Effect Of Altered Expression Of CCND1 On Biological Behavior And Temozolomide Sensitivity Of Glioma

Gliomas are the most common malignant brain tumors. They account for more than 40-50% of all neoplasms of the central nervous system in China and vary considerably in morphology. Although in the past two decades, the understanding of glioma biology and genetics has been greatly improved, but still no effective way to cure gliomas; Treatment with cytostastic agents and radiotherapy still dominates first-line adjuvant therapy. The main problem of malignant gliomas is resistant to chemotherapy treatment restricted further effect.In preliminary study in the Department of Neurosurgery, Changzheng Hospital, Shanghai Institute of Neurosurgery, we have founded that 29 genes are associated with chemotherapy sensitivity. CCND1 gene is one of them. In previous studies, it has been demonstrated that CCND1 overexpression can enhance the fibrosarcoma cells more resistant to methotrexate. In pancreatic cancer cell lines, CCND1 overexpression can promote cell proliferation and prevent apoptosis induced by chemotherapeutic drugs, thus results in chemoresistance. In contrast, Knockdown CCND1 expression could downregulate multidrug resistance gene expression and promotes drug-induced apoptosis, thus enhancing the tumor cells more sensitive to 5-FU, cisplatin, mitoxantrone, VP16 and other chemotherapeutic drug. Sallinen SL et al have documented that cyclin D1 overexpression is associated with high proliferation of astrocytomas invasion, and pathological level . However, the exact mechanism of cyclin D1 involved in astrocytomas development and progression of the specific mechanism is not clear, whether the abnormal expression of astrocytomasis related to chemotherapeutic drug sensitivity has not been reported. Elevated cyclin D1 in human astrocyte gliomas correlates with a poor prognosis. The goal of this study is to examine the role of astrocyte gliomas, which are often resistant to chemotherapy, in carcinogenesis and to determine whether the expression of cyclin D1 is associated with chemoresistance. Human malignant glioma cells were stably transfected with antisense cyclin D1 and ectogenic cyclin D1 using Lentivirus-mediated transfection, followed by in-vitro growth assays, cell cycle analyses, and invasion assays. The temozolomide sensitivity and level of siRNA-transfection and overexpressing cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.The experiment is divided into three parts: the first part, human malignant glioma cells were stably transfected with antisense cyclin D1 and ectogenic cyclin D1 using Lentivirus-mediated transfection to build cell lines with different CCND1 expression levels. Then, these cells are followed by proliferation assay,, apoptosis assay and invasion assay. The second part, we performed research to determined whether the change of CCND1 expression levels have effects on glioma cell lines U87MG, U251MG chemotherapy sensitivity to Temozolomide in vitro. MTT assay, Clonogenic survival assays and Apoptosis assay determined by flow cytometry are performed. The third part, we aimed to study the therapeutic effect of Temozolomide combined with shRNA targeting CCND1 on xenografts in nude mice.PartⅠKnockdown of CCND1 inhibits proliferation,induces apoptosis in human malignant gliomas cellsObjective: Elevated cyclin D1 in human astrocyte gliomas correlates with a poor prognosis. The goal of this study is to examine the role of astrocyte gliomas, which are often resistant to chemotherapy, in carcinogenesis and to determine whether the expression of cyclin D1 is associated with chemoresistance.Methods: Human malignant glioma SHG-44 cells were stably transfected with antisense cyclin D1 and ectogenic cyclin D1 using Lentivirus-mediated transfection, followed by in-vitro growth assays, cell cycle analyses, and invasion assays. The proliferation assay were determined by 3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide. measured mRNA and protein expression levels of MDR1, MRP1, Bcl-2, Caspase-3, MMP-2, and MMP-9 were measured by RT-PCR and Western blot analysis.Results: Our results indicated that transfection of CCND1-shRNA can effectively reduce the CCND1 proteins expression in SHG-44 cell lines. And CCND1 RNAi in SHG-44 cells inhibited cell proliferation, induced apoptosis, and attenuated invasiveness. Decreased expression levels of MDR1, Bcl-2, and Caspase 3 were found in the CCND1 inhibition cells as determined by Q-PCR and Western blot analysis. Compared with the control groups, CCND1 inhibition in U251MG inhibited cell proliferation and apoptosis rates were also markedly increased (p <0.01) as determined by flow cytometry after CCND1shRNA transfection. Invasive test revealed that CCND1shRNA can inhibit the invasive ability in SHG-44 cells, and overexpression of CCND1 was able to promote the invasion. After inhibition of CCND1, decrease of MDR1, Bcl-2, MMP-2, MMP-9 in the mRNA expression was measured, and Caspase-3 mRNA expression was significantly increased, MRP1 of mRNA expression did not change significantly; Overexpression of CCND1 in the glioblastoma cell lines significantly increased MDR1, Bcl-2, MMP-2, and MMP-9 mRNA expression compared with the control. Further Western blot analysis