Studies On Effects Of All Trans Retinoic Acid On Proliferation, Apoptosis And BP1 Gene Expression In Human Breast Cancer MDA-MB-468 And MCF-7 Cells

Breast cancer is a most popular cancer for women in modern times and gives a great menace to women health even their life.Although the way of operation,chemical therapy and radio therapy have achieved a lot of advance,but most of breast cancer patients come to relapse, tumor metastasis and so on.The therapy of inducing and promoting differentiation in tumor cells has became a novel way in manner of anti-tumor therapy and the study focus on this field became active recently. Retinoids is able to reverse the malignant cells which is infinite proliferative into the normal functional cells.These chemicals have been taken as targeted drugs for researchs.It is well known, Retinoids, chemicals of preonunced pro-differentiated,have a great effects on therapy of cancer such as leukecythemia.But there is few reports on entity tumor therapy. So it is urgency to carry out pilot researchs on high performance and harmfulless chemicals in vitro.In this study,the breast cancer cells,MDA-MB-468 and MCF-7 which is estrogen receptor negative and positive relatively,were treated with all-trans-retinoic-acid (ATRA). The techniques of flow cytometry, immunochemistry,TUNEL and PCR etc were applied to detect the changes of cell proliferation rates,apoptosis and cell cycle distribution among these two breast cancer cells.Furthermore,the expression of BP1 and some key members of IAPs,STATs were detected either.The relationship of them was explored and analysised.so as tomaking a pavement for the breast cancer research.The study inclues three parts:Part 1 Effects of all trans retinoic acid (ATRA)on proliferation and apotosis, cells cycle arrest in human breast cancer cells of MDA-MB-468, MCF-7Objects:To study the effects of all trans retinoic acid(ATAR) on biological behavior of human breast cancer cells lines of MDA-MB-468, MCF-7Methods:Human breast cancer cells lines of MDA-MB-468, MCF-7 were divided into 7 groups randomly:group of blank control and another groups treated by 10-3mol/L,10-4mol/L,10-5mol/L,10-6mol/L,10-7mol/L of ATRA respectively.Cells were collected when treated with ATRA for 24h,48h,72h,96h,120h and 144h.Inhibition rate was detected by way of tetrazolium-based colorimetric assay (MTT assay); the morphology appearance of cells was observed through inverted microscope and TUNEL(TdT-mediated dUTP nick end labeling kit); Cell cycle distribution and apoptosis were assessed with flow cytometry.Results:1. Treated by ATRA,all human breast cancer cell lines show decreased proliferation significantly(P<0.05)which depend on the drug conceration and time. Contrast to the control group,groups treated by different concentration of ATRA show the slow down of the proliferation rate,the increasted of inhibition rate.With the same level of drug conceration and time,the inhibition rate (IR) of ER positive(ER-), MDA-MB-468 appeared significantly higher than the one of ER positive(ER+) MCF-7 (P<0.05).IR was found positive related to the concentration and time of ATRA among the two cell lines with different ER status. 2. Experimental groups show morphological changes including dead cells,changing of cell shape under the inverted microscope.The apoptosis phenomenon was intensified at the condition of higher drug concentration,longer exposure time.3. Apoptosis phenomenon was detected by TUNEL.The control groups show the milder appearance than groups treated with 10-5mol/L ATRA.In MDA-MB-468 and MCF-7 cells,the apoptosis index(AI) in 72h were (32.93±1.512)% and (12.38±1.728)% relatively and all of them were significantly higher that the controls(P<0.05). Furthermore,AI of MDA-MB-468 cells was significantly higher than MCF-7 cells(P<0.05).4. Flow cytometry experiment showed,cell cycle distribution and apoptosis of the two human breast cancer cells lines were altered with the treatment of 10-5mol/L ATRA:compared with control groups,experimental groups showed increased population of cells in phase Go/G1 and decreased ones in phase G2/M and S correspondingly.Meanwhile,apoptosis rates of breast cancer cells treated with 10-5mol/LATRA stepped up.The early-stage apoptosis rate of MDA-MB-468 and MCF-7 cells treated with 10-5mol/L ATRA in 72h were (11.53±1.618)%, (6.22±0.731)%relatively which were significantly higher than the control groups(P<0.05).Furthermore,both of the two index were found positive relation with exposure time of 10-5mol/L ATRA.The alteration of apoptosis rate in ER(-) cells MDA-MB-468 was significantly higher than the ER(+) MCF-7 cells.Conclusions:1. ATRA is able to inhibit the proliferation of human breast cancer cells MDA-MB-468 and MCF-7 in manner of time-and dose-dependent in vitro.2. The cell cycle were arrested in phase Go/G1 in MDA-MB-468 and MCF-7 cells treated by ATRA and the apoptosis was detected.which lead to the inhibition of cell prolifitration.lt is predicted that ATRA inhibit tumor cell through cell cycle block and inducing apoptosis in manner of time-depend.Ability of ATRA anti-tumor was enhanced in the way of increasing drug dose and prolong exposure time. MDA-MB-468 cells was more sensitive than MCF-7 for ATRA. 3. ATRA is expected to take use as the anti-tumor drug in breast cancer therapy.Part 2 Effects of ATRA on BP1, Survivin and Stat3 expression alterations in human breast cancer cell lines of