Involvement Of SATB1in Regulation Of Multidrug Resistance In Human Breast Cancer And The Related Mechanisms Exploration

IntroductionAs a major adjuvant therapy employed before and/or after operative treatment for breast cancer, chemotherapy utilizes chemical agents to kill tumor cells or inhibit the proliferation of tumor cells to achive theraputic effects. But chemotherapy cannot prevent recurrence and metastasis of breast cancer in every individual patient, as there existing primary drug resistance (PDR) and secondary drug resistance (SDR, or multidrug resistance, MDR). PDR means the innate capability of drug resistance to a given chemical agent and MDR means the acquired drug resistance to many kinds of chemical agents with different structures and functions after exposured to previous chemotherapeutics. The multidrug resistance gene1(MDR1) encodes P-glycoprotein (P-gp), a transmembrane,170kD, ATP-dependent transporter, which can pump chemotherapeutic agents with diverse structures and functions from intracellular to extracellular, and decrease the intracellular drug accumulation to induce MDR. This is the classical molecular mechanism of MDR, which involves a complex and multifactorial process. Also, the suppression of apoptosis from stimulating facts and the cancer stem cells characteristics help cancer cells to escape the killing effects of chemotherapeutic agents.As one kind of tissue-specific nuclear matrix attachment regions (MARs) binding proteins, Special AT-rich sequence-binding protein1(SATB1) plays a role as genome organizer. SATB1facilitate breast cancer cells to infiltrate and metastase by remodeling the structure of chromosomes and changing the gene expression. SATB1is closely correlated with the growth, metastasis and prognosis of breast cancer.Our previous works indicated that there was a functional linkage between tumor invasion and MDR in breast cancer. But there is no description about the relation between SATB1and MDR. So our current works aim to explore the relationship between SATB1and MDR, and investigate the mechanisms underlying the phenomenon. Part Ⅰ The relationship between SATBl expression and multidrug resistance phenotype in human breast cancer cellsObjectives To observe the relations between SATB1and MDR, providing a base for the further research.Methods Real-time PCR and western blot were used to analyze gene transcription and protein expression of SATB1in MCF7and MCF7/ADR cells. Western blot was used to detect the SATB1expression in MCF7cells during the inducing of MDR by ADM and in MCF7, Hs578T, MX-1cells along with their drugresistant counterparts MCF7/mitoxantrone (MCF7/MX), Hs578T/doxorubicin (Hs578T/DOX), MX-1/Taxol (MX-1/T) cells. pEGFP-N1-SATB1-GFP eukaryon expression plasmid and SATB1siRNA duplex oligoribonucelotides were transfected into MCF7and MCF7/ADR cells, respectively. MTT assay was used to determine the inhibitory effects of various concentrations of ADM on MCF7/ADR-SATB1RNAi. MCF7, MCF7/ADR and their SATB1-related transfected counterparts were implanted into nude mice. ADM was injected into mice through caudal vein. The exact length and width of implanted tumors were mesured as the relative growth rate to valuate the sensitiveness of tumor cells to ADM in vivo.Results The SATB1expressions at mRNA and protein levels in MCF7/ADR cells were both higher than thoses in MCF7cells. The drug sensitive cells MCF7, Hs578T and MX-1expressed low level of SATB1than their drug resistant counterparts. The level of SATB1expression increased along with the quantity of ADM during the induction of multidrug resistant subline of MCF7cells. The inhibitory effects of various concentration of ADM on MCF7/ADR-SATB1RNAi cells were obviously higher than those in MCF7/ADR-con cells. Mouse model showed that MCF7-SATB1and MCF7/ADR-con cells showed higher drug resistance than MCF7-con and MCF7-SATB1-siRNA cells in vivo.Conclusions SATB1was closely correlated with the multidrug resistant phenotype in human breast cancer cells. Part Ⅱ Involvement of P-gp in SATB1-associated multidrug resistance in human breast cancerObjectives To investigate the regulating action of SATB1on multidrug resistant phenotype mediated by transmembrane transporter in human breast cancer.Methods pEGFP-N1-SATB1-GFP eukaryon expression plasmid and SATB1siRNA duplex oligoribonucelotides were transfected into MCF7and MCF7/ADR cells, respectively. The expression of P-gp, BRCP, and MRP1were determined by western blot analysis. MDR1mRNA level was evaluated by quantitative RT-PCR. MTT assay was used to determine the inhibitory effects of P-gp-related drugs and non-P-gp-related drugs in vitro. Laser cytometry was used to determine the intracellular concentration of ADM to evaluate the pumping effects of P-gp. Having been incubated with P-gp monoclonal antibody, MCF7-SATB1cells were treated with ADM. MTT assay was used to evaluate the sensitivity of cells to ADM (P-gp substrate) and MTX (non-P-gp substrate). Immunohistochemical analysis was used to determine the correlation between SATB1and P-gp expression in breast cancer specimens.Results P-gp expression levels in MCF7-SATB1and MCF7/ADR-con cells were higher than those in MCF7-con and MCF7/ADR-SATB1RNAi cells. Such changes did not happen to MRP1and BCRP. MCF7-SATB1and MCF7/ADR-con cells had higher MDR1mRNA level than MCF7-con and MCF7/ADR-SATB1RNAi cells. MCF7-SATB1cells showed significantly increased resistant to the P-gp-related drugs, but slightly to the non-P-gp-related drugs, compared with MCF7cells. MCF7/ADR-SATB1RNAi showed significantly increased sensitive to all drugs except BLM and CDDP, compared with MCF7/ADR-con. Both MCF7-SATB1and MCF7/ADR-con cells have significantly lower intracellular ADM concentrations than MCF7-con and MCF7/ADR-SATB1RNAi cells. P-gp mAb could partially reverse ADM resistance, but exerted no effects on MTX resistance in MCF7-SATB1cells. Immunohistochemical analysis showed that the expression of SATB1was highly correlated with P-gp in most breast carcinoma tissues.Conclusions SATB1enhance the drug resistance of breast cancer by upregulating the expression of P-gp. Part III Effects of SATBl on the breast cancer stem cell phenotype CD44+/CD24-and apoptosis induced by ADMObjectives To clarify the effects of SATB1on drug resistences mediated by cancer stem cells and involvement of SATB1in antiapoptosis, explore the underlying molecular mechanism.Methods SATB1siRNA duplex oligoribonucelotides were transfected into MCF7/ADR and MDA-MB-231cells. pEGFP-N1-SATB1-GFP eukaryon expression plasmids were transient transfected into MCF7. Flow cytometry assay was used to evaluate the effects of SATB1on the expression of cancer stem cells phenotype CD44+/CD24-. MCF7, MCF7/ADR and their SATB1-related transfectants were treated with ADM, western blot was used to analysis the expression of SATB1, for the purpose to understand the cleavage kinetics of SATB1, flow cytometry and caspase activity assay were used to analysis the apoptosis induced by ADM.Results The population of breast cancer cells with CD44+/CD24-phenotype was significantly increased by inducing SATB1into MCF7cells. Conversely, it was obviously decreased by knocking down SATB1in MDA-MB-231cells. In MCF7/ADR-SATB1RNAi cells, cleavage of SATB1occurred after treatment with ADM and progressed with ongoing apoptosis.The ADM-induced caspase activity was largely reduced in MCF7cells with ectopic SATB1expression, but significantly higher in SATB1-depleted MCF7/ADR cells, compared with control cells.Conclusions SATB1-related MDR may partially due to the involvement of SATB1in formation and maintaining of the cancer stem cell characteristics and the anti apoptosis effects induced by SATB1.