| BackgroundIt has been verified that CD147, a member of immunoglobulin surperfamily, is overexpressed in the drug-resistant breast cancer cells and mediates multidrug resistance (MDR) via multidrug resistance-related protein such as CD44, monocarboxylate transporters (MCTs). ABCG2, also named as BCRP, is originally cloned from drug-resistance breast cell line MCF-7/AdrVp and regulates MDR through expelling a variety of chemotherapeutic drugs from cells. Recent study demonstrates that CD147can form a complex with ABCG2on the cell membrane to modulate drug sensitivity in primary effusion lymphoma. However, whether these two molecules regulate each other in the breast cancer cell and result in MDR is not clear. The aim of the present study is to determine whether CD147could regulate ABCG2’s expression and function, and to reveal its exact regulatory mechanism and role in breast cancer chemo-resistance to provide a new target to reverse breast cancer chemo-resistance.Objective1. Investigate whether CD147could regulate ABCG2’s expression and function. 2. Investigate the mechanism of CD147involving in regulating ABCG2’ function.3. Investigate whether CD147involving in regulating ABCG2contributes to breast cancer chemoresistance.Methods1. We established four MCF-7cell lines transfected with CD147and/or ABCG2, MCF-7/Control, MCF-7/CD147, MCF-7/ABCG2, and MCF-7/CD147+ABCG2(briefly named as MCF-7/Con, MCF-7/C, MCF-7/B and MCF-7/C+B).2. Western Blot and RT-PCR were used to determine the expressions of CD147and ABCG2in these four cell lines. And the correlation between expressions of ABCG2and CD147in these four cell lines was analyzed.3. MTT assay was used to measure the sensitivities of these transfected cells to epirubicin (an ABCG2substrate) and5-fluorouracil (not ABCG2substrate).4. Chose proper concentration of mitoxantrone to induce ABCG2’s expression of MCF-7/Con and MCF-7/C cells, and analyzed the expressions of ABCG2protein and RNA between these two cell lines at each time points.5. ABCG2homodimers were detected by Western Blot and its expression levels in each cell lines was analyzed. 6. We used immunofluorescence to observe CD147and ABCG2’s cellular localization and coexistence in four cell lines and analyzed CD147’s impact on ABCG2cellular location.7. Co-immunoprecipitation was used to confirm whether CD147could form a complex with ABCG2.8. We detected the expression levels of these two molecules in clinical samples of chemotherapy-sensitive/resistant breast invasive ductal cancer and normal breast tissue by immunohistochemistry and analyzed correlation between these two molecules and their relationships with drug resistance.Results1. We successfully established MCF-7cells transfected with CD147and/or ABCG2(MCF-7/Con, MCF-7/C, MCF-7/B, MCF-7/C+B) for further investigation.2. Compared with MCF-7/B, there was a significant increase in ABCG2protein in MCF-7/C (P<0.05), but the mRNA levels of ABCG2in these two cells had no significant difference.3. The sensitivities to epirubicin between MCF-7/C+B and MCF-7/B, MCF-C and MCF-7/Con, were significantly different (P<0.05). The IC50values were17.09±1.7uM,10.13±0.55uM and2.76±0.07uM,0.98±0.06uM respectively. There was no significant difference between MCF-7/Con and MCF-7/B, MCF-7/C and MCF-7/C+B in sensitivities to5-fluorouracil (P>0.05). The IC50values were respectively413.95±17.55uM,478.94±42.13uM and1275.4±19.89uM,1339.33±37.68uM. These indicated that CD147could enhance breast cancer cells’chemoresistance to epirubicin via ABCG2.4. Under the mitoxantrone induction, the expression of ABCG2protein and mRNA of MCF-7/Con and MCF-7/C cells displayed a significant time-dependent increase and the protein levels (P<0.05) at all time points tested between these two cell lines had significant differences. But the RNA levels of ABCG2in MCF-7/Con and MCF-7/C had no difference at the corresponding time points. These indicated that CD147could regulate ACBG2protein expression without affecting its RNA level.5. ABCG2homodimers was much higher in MCF-7/C+B than that in MCF-7/B (P<0.001), which supported that CD147could affect ABCG2’s dimerization.6. More ABCG2proteins were seen localized in the cytoplasm in MCF-7/C+B than in MCF-7/B. CD147and ABCG2displayed a similar colocalization on membrane in MCF-7/C+B.7. Co-immunoprecipitation showed CD147formed a complex with ABCG2in MCF-7/C+B.8. Expression levels of CD147and ABCG2in chemotherapy-resistant group were higher than in chemotherapy-sensitive group. More ABCG2proteins localized on the cell membrane in the chemotherapy- resistant group. The expression of CD147significantly correlated with that of ABCG2(P<0.05).Conclusion1. In breast cancer cells, CD147regulates ACBG2’protein expression and function, and may influence ABCG2’s transport function via affecting its cellular localization and dimerization.2. The expression of CD147significantly correlates with that of ABCG2in breast invasive ductal cancer and they jointly regulate chemotherapeutic resistance of breast cancer. |
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