The Inhibitory Effect Of Thalidomide On Proliferation & The Expression Of Vascular Endothelial Growth Factor (VEGF) Of Breast Cancer Cell

Breast cancer is one of the most common malignant diseases in female. Although the different available multi-modalities of therapies, including radiotherapy,surgical operation,chemotherapy,endocrinotherapy and intervention therapy et al, have currently been used in the treatment for breast cancer, but the prognosis of breast cancer has not been markedly improved. With the development of the basic research on oncological fields, it was found that many tumor cells have the over expression in growth factors, the vascular endothelial growth factor (VEGF) is one of the importantest factor, which not only elicits the angiogensis in tumors, but also takes an important role in the growth,differentiation,proliferation of tumor cells. The research regard VEGF or VEGFRs as the anti-tumor make progress quickly, there has been various medicine enter cancers clinical, among them also have medicines used for the research of breast cancer treatment. Thalidomide enters the clinical after 2001. By now, its mechanism is not understood very clearly. There are no reports on whether thalidomide can repress expresses of VEGF in breast cancer cell and affect the proliferation of breast cancer cells. And this study contains two related research: The first, the expression of VEGF gene in two-stub classic human breast cancer cell lines with different biology characteristic. The second, the inhibitory effect of thalidomide on proliferation and the express of VEGF in breast cancer cell lines. PartⅠ: Study on the expression of VEGF in human breast cancer cell lines Objective: To study expression of VEGF in human breast cancer cells(MCF-7,MDA-MB-231). Methods: The protein expression of VEGF in human breast cancer cell lines was studied by immunohistochemistry staining. Expression of VEGFmRNA in human breast cancer cell lines was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) method. Results: 1 We confirmed the over expression of VEGFmRNA in breast cell lines by RT-PCR analysis and imunohistochemistry;VEGF165 and VEGF121were identified as the predominant species produced in breast cancer cell lines (MCF-7,MDA-MB-231). 2 The expresses level of VEGF165 in MDA-MB-231 cells is higher than MCF-7 cells(P<0.01). Conclusions: Breast cancer cell lines(MCF-7,MDA-MB-231) related to higher expression of VEGF. And the level of VEGF165 expresses has something to do with the biology characteristic of the cell stub. The study provides significant basis of theory andexperiment for go into researching the inhibitory effect of thalidomide on the express of VEGF. Part Ⅱ: The inhibitory effect of thalidomide on proliferation and the express of VEGF in breast cancer cell lines. Objective: To investigate the effect of thalidomide on proliferation and the express of VEGFmRNA in breast cancer cell lines. Methods: 1 Establishing the melting agent checks against and different densit(y10μg/ml,50μg/ml,100μg/ml,200μg/ml), Establishing different times(24h,48h,72h)at the same time, in order to observes the relation between time and efficacy, The growth rate among these groups was detected by MTT assay. 2 The influence of thalidomide on apoptotic cells and cell period were detected in treated cells by FCM assay. 3 According to the result of MTT, choice medicine density with function time, apply the technique of RT-PCR, examine the inhibitory effect of thalidomide on the express of VEGFmRNA in MCF-7 and MDA-MB-231. Results: 1 MTT assay results:The growth rate in these groups had differences, thalidomide can inhibit the proliferation of MCF-7 and MDA-MB-231 among 10μg/ml～200μg/ml. The beast inhibitory effect is occurring after thalidomide treated 48h. 100μg/ml have the beast inhibitory effect after treated 48h. 2 FCM assay results:Apoptotic cells were found in treated cells, the breast cancer cell presents the second period of G1 in theDNA histogram of FCM. 100μg/ml thalidomide treated cells for 48h, the MCF-7 cell apoptotic index can amount to 9.68%, MDA-MB-231 cell apoptotic index can amount to 11.18%, comparing with control groups, the difference show significant difference (P<0.01). 3 The RT-PCR result analyzes: The control group,50μg/ml group and 100μg/ml group thalidomide treated cells for 48h respectively, There were significant difference in VEGFmRNA expression of MCF-7 among control group and each density groups ( P <0.01). There was no significant difference in the VEGFmRNA expression of MDA-MB-231 between the control group and 100μg/ml group (P>0.05). The VEGFmRNA opposite value of 50μg/ml group was significantly lower than the control group and 100μg/ml group(P<0.01). Thalidomide obvious repress the VEGFmRNA express of MCF-7 and MDA-MB-231 at 50μg/ml, but measure continuously enlarge to the 100μg/ml, the repress function nonexistent measure the effect. VEGF121 and VEGF165 have the same change trend, but the analysis shows that two types (VEGF121 and VEGF165) has no relativity(r=0.429,P=0.28). Conclusions: 1 Thalidomide could inhibit the proliferation of breast cancer cells in the scope of 10μg/ml～200μg/ml. within the scope of 10μg/ml～100μg/ml, the effect relate to the medicine density, the repress rate have no along with the medicine density over 100μg/ml. The represses function of thalidomide attains the high peak at 48h. 2 Thalidomide can induce the breast cancer cell apoptotic, expressing the...