FTIs that occurs following radiotherapy and elucidate some of the reasons for the complexity underlying FTI-induced radiosensitization.     

Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy

Autophagy, or programmed cell death type II, is one of the responses of cancer cells to various therapies, including ionizing radiation. Recently, we have shown that radiation induces autophagy, but not apoptosis, in various malignant glioma cell lines. Autophagy is mainly regulated by the mammalian target of rapamycin (mTOR) pathway. The Akt/mTOR pathway also mediates oncogenesis and radioresistance. Thus, we hypothesized that inhibiting this pathway has both an anticancer and radiosensitizing effect by activating autophagy. The purpose of our study was therefore to determine whether and by which mechanisms an Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, had anticancer and radiosensitizing effects on malignant glioma U87-MG and radioresistant U87-MG cells with a consistitutively active form of epidermal growth factor receptor (U87-MGDeltaEGFR). Treatment with the Akt inhibitor successfully inhibited Akt activity and reduced cell viability in both cell lines. In terms of the mechanism, the Akt inhibitor decreased phosphorylated p70S6 kinase, a downstream target of Akt, and induced autophagy, but not apoptosis. Furthermore, the Akt inhibitor radiosensitized both U87-MG and U87-MGDeltaEGFR cells by enhancing autophagy. Specific inhibition of Akt using the dominant-negative Akt plasmid also resulted in enhanced radiation-induced autophagy. In conclusion, an Akt inhibitor showed anticancer and radiosensitizing effect on U87-MG and U87-MGDeltaEGFR cells by inducing autophagy. Thus, Akt inhibitors may represent a promising new therapy as a single treatment or used in combination with radiation for malignant gliomas, including radioresistant ones that express DeltaEGFR.     

Radiation-Enhanced Vascular Endothelial Growth Factor (VEGF) Secretion in Glioblastoma Multiforme Cell Lines – A Clue to Radioresistance?

Summary Objective: Postoperative radiotherapy is standard treatment for patients with a glioblastoma multiforme (GBM). However, a GBM is radioresistant and almost always recurs, even after a high dose of radiation. A GBM is characterized by its extensive neo-angiogenesis, which can be attributed to the high levels of vascular endothelial growth factor (VEGF). The scope of this study is to investigate the VEGF secretion by GBM cells with different radiosensitivity after irradiation. Methods:Three human GBM cell lines (U251, U251-NG2 and U87) were irradiated with single doses of 0, 5, 10 and 20 Gy of γ-rays from a ¹³⁷Cs source. VEGF levels in medium were measured by ELISA at 24, 48 and 72 h after radiation. Cell survival was measured by the XTT assay 7 days after irradiation. Results:Following single dose radiation, the VEGF levels showed a dose dependent increase in U251, U251-NG2 and U87 glioma cells. Both base-line and radiation-enhanced VEGF levels were about 10-fold higher in U87 compared to U251 and U251-NG2 cells. In addition, in the XTT assay, the U87 was more radioresistant than both U251 and U251-NG2 cell lines (dose modifying factor (DMF) = 1.6 and 1.7 resp). Conclusion:Irradiation enhanced VEGF secretion in all three tested glioma cell lines (up to eight times basal levels). It is tempting to associate the radiation-enhanced VEGF secretion with an increased angiogenic potential of the tumor, which may be a factor in radioresistance.     

Cycling hypoxia increases U87 glioma cell radioresistance via ROS induced higher and long-term HIF-1 signal transduction activity

Glioblastoma multiforme (GBM) tumors are the most common type of brain tumors and resistance to radiotherapy. This study aimed to investigate the differential effect and mechanism of tumor microenvironments, cycling hypoxia and non-interrupted hypoxia, on tumor cell radiosensitivity in the human U87 glioblastoma tumor model. We exposed U87 cells and mice bearing U87 glioma to experimentally imposed cycling or non-interrupted hypoxic stress in vitro and in vivo prior to treatment with ionizing irradiation. Clonogenic survival assay and tumor growth measurements were performed to determine tumor radiosensitivity. The differential regulation of non-interrupted vs. cycling hypoxia by hypoxia-inducible factor-1 (HIF-1) and the impact of HIF-1α on hypoxia-induced radioresistance were assessed by molecular assay and RNAi-knockdown technology. Our results demonstrated that cycling hypoxia induced higher and longer term HIF-1 signal transduction activity via reactive oxygen species (ROS) in U87 cells compared with non-interrupted hypoxia. Cycling hypoxia-induced HIF-1α activation reflected ROS mediated HIF-1α synthesis and stabilization, whereas non-interrupted hypoxia-induced HIF-1α activation was due to decreased HIF-1α degradation resulting from decreased prolyl hydroxylation. With regard to tumor radiosensitivity, cycling hypoxia induced more tumor cell radioresistance and a decreased response to radiotherapy in U87 cells compared with non-interrupted hypoxia. HIF-1 knockdown during in vitro and in vivo hypoxic stresses combined with radiotherapy suppressed cycling and non-interrupted hypoxia-induced radioresistance while increasing overall tumor radiosensitivity. Our results suggest that cycling hypoxia induces more radioresistance than non-interrupted hypoxia in U87 gliomas, and ROS mediated HIF-1α activation is a crucial mechanism involved in hypoxia-induced differential radioresistant in U87 gliomas.     

