Galectin-1 Knockdown Improves Drug Sensitivity Of Breast Cancer Through Inhibiting The Raf-1/AP-1 Signaling Pathway

Galectin-1(Gal-1),a member of the galectin family of carbohydrate binding proteins,plays a pivotal role in various cellular processes of tumorigenesis.The regulatory effect of Gal-1 on multidrug resistance(MDR)breast cancer cells is still unclear.This research was divided into three parts: Part One: The expression of Gal-1 and MDR1 in human breast cancer tumor tissues and cell lines;Part Two: The effects of Gal-1 knockdown on sensitivity to PTX and ADR in breast cancer resistant cell lines MCF-7/PTX and MCF-7/ADR;Part Three: The potential mechanism by which Gal-1 knockdown enhances sensitivity to PTX and ADR in breast cancer resistant cell lines MCF-7/PTX and MCF-7/ADR.Part One: The expression of Gal-1 and MDR1 in human breast cancer tumor tissues and cell lines Methods1.The mRNA expression levels of Gal-1 and MDR1 in human breast cancer tissue was measured by using real-time PCR analysis.2.Real-time PCR was performed to measure the mRNA expression of Gal-1 and MDR1 in human breast epithelial cell line MCF-10 A,breast cancer cell line MCF-7 and MCR-7 derived Adriamycin(ADR)resistant cell line MCF-7/ADR and paclitaxel(PTX)resistant cell line MCF-7/PTX.3.Western blot analysis was conducted to examine the protein levels of Gal-1 and P-glycoprotein(P-gp)in human breast epithelial cell line MCF-10 A,breast cancer cell line MCF-7 and MCR-7 derived Adriamycin(ADR)resistant cell line MCF-7/ADR and paclitaxel(PTX)resistant cell line MCF-7/PTX.Results1.qRT-PCR results indicated that Gal and MDR1 were significantly upregulated in breast tumor tissues compared with that in normal tissues.In addition,the mRNA expressions of Gal-1 in breast cancer patients were positively correlated with MDR1.2.The mRNA expression levels of Gal and MDR1 were significantly increased in breast cancer cell line MCR-7,compared with that in human breast epithelial cell line MCF-10 A.Addtionally,the mRNA expression levels of Gal and MDR1 in MCF-7/PTX and MCF-7/ADR cells were strikingly higher than that in MCF-7 cells.3.The protein levels of Gal-1 and P-gp were significantly increased in breast cancer cell line MCR-7,compared with that in human breast epithelial cell line MCF-10 A.Addtionally,the mRNA expression levels of Gal and MDR1 in MCF-7/PTX and MCF-7/ADR cells were strikingly higher than that in MCF-7 cells.Part Two: The effects of Gal-1 knockdown on sensitivity to PTX and ADR in breast cancer resistant cell lines MCF-7/PTX and MCF-7/ADR.Methods1.MTT assay was performed to determine the survival rates of breast cancer cell line MCF-7 and PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR.2.Western blot analysis was carried out to detect the expression of Gal-1 in PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR transfected with si RNA-Gal-1 and si RNA-Gal-2.3.MTT proliferation assay was conducted to determine the effect of Gal-1 knockdown on the cell viability of PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR.4.The effect of Gal-1 knockdown on the cell apoptosis of PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR was determined by the flow cytometry analysis.Results1.The results of MTT assay showed that the cell survival rates of PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR were significantly increased,as compared to the breast cancer cell line MCF-7.2.Western blot analysis confirmed Gal-1 si RNA1 and Gal-1 si RNA2 significantly decreased the levels of Gal-1 in PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR,as compared to Con si RNAs.3.MTT assay showed that Gal-1 knockdown or(PTX or ADR)treatment resulted in an obvious decrease in cell viability;however,combination of Gal-1 knockdown with PTX or ADR resulted in a lower cell viability than chemotherapy group alone in MCF-7/PTX and MCF-7/ADR cells.4.The results of flow cytometry analysis indicated that Gal-1 knockdown or(PTX or ADR)treatment led to a marked increase in cell