Experimental Study On Combining Triplex-forming Oligonucleotide Of PDGF-B Chain With Antisense Oligonucleotide Of VEGF Inhibiting The Tumor Growth Of Rat Glioma

Clinic and experimental studies suggest that angiogenesis is a prerequisite for solid tumor growth. Angiogenesis is a process by which new vascular networks are formed from pre-existing capillaries. In the absence of neovascularization most tumors might become dormant at a tiny diameter , perhaps 2~3mm. At the time of clinic diagnosis solid tumors are already well vascularized and contain vessels at different degrees of maturity. Gliomas , the most malignant brain tumors, have migrated from the primary site of malignant gliomas by the time of diagnosis in the majority of cases and are responsible for the local recurrence and tumor progression seen clinically. Most available treatment modalities fail to improve survival time and quality of life in patients with gliomas. To improve the prognosis for primary malignant tumors of the central nervous system, we need to expand the focus of our effects. The molecular analysis of growth regulation in gliomas may provide an opportunity for the development of improved therapy. Among targets suitable for new therapies are regulators of angiogenesis. The strategy has 3 advantages : (a) it should be applicable to many types of solid tumors because all require a blood supply for survival and growth;(b) the target endothelial cells are directly accessible through the blood and are normal cells, making the outgrowth of resistant mutants unlikely;(c) there is an in-bulit amplification mechanism because thousands of tumor cells are reliant on each capillary for nutrients and oxygen. Studies show VEGF (vascular endothelial growth factor) plays a critical role in tumorangiogenesis. Therefore, Blocking the effects of VEGF may be enough to suppress most angiogenesis, not only directly but also by suppressing its synergistic interaction with other mitogens. So VEGF is a valuable mark of antiangiogenesis. Inhibition of angiogenesis may therefore be an approach to destroying tumors that are highly vascularized and not treatable by conventional methods. These molecules are especially important in gliomas because hypervascularization is a major feature of the tumors. So gliomas are suitable for the treatment of antiangiogenesis . But antiangiogenesis do not kill tumour cell directly .Effective antiangiogenesis just turn tumour into dormancy and maintenance of tumour dormancy may require continuous life-long treatment. The heterogenous cell population of most glioblastomas multiforme may require the coadministration of several AONs or their combination with targeted toxins or conventional chemotherapeutic agents may be necessary to achieve the greatest therapeutic effect and to decrease in dose-limiting systemic toxicity. Upon this, after we haved finished the experimental study on the inhibition of angiogenesis and tumor growth of glioma with VEGF antisense Oligonucleotide , explore the gene therapy combining antiangiogenesis of VEGF AON inhibiting VEGF expression with the method of directly killing tumor cells. Because human glioma is resulted from transformation and depravation of gliocyte. PDGF(platelet-derived growth factor) is a ubiquitous, potent mitogen and chemotactic factor. The finding that the v-sisoncogene of simian sarcoma virus (SSV) is a retroviral homologue of the PDGF-B chain. Among the sjs family, csis encodes the B chain of PDGF . Normal astrocytes do not express the PDGF genes. Therefore, the inappropriate expression of genes encoding PDGF and the coexpression of PDGFR by astrocytes may represent important steps in the development, proliferation and maintenance of the malignant astrocyte. The possibility is strengthened by the findingthat the intracranial injection of SSV , carrying the v-sis oncogene which encodes the PDGF-B chain, can induce a high frequency of gliomas in newborn marmosets. TFO (Triplex forming oligonucleotide) of offer the potential to block the expression of specific genes within cells. We therefore emploied TFO complementary to PDGF-B chain to observe their effects on the inhibition of PDGF-B chain expression and tumor growth of C6 glioma in vitro and in vivo .We also observed the effects of cell apoptosis, proliferation and cell cycle in vitro and in vivo. Upon this , to detect the feasibility and the possibility of combined anti-gene treatment, the experimental study on combining PDGF-B chain TFO and VEGF AON inhibiting the tumor growth of glioma was performed. The study can be divided into two parts briefly described as below:Part 1. In Vitro study1. Inhibiting the expression of PDGF-B chain and the growth of C6 glioma cells with triplex-forming oligonucleotide (TFO)Objective To clarify the inhibition of the expression of PDGF-B chain and the growth of C6 glioma cells by triplex-formingoligonucleotide (TFO) .Methods Q glioma cell growth was assessed by microculturetetrazolium dye (MTT) assay two days after adding different dosage of triplex-forming oligonucleotide (TFO) . The expression of PDGF-B chain was quantified by immunofluorescence flow cytometric analysis.Results triplex-forming oligonucleotide (TFO) can obviously inhibit the expression of PDGF-B chain and the growth of Ceglioma cells in concentration-dependent fashion.Conclusion Triplex-forming oligonucleotide(TFO)can effectively suppress the expression of C6 glioma cell PDGF-B chain and the growth of C6 glioma cell .2. Effects of proliferation and cell cycle of Ce glioma cellswith triplex forming oligonucleotide (TFO)Objective To clarify effects of proliferation and cell cycle ofC6 glioma cells by triplex forming oligonucleotide (TFO).Methods The Q glioma cell expression of PCNA after adding different dosage of TFOs was quantified by immunofluorescence flow cytometric analysis. Cell cycle of C6 glioma cells after adding different dosage of TFOs was detected by flow cytometric analysis.Results TFOs can obviously inhibit the