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Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells.

Zhang HC, Zhang F, Wu B, Han JH, Ji W, Zhou Y, Niu RF - Cancer Biol Med (2012)

Bottom Line: The expression of P-gp was detected by Western blot.There was a close interaction between Anxa2 and P-gp.The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Public Laboratory, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.

ABSTRACT

Objective: To explore the interaction of Anxa2 with P-Glycoprotein (P-gp) in the migration and invasion of the multidrug-resistant (MDR) human breast cancer cell line MCF-7/ADR.

Methods: A pair of short hairpin RNA (shRNA) targeting P-gp was transfected into MCF-7/ADR cells, and monoclonal cell strains were screened. The expression of P-gp was detected by Western blot. Transwell chambers were used to observe the cell migration capacity and invasion ability. The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses.

Results: P-gp expression was significantly knocked down, and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells (P<0.05). There was a close interaction between Anxa2 and P-gp.

Conclusions: MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities. The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells. The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells.

No MeSH data available.


Related in: MedlinePlus

Screening of stable clones down-expressing P-gp. A: Western blot analysis of P-gp expression in MCF-7/ADR cells, 3 stable clones of shMDR1/MCF-7/ADR cells and control cells (transfected with a shRNA plasmid containing scrambled targeting sequence). P-gp expression was successfully inhibited in the knockdown experiments. B: The 3 P-gp knockdown clones were significantly more sensitive to adriamycin treatment than the MCF-7/ADR wild-type and control cells. The experiments were repeated at least 3 times. The statistical significance was assessed by one-way ANOVA, *P<0.05 vs. control.
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f1: Screening of stable clones down-expressing P-gp. A: Western blot analysis of P-gp expression in MCF-7/ADR cells, 3 stable clones of shMDR1/MCF-7/ADR cells and control cells (transfected with a shRNA plasmid containing scrambled targeting sequence). P-gp expression was successfully inhibited in the knockdown experiments. B: The 3 P-gp knockdown clones were significantly more sensitive to adriamycin treatment than the MCF-7/ADR wild-type and control cells. The experiments were repeated at least 3 times. The statistical significance was assessed by one-way ANOVA, *P<0.05 vs. control.

Mentions: Three stable transfectants of shMDR1/MCF-7/ADR cells were screened and maintained. As shown in Figure 1A, Western blot analysis was used to screen the stable clones for knocking down P-gp expression. The P-gp expression levels showed a decrease of more than 10 folds in the three selected clones compared with MCF-7/ADR and control cells. The MTT assay was used to evaluate the adriamycin cytotoxic effect on the screened stable transfectants and control cells. As depicted in Figure 1B, the transfectants of shMDR1/MCF-7/ADR showed significantly different IC50 values of adriamycin (the IC50 values of clones 1 to 3 were 11.25±2.96, 13.22±3.68, and 2.13±4.72) compared with MCF-7/ADR cells (the IC50=34.29±2.47) and control cells (IC50=30.80±5.05) (P<0.05).


Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells.

Zhang HC, Zhang F, Wu B, Han JH, Ji W, Zhou Y, Niu RF - Cancer Biol Med (2012)

Screening of stable clones down-expressing P-gp. A: Western blot analysis of P-gp expression in MCF-7/ADR cells, 3 stable clones of shMDR1/MCF-7/ADR cells and control cells (transfected with a shRNA plasmid containing scrambled targeting sequence). P-gp expression was successfully inhibited in the knockdown experiments. B: The 3 P-gp knockdown clones were significantly more sensitive to adriamycin treatment than the MCF-7/ADR wild-type and control cells. The experiments were repeated at least 3 times. The statistical significance was assessed by one-way ANOVA, *P<0.05 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643660&req=5

f1: Screening of stable clones down-expressing P-gp. A: Western blot analysis of P-gp expression in MCF-7/ADR cells, 3 stable clones of shMDR1/MCF-7/ADR cells and control cells (transfected with a shRNA plasmid containing scrambled targeting sequence). P-gp expression was successfully inhibited in the knockdown experiments. B: The 3 P-gp knockdown clones were significantly more sensitive to adriamycin treatment than the MCF-7/ADR wild-type and control cells. The experiments were repeated at least 3 times. The statistical significance was assessed by one-way ANOVA, *P<0.05 vs. control.
Mentions: Three stable transfectants of shMDR1/MCF-7/ADR cells were screened and maintained. As shown in Figure 1A, Western blot analysis was used to screen the stable clones for knocking down P-gp expression. The P-gp expression levels showed a decrease of more than 10 folds in the three selected clones compared with MCF-7/ADR and control cells. The MTT assay was used to evaluate the adriamycin cytotoxic effect on the screened stable transfectants and control cells. As depicted in Figure 1B, the transfectants of shMDR1/MCF-7/ADR showed significantly different IC50 values of adriamycin (the IC50 values of clones 1 to 3 were 11.25±2.96, 13.22±3.68, and 2.13±4.72) compared with MCF-7/ADR cells (the IC50=34.29±2.47) and control cells (IC50=30.80±5.05) (P<0.05).

Bottom Line: The expression of P-gp was detected by Western blot.There was a close interaction between Anxa2 and P-gp.The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Public Laboratory, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.

ABSTRACT

Objective: To explore the interaction of Anxa2 with P-Glycoprotein (P-gp) in the migration and invasion of the multidrug-resistant (MDR) human breast cancer cell line MCF-7/ADR.

Methods: A pair of short hairpin RNA (shRNA) targeting P-gp was transfected into MCF-7/ADR cells, and monoclonal cell strains were screened. The expression of P-gp was detected by Western blot. Transwell chambers were used to observe the cell migration capacity and invasion ability. The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses.

Results: P-gp expression was significantly knocked down, and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells (P<0.05). There was a close interaction between Anxa2 and P-gp.

Conclusions: MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities. The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells. The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells.

No MeSH data available.


Related in: MedlinePlus