Study Of The Molecular Mechanism And The Effects Of Chloride Channel CLC-3 And Mitochondrion On Apoptosis Induced By Tamoxifen In The MCF-7 Breast Cancer Cell Line

The breast cancer is a common malignant tumor which threaten to thehealth of women.Whether in developed country or in developing country,themorbility of breast cancer increases. In America ,ever since 1960,themorbility of breast cancer rised stably at the rate of 1-2% per year.InJapan,from1960 to 1985,the morbility of breast cancer doubled.But,China,asa developing country, her breast cancer`s morbility is similar to thedeveloped country,and has a rising tendency.Somestudies have showed thatin recent 20 years ,China`s breast cancer morbility had increased 37.6% at arate of 2.3% per year.In our country the morbility had been 2.88/million atthe year 2000 , and had taken the first place of the female malignanttumor.Generally,the morbility and the epidemic goes up with the increase ofage.With the best knowledge, breast cancer morbility increases rapidlybetween 30 and 40 years old,and increases persistently,but between 40,50and 60 years old,the speed of increase fall-off.In the next decade,there willbe 5 millon breast cancer patients all over the world.Now breast cancer has been identified as a kind of systemic diseasewhich has the local symptoms.Up to date, the therapy of breast cancer is totake an operation together with the chemotherapy, radiotherapy,biotherapyand traditional Chinese medicine.With the development of molecularbiology and immunology, endocrine therapy and biotherapy graduallybecome the important part of the combined therapy.The relationship between the ion channel and the every kind of tumors hasbeen studied in the last two decades.There are some different kinds of ionchannel relate with the breast cancer, MCF-7 cells have been studied morecarefully.CLC-3 is a kind of the chloride channels,CLC-3 express mostly in theneuroepidermal layer,and in the breast cancer the report is seldom.Recentlythe studies have found that CLC-3 may be the molecular foundation ofvolume regulatory chloride channel,it plays the important role in the cellularvolume regulatory ,proliferation and the differentiation.Up to date there aresome reports to identified that the chloride channels locate at the outermitochondrial membrane and it plays a crucial role in the cellular apoptosisprocess regulated by the mitochondria.The objective of our study is to determine the molecular mechanismsand the effects of mitochondria and chloride channel CLC-3 on apoptosisinduced by tamoxifen in the MCF-7 cell line, to find the new theory to guidethe clinical practice of CLC-3antisense in the treatment of breast cancer.We divide the experiment into four partsThe part 1;Expression of the CLC-3 in the breast cancerWe carry out the immunohistochemical staining in the breast cancertissue and the same perifocal benign tissue of the 27 patients who had beentaken operation. We found that the ClC-3 protein mainly expressed in thecytoplasm and on the cellular membrane.The study showed that the21(77.77%) breast cancer tissue in 27 patients were positive and the6(22.33%) were negative;the 3 (11.1%%) perifocal benign tissue in 27werepositive,24(88.9%)were negative. x2 was 24.3, P<0.05.The result implieedthat there was a obvious difference between the expression of CLC-3 in thebreast cancer and the benign perifocal tissue P<0.05.This implied that theCLC-3 had some pathological and physiological significance in the processof breast cancer`s genesis and growth.This became the clinical foundation tomake a further study.The part 2 Design and the identification of the CLC-3 antisense strandTo obtain the tools which were applied in the following steps studies ,wedesigned and identified the CLC-3 antisense strand with the method ofRT-PCR.The part 3;Effects of the CLC-3 antisense strand and TAM on theproliferation of MCF-7 cell linesTo add the different concentration TAM in each group MCF-7 cells thento incubate the cells for 24 huors ,to conllect the cells and then with MTTchromatometry,we found that TAM could enhance the apoptosis induction inthe MCF-7 cells vs the control,and this effect can be improved with theconcentration increasing(from 0 to 25 of the).shows the dose dependent 0-10μM(P<0.05),10-20μM(P<0.01).Then we divided the MCF-7 cell into 6 gruops, control group;group 1:CLC-3 antisense;group 2:TAM 10μM;group 3:TAM 20μM;group4:CLC-3 antisense+TAM 10μM;group 5:CLC-3 antisense+TAM 20μM.To transfect CLC-3 antisense strand into the cells for 4 huors and then toincubated the cells for 24 huors ,to conllect the cells and then with MTTchromatometry.We found that the proliferation inhibition of MCF-7cells hadnot been affected clearly only by CLC-3 antisense strand.There were adifference of proliferation inhibition in the MCF-7cells between CLC-3antisense+TAM 10μand the control(p<0.05);CLC-3 antisense+TAM 20μM vs the control had a big difference(p<0.01)。