P-glycoprotein is not involved in pathway of anti-Fas/Fas-induced apoptosis in KBv200 cells

Abstract

Aim: To verify whether P-glycoprotein (P-gp) could induce cell resistance to apoptosis by inhibiting caspase-8 and caspase-3.

Methods: Human KB cells, either drug-sensitive or with multidrug resistance (MDR) phenotype caused by overexpression of P-gp (KBv200 cells), were treated with anti-Fas (CH-11 monoclonal antibody) to induce apoptosis. Cytotoxicity was detected by MTT assay. Symptoms of cell death were assessed by morphological observation after Hoechst33258 staining, activation of caspase-8 and caspase-3 was measured by Western blotting.

Results: Compared with KB cells, the resistance of KBv200 cells to VCR (vincristine) was about 51-fold higher. Anti-Fas (CH-11) induced cytotoxicity and apoptosis in both sensitive KB cells and MDR phenotype KBv200 cells. The IC50 of CH-11 in KB cells was similar to that in KBv200 cells. CH-11 induced similar apoptotic rates in both KB cells and KBv200 cells, which could be classified as caspase-dependent apoptotic pathway. Verapamil (VRP) did not affect CH-11-mediated apoptosis in KBv200 cells.

Conclusion: Expression of P-glycoprotein does not induce resistance to caspase-8 and -3 activation or anti-Fas-induced cell apoptosis.

Figure 1

Western blot analysis of P-gp and Fas expression in KBv200 cells and KB cells.

Figure 2

VRP enhanced cytotoxicity of VCR to KB cells (A) and KBv200 cells (B).

Figure 3

VRP did not enhance cytotoxicity of anti-Fas to KB cells (A) and KBv200 cells (B).

Figure 4

Cell morphology and DNA fragmentation.

Figure 5

Western blot analysis of caspase-8 and -3 in KBv200 cells after treatment with anti-Fas antibody (CH-11) in the presence (+) or absence (-) of VRP for 24 h at 37 °C.