Radiation-induced mitotic cell death and glioblastoma radioresistance: A new regulating pathway controlled by integrin-linked kinase,hypoxia-inducible factor 1alpha and survivin in U87 cells

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity.In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.     

The farnesyltransferase inhibitor R115777 (ZARNESTRA) enhances the pro-apoptotic activity of interferon-alpha through the inhibition of multiple survival pathways

Interferon alpha (IFNalpha) induces an EGF-Ras-->Raf-1-->Erk dependent survival pathway counteracting apoptosis induced by the cytokine. In this paper we have evaluated the effects of the combination between farnesyl-transferase inhibitor (FTI) R115777 and IFNalpha on the growth inhibition and apoptosis of cancer cells. Simultaneous exposure to R115777 and IFNalpha produced synergistic both antiproliferative and proapoptotic effects. In these experimental conditions, IFNalpha and R115777 completely antagonized the increased activity of both Ras and Erk-1/2 induced by IFNalpha and strongly reduced Akt activity. Furthermore, treatment with R115777 in combination with IFNalpha regimen induced tumor growth delay on established KB cell xenografts in nude mice, while the single agents were almost inactive. R115777 was again able to antagonize the Ras-dependent survival pathway induced by IFNalpha also in vivo. Raf-1, one of the downstream targets of Ras, has been reported to activate bcl-2 through displacement and/or phosphorylation of Bad. We have found that IFNalpha induced mitochondrial localization of Raf-1 that was antagonized by R115777. Moreover, IFNalpha increased Raf-1/bcl-2 immuno-conjugate formation and intracellular co-localization and enhanced phosphorylation of Bad at Ser 112 and again R115777 counteracted all these effects. Moreover, the use of plasmids encoding for dominant negative or dominant positive Raf-1 antagonized and potentiated, respectively, the co-immunoprecipitation between Raf-1 and bcl-2. In conclusion, FTI R115777 strongly potentiates the antitumor activity of IFNalpha both in vitro and in vivo through the inhibition of different survival pathways that are dependent from isoprenylation of intracellular proteins such as ras.     

Fas (APO-1/CD95) signaling pathway is intact in radioresistant human glioma cells

Radiation-induced apoptosis can be mediated through pathways initiated by either DNA damage or ceramide-induced Fas signaling. Glioblastoma multiforme is a primary brain tumor that is highly resistant to irradiation, and U-87 MG, SF126, and T98G are glioblastoma-derived cell lines that mimic this characteristic. We found that these radioresistant glioma cells are susceptible to Fas-mediated cell death induced by treatment with either anti-Fas antibody or exogenous ceramide. Fas-mediated cell death in these cell lines is p53-independent. These data demonstrate that apoptosis can be induced by ceramide and mediated through the Fas pathway in glioma cells, although high-dose ionizing radiation fails to trigger this pathway.     

Radioresponsiveness, sublethal damage repair and stem cell rate in spheroids from three human tumor lines: comparison with xenograft data.