apoptosis;however,combination of Gal-1 knockdown with PTX or ADR induced a higher cell apoptosis than chemotherapy group alone in MCF-7/PTX and MCF-7/ADR cells.Part Three: The potential mechanism by which Gal-1 knockdown enhances sensitivity to PTX and ADR in breast cancer resistant cell lines MCF-7/PTX and MCF-7/ADR Methods1.Western blot analysis was used to detect the expression of P-gp protein level in breast cancer cell lines MCF-7/PTX and MCF-7/ADR transfected with si RNA-Gal-1 and pc DNA-MDR1.2.MTT assay was performed to detect the effect of Gal-1 knockdown or MDR1 overexpression on the cell viability of PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR.3.The effect of Gal-1 knockdown or MDR1 overexpression on the cell apoptosis of PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR was determined by the flow cytometry analysis.4.The levels of p-Raf-1(Ser338),Raf-1,p-c-Jun(Ser73),c-Jun and c-Fos were determined by western blot analysis to investigate the effect of Gal-1 knockdown on the Raf-1/AP-1 signaling pathway.5.Western blot analysis was performed to invesitagate the effect of Raf-1 si RNAs and Raf-1 inhibitor GW5074 on the expression levels of related proteins of Raf-1/AP-1 signaling pathway and P-gp protein in breast cancer cell lines MCF-7/PTX and MCF-7/ADR.6.MTT assay was performed to detect the effect of Raf-1 si RNA1/GW5074 or Raf-1 si RNA1/GW5074 + pc DNA-MDR1 treatment on the cell viability of PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR.7.The effect of Raf-1 si RNA1/GW5074 or Raf-1 si RNA1/GW5074 + pc DNA-MDR1 treatment on the cell apoptosis of PTX and ADR resistant breast cancer cell lines MCF-7/PTX and MCF-7/ADR was determined by the flow cytometry analysis.Results1.The western blot results indicated that Gal-1 si RNA1 and Gal-1 si RNA2 both significantly decreased the levels of P-gp in MCF-7/PTX and MCF-7/ADR cells compared with corresponding control groups,while MDR1 overexpression alleviated the inhibition effects of Gal-1 si RNA1 or Gal-1 si RNA2 on P-gp.2.The MTT assay showed that combination of Gal-1 si RNA1 and PTX or Gal-1 si RNA1 and ADR strikingly suppressed the relative cell viability of MCF-7/PTX and MCF-7/ADR cells compared to(Con si RNA + drug)group,whereas MDR1 overexpression reversed these negative effects.3.Flow cytometry assay indicated that Gal-1 si RNA1 and PTX or Gal-1 si RNA1 and ADR obviously induced apoptosis of MCF-7/PTX and MCF-7/ADR cells compared with(Con si RNA + drug)group,while MDR1 overexpression attenuated the induction effects.4.Western blot analysis indicated Gal-1 si RNA1 and Gal-1 si RNA2 significantly decreased the levels of p-Raf-1(Ser338),p-c-Jun(Ser73),c-Jun and c-Fos in MCF-7/PTX and MCF-7/ADR cells,as compared with Con si RNA group.5.The western blot results showed that Raf-1 si RNA1 and Raf-1 si RNA2 dramatically decreased the levels of P-gp,p-Raf-1(Ser338),Raf-1,p-c-Jun(Ser73),c-Jun and c-Fos in MCF-7/PTX cells,and Raf-1 inhibitor GW5074 obviously reduced the levels of P-gp,p-Raf-1(Ser338),p-c-Jun(Ser73),c-Jun and c-Fos in MCF-7/ADR cells.6.The results of MTT assay showed that combination of Raf-1 si RNA1 and PTX or GW5074 and ADR dramatically inhibited cell viability in MCF-7/PTX and MCF-7/ADR cells compared with PTX or ADR treatment group,whereas MDR1 overexpression abated these effects.7.Flow cytometry showed that combination of Raf-1 si RNA1 and PTX or GW5074 and ADR significantly promoted apoptosis in MCF-7/PTX and MCF-7/ADR cells compared with PTX or ADR treatment group,while MDR1 overexpression overturned these effects.Conclusions1.Gal-1 and MDR1 levels were both upregulated in breast tumor tissues and cell lines.2.Gal-1 knockdown enhanced sensitivity to PTX and ADR in MCF-7/PTX and MCF-7/ADR cells.3.Overexpression of MDR1 reduced the sensitivity to PTX and ADR induced by Gal-1 knockdown in MCF-7/PTX and MCF-7/ADR cells.4.Gal-1 knockdown enhanced sensitivity to PTX and ADR by reducing P-glycoprotein expression through inhibiting the Raf-1/AP-1 signaling pathway.