Down-regulation Of Annexin A5 With ShRNA To Reverse Adriamycin Resistant In MCF-7/ADR Cells

Background:Breast cancer is one of the most common malignant tumors among women,caused serious damage to women’s health, accounting for 7%-10% of the total cancer cases,accounting for 34.17% mortality rate of diagnosis. Advances in early detection and improved treatment largely decreased the breast cancer death rates.Chemotherapy plays an important role in the treatment of breast cancer. However,resistance to chemotherapeutic agents is one of main causes in tumor chemotherapy failure. Tumor cells become resistance to chemotherapy drugs can be divided into inherent drug resistance and acquired drug resistance. Inherent resistance refers to tumor cells not sensitive to anti-cancer drugs intrinsically. Acquired resistance refers to tumor cells sensitive to chemotherapy initially, but after treatment for some cycles,drug efficacy decreased gradually, then tumor cells become more and more tolerant to drugs, and finally tumor recurrence can be found. According to drug-resistant phenotype, cells can be divided into primary drug resistance(PDR) and multidrug resistance(MDR). PDR refers to tumor cells only resistant to a single cytotoxic drug after exposure to it. MDR means that tumors acquire cross chemoresistance to a number of chemotherapeutic agents with unrelated function and structure after exposure to a single cytotoxic drug. MDR is the main reason for the failure in tumor chemotherapy, and is also an urgent problem to be solved. The primary mechanisms of MDR can be divided into two aspects: pump resistance and non-pump resistance.The generation of drug efflux mainly because of the abnormal expression of ABC transporter protein family. They can pump the drug from the intracellular to the extracellular by using the energy of ATP hydrolysis,resulting in the decrease of intracellular drug concentration, so that the tumor cells is not sensitive to chemotherapy drugs. P-glycoprotein(P-gp) is one of the most common ABC transporters, other common ABC transporters are Multidrug resistance-associated protein(MRP) and breast cancer resistance protein(BCRP). The main reason of non-pump resistance is anti-death defense, cells can active anti-apoptotic program through various ways, enhance the survival of tumor cells, delay apoptosis, obtain apoptosis resistance, leading to the formation of multidrug resistance of tumor cells.The clinical application of reversal agents has not got significant progress,because of reversal agents focus on one of tumor multidrug resistance factors., thus they can’t reverse tumor drug resistance completely. Therefore it is essential to discover new multi-drug resistance related proteins, and provide new therapeutic targets.Annexins are thought of as calcium-dependent phospholipid-binding proteins,regulation of membrane organization, membrane traffic, membrane cytoskeleton linkage, and ion conductance across membranes. Recently, with the thorough study of Annexins, researchers found that Annexin not only play important role in the development of tumor, but also related to the drug resistance. For example, Annexin A1 was reported over-expression in ovarian cancer cells, Down-regulation of Annexin A1 restored the chemosensitivity of ovarian cancer cells. The expression of Annexin A2, A3, A4, and A11 were abnormal in resistant tumor cell strains, down or up regulation of Annexin expression, can reverse drug chemosensitivity t o a certain extent. Annexin A5 is one of the most widespread calcium-dependent phospholipid-binding proteins. Recently, Annexin A5 was shown up-regulated in several resistant cell strains such as CNE2/c DDP, SGC-7901/DDP, and MCF-7/ADR cells. However, Annexin A5 remained unchanged in MCF-7/Vp, MCF-7/MX, and Mel R/MCF-7.RNA interference is a double-stranded RNA, which targets homologous gene sequences, and induces a specific post-transcriptional gene silencing. It is an effective way to study gene function.MCF-7/ADR cell strain was developed via stepwise increments of concentration of ADR.Objective:1. Compare the difference expression of Annexin A5 between