Dose-control curves after fractionated irradiation were generated for small oxic spheroids from the two human glioma cell lines, U87 and A7, as well as the squamous cell carcinoma line FaDu. These data were fitted by the linear quadratic model assuming Poisson statistics. The alpha/beta values of A7, U87, and FaDu spheroids, respectively were 10.3 (8.1-12.9) Gy, 17.8 (15.1-21.1) Gy, and 37.9 (29.1-51.5) Gy. These data were compared with those previously published by Suit et al. (31) and Zietman et al. (40) for 6 mm xenografts of U87 and FaDu after fractionated irradiation and for A7 after single dose irradiation under clamped conditions. A good agreement in the alpha/beta values was observed for U87 and Fadu xenografts and spheroids assuming an oxygen enhancement ratio (OER) of 2.7. In addition, the ranking according to the single doses needed to control 50% of the tumors agreed for xenografts and spheroids from the three cell lines. U87 was the most resistant line in both model systems, followed by A7 and FaDu. However, the absolute values of alpha and beta, obtained from the direct fit to the dose-control data were only about half as high for U87 and FaDu xenografts than for the spheroids. Monte Carlo simulations showed that this discrepancy can be explained by a greater tumor heterogeneity of the xenografts. While the number of critical stem cells or spheroid rescuing units equaled the number of cells per spheroid for the three cell lines, the percentage of tumor rescuing units for Fadu and U87 xenografts was estimated to be below 1%. In a next step, survival curves were generated for exponentially growing cells of the three lines. A7 cells were significantly more radioresistant when plated on tissue plastic than in soft agar. Using the most resistance-promoting colony assay conditions for each cell line, a good agreement was observed for the alpha and SF2Gy values calculated from the colony and spheroid control data. This study shows that the spheroid model can quantitatively predict the repair capacity of sublethal damage as well as the rank order of radiation sensitivity of in vivo tumors.     

Biomarkers of anticancer activity of R115777 (Tipifarnib, Zarnestra) in human breast cancer models in vitro

BACKGROUND: Farnesyltransferase inhibitor R115777 (Tipifamib, Zarnestra) is active in breast cancer, but its efficacy in drug combinations has not been extensively investigated. MATERIALS AND METHODS: The activity of R115777 and paclitaxel, alone and in combination, was studied in the human breast cancer cell lines, BT-474 (overexpressed HER2/neu) and MDA-MB-231 (low HER2/neu), with cell viability and biomarkers for farnesylation (HDJ-2, Rho B), tumor growth (Raf/MEK/ERK), survival (PI3K/Akt) and angiogenesis (VEGF, FGF-2, MMP-1, MMP-2, MMP-9) as the endpoints. RESULTS: The drug combination resulted in additive cytotoxicity. R115777 +/- paclitaxel inhibited HDJ-2 farnesylation, up-regulated RhoB, transiently lowered (P)ERK/ERK and (P)Akt/Akt, reduced Raf-1 and MEK and inhibited secretion of VEGF and MMP-1. CONCLUSION: The effect of R115777 on prenylation biomarkers is consistent with its mechanism of action. The drug interfered with tumor growth, survival and angiogenesis pathways in breast cancer models with low or overexpressed HER2/neu receptor. The combination of R115777 with paclitaxel might offer clinical advantage over monotherapies.     

Relationship between telomere length and radiosensitivity in various human cancer cell lines

Objective： To investigate the relationship between telomere length and radiosensitivity in various human cancer cell lines with the expectation to find a valid and common predictor of radiosensitivity for different cancers. Methods： Eight human cancer cell lines were used, including five human breast cancer cell lines （ZR-75-30, MCF-7, MDA-MB-435S, T-47-D, F539-1590）, two human larynx squamous carcinoma cell lines （Hep-2 and Hep-2R） and a human malignant glioma cell line （U251）. Among them, the radioresistant cell line Hep-2R was isolated and established from a radiosensitive human larynx squamous carcinoma cell line Hep-2 by our center. The radiobiological characteristics of the eight lines were analyzed by the method of colony-forming assay and the radiosensitivity parameters were calculated. Telomere length was analyzed by TRF （mean Telomere Restriction Fragments） length assay. Results： The radioresistance of Hep-2R cell line proved to be stable in long-term passaged cultures as well as in frozen samples. Radiosensitivity parameters are different among those lines. The SF2 values of Hep-2 and U251 are 0.4148 and 0.7520, respectively; The SF2 values of breast cancer cell lines are between those of Hep-2 and U251. The TRF of Hep-2R is 11.12Kb, longer than three times that of its parental counterpart. There is a positive correlation both between SF2 and TRF （r=-0.786, P〈0.05）, and between Do and TRF （r=0.905, P〈0.01）. Conclusion： It is concluded that radiosensitivity and telomere length （TRF） are negatively correlated, TRF could be a valid predictor for radiosensitivity.     