MDA-MB-468, MCF-7Objects:To study the effect of all trans retinoic acid (ATRA) on expression of BP1, Survivin and Stat3 mRNA and their protein in cultured human breast cancer cells and explore the mechanism of ATRA on growth inhibition of human breast cancer cells.Methods:Prtotocol of experimental grouping and cells culture was the same as the Part 1.Cells were collected when treated with 10-5mol/LATRA for 0 hours,24h,48h and 72 h.The mRNA expression of BP1, Survivin and Stat3 were detected by RT-PCR and the immunochemistry was applied to detect the expression of BP1 protein, Survivin and Stat3.Results:1. There was significantly difference of BP1 mRNA expression between untreated human breast cancer cells of MDA-MB-468 and MCF-7.Expression of BP1 mRNA was decreased in all human breast cancer cells treated with ATRA compared with control groups.Optical density ratio(ODR) of each ATRA treated group was decreased.ODR was found decreased along with 24h,48h,72h.It was found significantly difference amoung different time groups.Changes of decreased ODR in ER(-) cells MDA-MB-468 was significantly higher than the ER(+) MCF-7(P<0.05). 2. The expression of Survivin and Stat3 mRNA were decreased significantly in MDA-MB-468, MCF-7 cells treated with 10-5mol/L ATRA(P<0.05).3. The expression of Survivin and Stat3 protein were decreased treated by 10-5mol/LATRA.Compared with the controls,the expression of BP1,Survivin and Stat3 protein were decreased significantly in 24h,48h and 72h(P<0.05).The down-regulation of Survivin and Stat3 protein in ER negative cells were higher than the ones in ER positive cells.4. ATRA was able to inhibit the mRNA and protein expression of BP1, Survivin and Stat3 and the down-regulation of mRNA and protein is synchronism.Conclusion:1. Down-regulated expression of BP1 in human breast cancer cells of MDA-MB-468, MCF-7 may contribuate to the apoptosis and proliferation inhibition induced by ATRA.To some extent,increasing drug concentration and prolong drug induced time may enhance these effects.2. Survivin and Stat3 play a role in cell inhibition and apoptosis in human breast cancer cells.3. Survivin and Stat3 pathway is important in the regulation of BP1 in human breast cancer cells treated with ATRA.4. Further study is needed to explore the mechanism of BP1 gene's oncogene role and may be the theraphy target in breast cancer treatment.Party 3 Correlation between expression of BP1, C-erbB-2 and invasion and metastasis in breast cancerObjects:To detect the expression of BPlmRNA, BP1 and C-erbB-2 protein in breast cancer tissues and to better explain the pathogenesis of breast cancer and to provide more experimental evidence for clinical therapy of breast cancer.Methods:RT-PCR and double immunochemistry were applied to detect the expression of BP1 and C-erbB-2 in 74 cases of breast cancer,peri-cancerous and pair normal breast tissues. The relationship between these proteins expression and pathobiology behavior was analysised and the correlation of two protein was studied.Results:1. RT-PCR results indicated:The expression rate of BP1 in 74 cases of breast cancer, peri-cancerous and normal breast tissue was 64.8%(48/74),8.1%(6/74) and 1.3%(1/74) respectivley. The expression rate of BP1 in breast cancer is significantly higher than that in peri-cancerous and normal breast tissue(P<0.05). There was a close correlation between the expression of BP1 and the patients clinical features such as clinical stage,ER.The positive expression rate of BP1 in the breast cancer with negative ER was significantly higher than that in breast cancer with positive ER. The expression level of BP1 was in rising with the increase of the tumor clinical stage. There was a coincident result between double immunohistochemistry and RT-PCR about BP1.2. The positive ratio of C-erbB-2 protein in breast cancer was significantly higher than peri-cancerous and normal breast tissues(P<0.05). The positive ratio of C-erbB-2 in metastatic, clinical grade III groups was significantly higher than that in non-metastatic, clinical gradeⅠorⅡgroups(P<0.05). The expression rate of C-erbB-2 in the invasive carcinoma was higher than that in the carcinoma in situ and normal breast tissue.3. Analysis of correlation indicated:There was a significant correlation in BP1/ C-erbB-2 expression (P<0.01).Conclusion: 1. There is a close correlation between the expression level of BP1 and the devel-opment of breast cancer and the clinical pathology features such as tumor clinical stage, pathology grade and ER status. There was a coincident result between double immunohistochemistry and RT-PCR for BP1. It indicated that the popularity and price of immunohistochemistry technique is better than that of RT-PCR in the detection of BP1.2. The positive expression of C-erbB-2 protein has a closely correlation with the the clinical pathology features of breast cancer such as clinical stage, pathology grade and the metastasis of lymph node. It indicated there was positive correlation between the expression of C-erbB-2 protein and the metastatic ability of breast cancer.3. There is a significant correlation between BP1 and C-erbB-2.It indicates that BP1 plays an important role in the generation and development of breast cancer by C-erbB-2.4. The united detection of BP1, C-erbB-2 expression can predict the ability of invasion and metastasis of breast cancer and supply the evidence for clinical therapy and judgement of prognosis.