confirm the similar changes of these genes.Conclusions: In this study we provide data showing that, in addition to the inhibition of cell proliferation, the promotion of apoptosis, and attenuation of invasiveness, siRNA-mediated inhibition of cyclin D1 in a human glioma SHG-44 cell line significantly downregulates the mRNA and protein level of MDR1. Conversely, the overexpression of cyclin D1 results in an increased expression of MDR1. Collectively, these findings demonstrate that cyclin D1 performs multiple functions as an oncogene through enhancement of abnormal growth and resistance to apoptosis, and may contribute to chemoresistance in gliomas.PartⅡEffect of abnormal expression of CCND1 in glioblastoma cell lines on Temozolomide sensitivityObjective: To investigate the effect of abnormal expression of CCND1 in glioblastoma cell lines U87MG and U251MG on Temozolomide sensitivityMETHODS: Human malignant glioma U87, U251 cells were stably transfected with antisense cyclin D1 and ectogenic cyclin D1 using Lentivirus-mediated transfection. The glioma cell lines with different Cyclin D1 levels were cultured with different concentrations of Temozolomide for 72 hours, inhibition rate were determined by MTT assay.IC50 of U251 and U87 for Temozolomide were calculated. Clonogenic survival assays were performed on the parental U251 cells, U251 cells transfected with Lenti-GFP as cells as U251 cells transfected with Lenti-shRNA. Apoptosis of U251 cells, U251 cells transfected with Lenti-GFP as cells as U251 determined by flow cytometry were performed on 24 h, 48 h, 72h, 96 h, 120 h, 144h after incubation with temozolomide. Western blot analysis was performed on 96h after incubation with temozolomide.Results: The IC50 of U87, U87-CCND1-GFP (negative control), U87-CCND1 (overexpression), U87-GFP (shRNA negative control), U87-shRNA (inhibition) cells for Temozolomide was 7.13μg/ml,7.23μg/ml,21.32μg/ml, 7.01μg/ml, 3.90μg/ml respectively; The IC50 of U251, U251-CCND1-GFP, U251-CCND1, U251-GFP, U251-shRNA cells for Temozolomide was 9.09μg/ml, 9.35μg/ml, 30.55μg/ml, 8.98μg/ml,4.76μg/ml. Cloning survival tests and apoptosis assay revealed that significantly decreased colony formation and induced apoptosis after CCND1shRNA and temozolomide combination treatment compared with either treatment alone (P < 0.05).CONCLUSIONS: CCND1 expression levels of U251, U87 glioma cells may associate with the Chemoresistance of these cell lines to Temozolomide. CCND1shRNA combined with Temozolomide can significantly reduce U251 cell colony formation and induce apoptosis than any single treatment.. CCND1 shRNA combined with Temozolomide treatment may provide an effective method of treatment for malignant gliomas.PartⅢTherapeutic effect of Temozolomide and CCND1 shRNA combination on xenografts in nude mice.Objective: To observed the therapeutic effect of Temozolomide and/or CCND1 shRNA on U251 cell line xenografts in nude mice.Methods: A randomly selected group of 18 mice are divided into 3 groups ( n = 6) respectively: 1) U251 treated group; 2) U251-GFP treated group( U251 cell transfected with Lenti-GFP, negative control); 3) U251-shRNA treated group( U251 cell transfected with Lenti-shRNA). 5×106 cells of the three groups in 100μL of DMEM were injected subcutaneously (s.c.) into the right hind limb of nude mice. Animals were euthanized 4 weeks after treatment, and tumors were harvested. The tumor volume(length×width×thickness/2) and weights were calculated and recorded. Another 30 nude mice xenografts were established as described above. When tumors reached an average size of 150 mm3 (length×width×thickness/2), the tumor-bearing mice were randomized into 6 groups(n=3): (1) PBS-treated control, (2) Lenti-GFP alone, (3) Lenti-shRNA (4) PBS and temozolomide. (5) Lenti-GFP and temozolomide (6) Lenti-shRNA and temozolomide. Lenti-shRNA was injected i.t. at a dose of 106 TU(2ul)each day. 5 mg/kg/d of temozolomide were given by intraperitoneal (i.p.) injection 3 hours after vector. Four consecutive daily i.t. and i.p. injections were given, and control animals received i.t. and i.p. serum-free medium. Animals were euthanized on days 7 (i.e., 144 hours after treatment initiation), and tumors were harvested. after intracranial perfusion and fixed with 10% neutral buffered formalin for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) evaluation.Results: In the 4-week observation period, the average weights of tumor xenograft in U251 treated group,U251-GFP treated group and CCND1 shRNA treated group is (0.250±0.071 ) g, (0.193±0.052) g,(0.048±0.084) g respectively. The average size of the three groups is (0. 126±0.064 ) mm3 ,(0. 087±0.037 ) mm3, (0.0262±009 ) mm3 .Compared with the control groups ,the weights and volume of tumor xenograft in U251-shRNA treated group, was statistically significant (P = 0.00,P=0.01 respectively). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) evaluation of tumor sections revealed significantly increased TUNEL-positive cells after CCND1shRNA and temozolomide combination treatment compared with either treatment alone (P < 0.05).Conclusion: In conclusion, combination treatment with Lenti-shRNA targeting CCND1 and systemic temozolomide significantly induced apoptosis in an experimental glioblastoma multiforme model...