CpG ODN107 potentiates radiosensitivity of human glioma cells via TLR9-mediated NF-κB activation and NO production

Radiotherapy is a standard treatment for glioma patient with or without surgery; radiosensitizer can increase tumor sensitivity for radiotherapy. Herein, a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN107) as a radiosensitizer was investigated in vitro and in vivo, and the possible mechanisms were studied in vitro. In the present experiments, the human glioma U87 cell line used herein was resistant to 5?Gy of ??-ray irradiation. The results showed that 10???g/ml of CpG ODN107 in combination with irradiation significantly inhibited cell proliferation both in MTT assay and colony formation experiments. Tumor growth was inhibited by CpG ODN107 in combination with local irradiation but not by local irradiation or CpG ODN107 alone in human glioma xenograft model in nude mice. The inhibition ratio of tumor growth produced by CpG ODN107 (1.7, 5, and 15?mg/kg) in combination with irradiation was 27.3, 67.0, and 65.5?%, respectively. Further molecular mechanisms were studied in vitro. The results showed that the expressions of iNOS, NO, TLR9 mRNA, and NF-??B p50/p65 increased in the cells treated with CpG ODN107 in combination with irradiation. CpG ODN107 in combination with irradiation did not induce apoptosis but induced cell cycle arrest at G₁ phase. The said results demonstrated that CpG ODN107 possessed a radiosensitizing effect via TLR9-mediated NF-??B activation and NO production in the tumor cells, leading to cell cycle arrest. Therefore, CpG ODN107 is a potential candidate as radiosensitizer for human glioma.     

人脑胶质瘤细胞株MGR2放射抗拒性的诱导

背景与目的:放射治疗是脑胶质瘤重要辅助治疗手段之一,但脑胶质瘤放射敏感性存在很大差异。本研究通过对放射相对敏感的人脑胶质瘤细胞株进行放射诱导得到具有稳定放射抗拒性的后代细胞,从而为研究胶质瘤放射抗拒机制提供具有可比性的成对细胞。方法:选取对X射线相对敏感的人脑胶质瘤细胞株MGR2[2Gy照射下的存活分数(survivalfractionof2Gy,SF2)为0.180±0.05]进行间歇性大剂量X射线照射(每次2Gy,共3次,而后每次5Gy,共2次),每次照射后的细胞继续培养,待5～8周存活的细胞状态稳定后进行下一次照射,整个照射及培养过程历时11个月,最终得到的后代细胞命名为MGR2R(MGR2radiationinduction)。采用MTT法检测MGR2和MGR2R细胞生长的倍增时间,平板集落形成法和线性二次模型(L-Q模型)拟合MGR2和MGR2R细胞的存活曲线并计算相关放射生物学参数和SF2值。血清饥饿细胞周期同步化方法检测MGR2和MGR2R的细胞周期变化。结果:MGR2的倍增时间为3.6天,MGR2R的生长速度减慢,倍增时间为4.0天。MGR2和MGR2R的α值分别为0.447和0.089(t=4.524,P=0.011),β值分别为0.177和0.141(t=1.562,P=0.193),SF2值为0.208和0.478(t=-6.062,P=0.040),MGR2R的放射抗拒性增加。细胞周期同步化后MGR2的细胞周期分布为G1期54.8%,S期30.9%,G2期14.3%,去同步化后G1期35.9%,S期51.2%,G2期12.8%,MGR2R正常生长时存在G2期阻滞(81.7%),同步化后G1期55.7%,S期27.8%,G2期16.6%,去同步化后分别为G1期56.4%,S期26.7%,G2期16.9%,MGR2R存在G1期阻滞,去同步化后两者细胞周期改变不同。结论:人脑胶质瘤细胞株MGR2经间歇性大剂量X射线多次照射后得到的后代细胞MGR2R具有稳定放射抗拒性。MGR2R生长速度减慢,倍增时间延长,同时存在G1和G2期阻滞,可能与其放射抗拒性的产生有关。     