CLC-3 antisense+TAM10μM vs TAM 10μM,had a difference(p<0.05);CLC-3 antisense+TAM20μM vs TAM 20μM, had a big difference(p<0.01)。Inorder to study the apoptosis ,we detected the each group cells with theTUNEL and the DNA ladder methods .The TUNEL result showed that theapoptosis rate of TAM 10μM was 38%,TAM 20μM was 66%,TAM 20μM+CLC-3antisense was 85%.From DNA ladder electropherogram, wecould see the ladders in the TAM 10uM , TAM 20uM and TAM20uM+CLC-3Antisense strand because of the DNA fragments,but thecontrol had no.From the date,we could find that TAM could enhance theapoptosis induction in the MCF-7 cells and this effect could be improved withthe concentration increasing. CLC-3 antisense strand could increase theeffects of TAM either.Part four.Study of the molecular mechanisms and the effects of mitochondriaand chloride channel CLC-3 on apoptosis induced by tamoxifen in the MCF-7cell line.By the method of cellular immunohistochemical staining with the estrogenreceptorα (ERα ) antibody ,we found that in MCF-7 cells of TAM20uM,theexpression of ERα vs the control reduced obviously,but the CLC-3antisensehad no clearly change .This could determind that TAM could inhibit theexpression of the ERα in MCF-7 cells,but CLC-3 antisense strand had nothis effect.With the RT-PCR method,we detected the mRNA of CLC-3,BCL-2and Bax,we found that TAM and/or CLC-3antisense made the low expressionof CLC-3,BCL-2 but the high expression of Bax,and the both had the jointaction.With the DCFH-DA fluorescent staining,we could detect the ROS in thecells,and we found the TAM20u and TAM20u+ CLC-3 antisense strand couldincrease the ROS in the cells.The both had the joint action.With the method ofisolation of mitochondria we found that TAM20u and TAM20u+CLC-3antisense strand gave rise to the decreased of GSH in themitochondria ,and the both had the joint action..By the method of western blotwe found that TAM20u and TAM20u+ CLC-3 antisense strand brought aboutcytochrome C increased in the cytoplasm, but there was no clear differencebetween the two groups.From the above date and discussion we can drew the following conclusions:1,There are a obvious difference between the expression of CLC-3 in thebreast cancer and the benign tissue P<0.05.This implies that the CLC-3 hassome pathological and physiological significance in the process of breastcancer`s genesis and growth2,The proliferation of MCF-7 cells treated with the CLC-3 antisense isnotinhibited significantly.This may be relate with the concentration which weapplied in the study or the chloride channel CLC-3 is not necessary in the cell.CLC-3 antisense srand can enhances the apoptosis induction of the TAM in theMCF-7 cells, and this effect can be improved with the TAM concentrationincreasing.This can be refered by the clinical Endocrine therapy and thebiotherapy3,Mitochondria plays a key role in the process of cellular apoptosis, TAMcan inhibit the estrogen receptor of the mitochondria.If the mitochondrialestrogen receptor directly take part in the expression of the mitochondrial DNAinduced by the estrogen, there will be a target point to regulate, prevent andtreat with the tumor in the signal pathway of mitochondrial estrogen receptor4,TAM maybe induce the apoptosis through the estrogen receptor of themitochondria and the chloride channel CLC-3 pathway,but mainly through theestrogen receptor pathway. Proper enhancement of the chloride channel`santagonism can improve the inducement capacity of the apoptosis. This impliesthat there are some many pathways and mechanisms involved together in theprocess of tumor growth, the breast cancer as the hormonal dependent tumordoes not exclude.