Breast cancer sensitive MCF-7 cells and Breast cancer adriamycin resistant MCF-7/ADR cells.Down-regulation of Annexin A5 expression in MCF-7/ADR cells using sh RNA, MTT testing whether adriamycin(ADR) resistance can be reversed or not.2. In order to investigated the mechanism of down-regulation of Annexin A5 expression reverseing drug susceptibility, After down-regulation Annexin A5 expression in MCF-7/ADR cells, the level of MDR1 m RNA, P-gp expression and activity were detected. Meanwhile, apoptosis-associated proteins PARP were also measured.Methods:1. Western-Blot and q RT-PCR were applied to compare the difference of m RNA and protein expression of Annexin A5 between Breast cancer sensitive MCF-7 cells and Breast cancer adriamycin resistant MCF-7/ADR cells.2. The stable transfection of MCF-7/ADR with p LKO.1-sh Annexin A5 construct was performed as follows:5-CCGGGCTGGAATTGATGAAGCTCAACTCGAGTTGAGCTTCATCAATTCCAGCTTTTT-3(Annexin A5-1),5-CCGGCCATGATTAAGGGAGATACATCTCGAGATGTATCTCCCTTAATCATGGTTTTT-3(Annexin A5-2),5-CCGGCGCGAGACTTCTGGCAATTTACTCGAGTAAATTGCCAGAAGTCTCGCGTTTTT-3(Annexin A5-3).then four cell strains were gained: MCF-7/ADR/si-A5-1, MCF-7/ADR/si-A5-2,MCF-7/ADR/si-A5-3, and MCF-7/ADR/si-A5-CTR. The level of Annexin A5 m RNA and protein expression were quantified by q RT-PCR and Western blot respectively to compare the efficiency.3. MTT assay was performed in MCF-7/ADR/si-A5-2 cells to investigate the effect of silencing of Annexin A5 on the chemosensitivity of MCF-7/ADR cells. Flow cytometry, q RT-PCR and Western blot were used to detect whether Annexin A5down-regulation influenced the MDR1 m RNA and P-gp expression or not.4. By the use of Western blot analysis of cleaved PARP, cell apoptosis was measured to investigate whether down-regulation of Annexin A5 expression influenced the susceptibility of MCF-7/ADR cells to apoptosis or not.Result:1. The expression level of Annexin A5 m RNA in MCF-7/ADR cells was 3-fold higher than that in MCF-7 cells and the Annexin A5 protein level in MCF-7/ADR was significantly higher than that in MCF-7 cells.2. Three targeting sequences of sh RNA were designed sh RNAs of Annexin A5 and negative control were cloned into vector PLKO.1 and then four cell strains were gained: MCF-7/ADR/si-A5-1, MCF-7/ADR/si-A5-2, MCF-7/ADR/si-A5-3, and MCF-7/ADR/si-A5-CTR. The level of Annexin A5 m RNA and protein expression were quantified by q RT-PCR and Western blot respectively to compare the efficiency.The results showed that Annexin A5 expression was successfully down-regulated at m RNA and protein level. The lowest levels of Annexin A5 m RNA were detected in MCF-7/ADR/si-A5-2 cells.3. Drug sensitivity of MCF-7/ADR cells was significantly increased by sh RNA transfection. The IC50 for Adriamycin in MCF-7/ADR/si-A5-2 cells was three times increased(p<0.05). There was no significant difference in chemosensitivity between MCF/ADR cells and MCF-7/ADR/si-A5-CTR cells(p>0.05).4. The expression level of P-gp was analyzed by flow cytometry. The expression level of P-gp in MCF-7/ADR/si-A5-2 cells was decreased by 9.11%, while the results of q RT-PCR displayed that MCF-7/ADR/si-A5-2, MCF-7/ADR/si-CTR, and MCF-7/ADR had the same amount of MDR1 levels. The intensity of fluorescence in cells was detected by Confocal laser scanning microscopy. MCF-7/ADR/si-A5-2 cells have strong fluorescence intensity than that in MCF-7/ADR/si-CTR cells and MCF-7/ADR cells.5. Cells were treated with Adriamycin for 24 h, and significantly higher levels of cleaved-PARP were observed. The level of cleaved-PARP was increased in dose-dependent manner in MCF-7/ADR cells.Conclusion:1. The Annexin A5 protein level in MCF-7/ADR was significantly higher than that in MCF-7 cells.2. Silencing of Annexin A5 restored the chemosensitivity of MCF-7/ADR cells.3. The mechanism of down-regulation of Annexin A5 expression increasing drug susceptibility include two aspects: firstly, silencing of Annexin A5 expression inhibited the P-gp expression and activity. Secondly, silencing of Annexin A5 expression affected the expression of apoptosis-associated proteins, and then promoted apoptosis in MCF-7/ADR cells.