胶质瘤干细胞样细胞中MGMT表达以及与替莫唑胺的耐药关系研究

背景与目的:O6甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltranferase,MGMT)是一种能将鸟嘌呤DNA第六位氧氧原子上的甲基加合物移除和修复损伤DNA的酶,临床上能影响甲基化类化疗药物的疗效。胶质瘤干细胞样细胞被认为是胶质瘤复发的根源之一。本研究旨在探讨MGMT在胶质瘤干细胞样细胞中的表达以及与替莫唑胺耐药的关系。方法:采用悬浮克隆球形成法自胶质瘤细胞株U251、SKMG-4、SF295、SKMG-1、U373、U87、MGR1和MGR2中富集胶质瘤干细胞样细胞。应用免疫荧光技术检测胶质瘤干细胞样细胞相关分子标志;裸鼠移植瘤试验检测胶质瘤干细胞样细胞的成瘤能力。RT-PCR和Western blot检测胶质瘤干细胞样细胞中MGMT的表达;甲基化特异性PCR分析胶质瘤干细胞样细胞MGMT启动子甲基化状况;CCK-8法检测不同浓度替莫唑胺对胶质瘤干细胞样细胞和胶质瘤细胞增殖的作用。结果:分别自8个胶质瘤细胞株中成功富集胶质瘤干细胞样细胞:U251G、SKMG-4G、SF295G、SKMG-1G、U373G、U87G、MGR1G和MGR2G。胶质瘤干细胞样细胞高表达CD133、Nestin和Sox-2等干细胞标志,而且低表达GFAP和TUJ1。胶质瘤干细胞样细胞均能在裸鼠移植成瘤。MGMT在8株胶质瘤细胞及U87G、MGR1G和MGR2G中为阴性,而在U251G、SKMG-4G、SF295G、SKMG-1G和U373G中为阳性表达。替莫唑胺对胶质瘤干细胞样细胞和胶质瘤细胞的抑制作用差异具有显著性。胶质瘤干细胞样细胞与胶质瘤亲代细胞相比更加耐药(P<0.05)。另外,替莫唑胺对MGMT阳性及MGMT阴性胶质瘤干细胞样细胞IC50间的差异无统计学意义(P>0.05)。结论:MGMT阴性表达的胶质瘤细胞经干细胞样培养后,MGMT表达可转为阳性;胶质瘤干细胞样细胞较胶质瘤亲代细胞更耐受替莫唑胺;MGMT的表达与胶质瘤干细胞样细胞对替莫唑胺的耐受之间无明显关联,提示胶质瘤干细胞样细胞对替莫唑胺的耐药可能还有MGMT以外的机制参与。     

Radioresistance is associated to increased Hsp70 content in human glioblastoma cell lines

Radiation therapy is routinely prescribed for high-grade malignant gliomas. However, the efficacy of this therapeutic modality is often limited by the occurrence of radioresistance, reflected as a diminished susceptibility of the irradiated cells to undergo apoptosis. Heat-shock proteins (Hsps) synthesis can be increased by cellular insults, such as radiation-induced damage. Inducible Hsp70 has been suggested to have multiple roles in cytoprotection against apoptosis. Accordingly, high levels of Hsp70 prevent stress-induced apoptosis. In the present study, we investigated whether the content of Hsp70 is associated to glioblastoma cell radioresistance. To this end, the U-87MG, U-251MG and MO59J human glioblastoma cell lines were irradiated at 2, 5 and 10 Gy and their relative radioresistance and Hsp70 were determined. Following 5 Gy irradiation, in MO59J and U-251MG a significant decrease in colony formation was found, whereas the U-87MG was relatively radioresistant. Three hours after the irradiation (at 5 Gy) Hsp70 contents increased 110% in the U-87 MG cells, but did not significantly change in the U-251MG and MO59J cells. Thus, our results suggest that Hsp70 protection against radiation-induced apoptosis might underlie glioblastoma radioresistance.     

Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells.

R115777, a nonpeptidomimetic farnesyl transferase inhibitor has recently demonstrated a significant antileukemic activity in vivo in acute myeloid leukemia. Multiple myeloma (MM) is a fatal hematological malignancy characterized by an accumulation of long-lived plasma cells within the bone marrow. In the present study, we have investigated the effect of the R115777 on growth and survival of myeloma cells. We have found that R115777 induced (1) a significant and dose-dependent growth inhibition of the three myeloma cell lines tested; and (2) a significant and time-dependent apoptosis. R115777 also induced apoptosis in the bone marrow mononuclear cell population of four MM patients, being almost restricted to the malignant plasma cells. Finally, we have investigated the effect of the R115777 in the Ras/MAPK and JAK/STAT pathways which are implicated in survival and/or proliferation in MM. The phosphorylation of both STAT3 and ERK1/2 induced by IL-6 was totally blocked at 15 microM of R115777 and partially blocked when R115777 was used at 10 and 5 microM. The induction of apoptosis by R115777 in myeloma cells and its implication in the regulation of JAK/STAT signalling suggest that R115777 might be an interesting therapeutical approach in MM.