Q glioma cell expression of PCNA in concentration-dependent fashion. TFOs can obviously inhibit the transit of cells from the GO/Gl phase to S phase in concentration-dependent fashion.Conclusion TFO can effectively suppress the C6 glioma cell proliferation. TFO also can effectively induce the C6 glioma cells arrest in the Gl phase of the cell cycle.3. Effect of apoptosis of Ce glioma cells with triplex formingoligonucleotides (TFO)Objective To clarify effects of apoptosis of C6 glioma cells bytriplex forming oligonucleotide (TFO).Methods Apoptosis of C6 glioma cells after adding different dosage of TFOs was detected by flow cytometric analysis.Results TFO can obviously induce the C6 glioma cell apoptosis in concentration-dependent fashion.Conclusion TFO can effectively induce the C6 glioma cell apoptosis.Part 2. In Vivo study1. Inhibition of glioma growth of tumor -bearing rats with triplex forming oligonucleotide of PDGF-B chainObjective To observe the inhibition effects of growth and tumorigenicity of rats with triplex forming oligonucleotide of PDGF-B chain.Methods 1 X 106 C6 glioma cells/20ul liquid with high-flow microinfusion were seed into righ caudate putamen of all rats with stereotactic technique. TFO was used in situ with sterotactic technique 8 days later after glioma cell inoculation. The treated group Iandllwere treated with 1. 5mg/20ul, 3. 0mg/20ul TFO 3 times at the eighth, eleventh, fourteenth day after cell inoculation, respectively. The control group I was just treated with 20ul liquid 3 times at the same time. Three weeks after cell inoculation,all rats were killed. Samples were detected with macroscopic and microscopic histology as well as immunofluorescence flow cytometric analysis.Results The treated groups had better survival effects. All control rats were in severe danger. The inhibition rate of tumor growth was66. 1% in the treated group I , 91. 8% in the treated group II. PDGF-B, VEGF and PCNA expressions of qualitative analysis of C6 glioma cells with immunohistochemistry were weak in treated group I and in the treated group II, strong in the control group. PDGF-B, VEGF and PCNA expressions of quantified analysis were detected by immunofluorescence flow cytometric technique of C6 glioma cells. From the flow cytometric analysis, it was demonstrated that TFO could specifically block these expressions of C6 glioma cells in concentration-dependent fashion. TFO could significantly block the converting of glioma cells from Go/G! phase to S phase and induce gliomacell apoptosis in concentration-dependent fashion.Conclusion PDGF-B is required for the maintenance and thedevelopment of tumor growth of glioma . PDGF-B is a very good mark of antigene treatment. TFO used in situ could commendably inhibit tumor growth of glioma well .132. Suppression of glioma growth and angiogenesis of tumor -bearing rats by combining TFO of PDGF-B chain and AON of VEGFObjective To detect the new strategy of combined anti-gene therapy so as to observe the inhibition effects of glioma growth and angiogenesis of tumor-bearing rats by combining TFO of PDGF-B chain and AON of VEGF.Methods lxlO6 C6 glioma cells/20ul liquid with high-flow microinfusion were seed into righ caudate putamen of all rats with stereotactic technique. Rats were treated in situ with sterotactic technique 8 days later after glioma cell inoculation. The treated group I was treated with 1.5mg/20ul TFO 3 times at the eighth, eleventh, fourteenth day after cell inoculation. The treated group II and III were treated with PDGF-TFO 1.5mg+VEGF-A0D 0. 125mg/20ul, PDGF-TFO 1.5mg+VEGF-A0D 0.250mg/20ul 3 times at the eighth, eleventh, fourteenth day after cell inoculation, respectively. The control group I was just treated with 20ul liquid 3 times. Three weeks after cell inoculation, all rats were killed. Samples were detected with macroscopic and microscopic histology as well as immunofluorescence flow cytometric analysis.Results The treated groups had better survival effects. All control rats were in severe danger. The inhibition rate of tumor growth was 53. 1% in the treated group I , 81.4% in the treated group II, 93. l%in the treated III. PDGF-B, VEGF and PCNA expressions of qualitative analysis of Q glioma cells with immunohistochemistry were weak in treated group I , in the treated group II and in treated group in treated group, strong in the control group. PDGF-B, VEGF and PCNA expressions of quantified analysis were detected by immunofluorescence flow cytometric technique of C6 glioma cells. From the flow cytometric analysis, it was demonstrated that TFO of the treated group I couldspecifically block these expressions of Cs glioma cells in concentration-dependent fashion. TFO the treated group I could significantly block the converting of glioma cells from G0/Gi phase to S phase and induce glioma cell apoptosis .When the treated group II and the treated III by combining TFO with AON, not only PDGF-B, VEGF and PCNA expressions more significantly decresed, but also could more significantly block the converting of glioma cells from G0/Gi phase to S phase and induce glioma cell apoptosis . These effects were relative AON in concentration-dependent fashion.Conclusion (1)PDGF-B is important key of tumor growth of glioma . TFO of PDGF-B through blocking PDGF-B expression inhibit tumor growth of glioma with inhibiting cell proliferation and inducing cell apoptosis. TFO of PDGF-B could inhibit tumor angiogenesis of glioma through decreasing VEGF expression. PDGF-B is relative to the tumor development and tumor angiogenesis. (2) When combining PDGF-TFO with VEGF-AON, VEGF-AON could suppress angiogenesis through Inhibiting VEGF expression resulting in more reducing PDGF-B expression , inhibiting cell proliferation and inducing cell apoptosis. These effects were relative AON in concentration-dependent fashion. So PDGF-B and VEGF to the tumor growth and tumor angiogenesis have cooperative effects. (3) PDGF-B and VEGF are good mark of combining gene therapy.