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.

Key Words: P-glycoprotein, Apoptosis, CH-11, Fas, Verapamil

INTRODUCTION

Multidrug resistance (MDR) is a kind of resistance of cancer cells to multiple classes of chemotherapeutic drugs. MDR is a major obstacle to treating patients with cancer and is often the result of overexpression of a 170-ku plasma membrane glycoprotein known as P-glycoprotein (P-gp)[1]. Tumor cells are susceptible to apoptosis in response to drugs, the susceptibility of cells to undergo apoptosis may ultimately determine its drug sensitivity. In other words, resistance to apoptosis is an important factor in drug resistance, ultimately determining the cellular response to drug treatment [2,3].

Many chemotherapeutic drugs function in a caspase-dependent manner, by activating the main effector caspase-3. Caspase-3 can be activated via two main apoptotic pathways described sometimes as receptor-dependent and mitochondrial-dependent pathways. Mitochondrial-dependent pathway, which is mainly triggered by stress and cytotoxic drugs, involves the release of cytochrome c from mitochondria. Stimulation of the mitochondrial membrane in this process is not yet fully understood. Cytochrome c in synergy with ATP allows a conformational change of Apaf-1 to occur. Apaf-1 binds to caspase-9, which in turn activates caspase-3. The receptor-dependent pathway is triggered by ligation of death receptors that are members of the tumor necrosis factor (TNF) superfamily (i.e., Fas receptor) and characterized by an intracellular death domain. The death domain attracts the intracellular adaptor protein FADD that in turn recruits procaspase-8 to the death-inducing signaling complex (DISC). At the DISC, procaspase-8 is cleaved and yields active caspase-8, activating in turn caspase-3[4-7]. Cytotoxic drugs have been shown to induce receptor, which then mediates cell death through the activation of receptor-dependent pathways such as Fas /Fas-Ligand[8].

Recently, it has been postulated that P-gp, apart from actively effluxing drugs from cells, may protect them against apoptosis by inhibiting caspase-8 and caspase-3, as P-gp-positive cells have been found to resist cell death induced by UV irradiation, and ligation of cell surface death receptors Fas and TNF[9-11]. Smyth et al[12], and Cuello et al[13], postulated that successful treatment of P-gp⁺ MDR tumors can be achieved using chemotherapeutic agents that can function in the absence of caspase-3 activation. However, the molecular mechanisms underlying this novel function of P-gp remain unknown.

Apoptosis by Fas/Fas L (anti-Fas) is triggered via activating caspase-8 and in turn activating caspase-3[8,14].

The present study was to verify the hypothesis that there is a correlation between P-gp overexpression and caspase-8 and caspase-3 inhibition.

MATERIALS AND METHODS

Materials

Verapamil (VRP), vincristine (VCR), Hoechst 33258 and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Company. RPMI 1640 was purchased from Gibco BRL. Mouse monoclonal antibodies (MAbs) against caspase-8 (Ab-3) and caspase-3 (E-8) and rabbit polyclonal antibody against P-gp (mdr1) were purchased from Santa-Cruz Biotechnology (Santa Cruz, CA). PARP (c-20) was purchased from Pharmingen (San Diego, CA). Peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased from Calbiochem (La Jolla, CA). Anti-Fas monoclonal antibody (CH-11) was obtained from Immunotech Co.

Cell lines and cell culture

KBv200 cells, a classic multidrug resistant human epidermoid carcinoma cell line expressing high levels of P-gp, were cloned from parental drug-sensitive KB cells by stepwise exposure to increasing doses of vincristine and ethylmethane sulfonate (EMS) mutagenesis. KBv200 cells and parental sensitive KB cells were obtained from Chinese Academy of Medical Sciences, Beijing. KBv200 cells and KB cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, benzylpenicillin (50 kU/L ), and streptomycin (50 mg/L) at 37 °C in a humidified atmosphere of 50 mL/L CO₂+95% air[15-17].

Cytotoxicity assay

Sensitivity to anticancer agents such as VCR was determined in triplicate using MTT cell viability assay as previously described[18]. The 50% inhibitory concentration (IC₅₀) was determined as the drug concentration causing 50% reduction in cell viability. The degree of resistance was calculated by dividing the IC₅₀ for MDR cells by that for parental sensitive cells. The fold-reversal of MDR was calculated by dividing the IC₅₀ for cells to anticancer drug in the absence of modulator (VRP) by that in the presence of modulator (VRP).

Western blot analysis

The cells were treated with desired Fas antibody in the presence or absence of VRP (10 μmol/L) for 24 h. After treatment, whole-cell lysates were extracted with lysis buffer containing 1% Triton-100, 50 mmol/L sodium chloride, 50 mmol/L sodium fluoride, 20 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium vanadate, 0.2 mmol/L phenylmethylsulfonyl fluoride and 0.5% Nonidet P-40. Western blotting was carried out as described previously [19]. In brief, equal amounts of cell lysate (25 μg) were solubilized in 6-12% SDS-PAGE and transferred onto nitrocellulose membranes. After being blocked with non-fat milk, membranes were incubated with the appropriately diluted primary antibody. Then, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody. Proteins were detected by the enhanced chemiluminescence detection system (Amersham, Aylesbury, UK).

Apoptosis measurement

Cells were treated with anti-Fas (CH-11) in the presence or absence of VRP (10 μmol/L) for 48 h, both floating and trypsinized adherent cells were collected, washed with phosphate-buffered saline, fixed with 10% paraformaldehyde for 30 min, and incubated in Hoechst 33258 (Sigma) at room temperature for 30 min (final concentration, 30 µg/mL). Nuclear morphology was examined using fluorescence microscopy with standard excitation filters. Apoptotic cells stained brightly and displayed condensed and fragmented nuclei, normal cells showed an even distribution of the stain throughout the nucleus with flocculated chromatin. The percentage of apoptotic cells was calculated, all cells from four random microscopic fields at 400× magnification were counted[20].

Statistical analysis

All experiments were repeated at least twice and differences were determined using Student’s t-test. P<0.05 was considered statistically significant.

RESULTS

Characterization of Fas and P-glycoprotein-expressing in KBv200 and KB cells

As drug resistance is correlated with decreased Fas receptor expression in hematopoietic MDR cell lines[21,22], Fas expression was examined in these cells. Drug sensitive KB cells and resistant KBv200 cells showed similar levels of Fas expression (Figure 1). A very strong band of P-gp expression was observed in MDR KBv200 cells, while there was no P-gp expression in their parental drug-sensitive KB cells.

Figure 1 Western blot analysis of P-gp and Fas expression in KBv200 cells and KB cells.

VRP effectively reversed resistance of KBv200 cells to VCR

To determine the resistance of KBv200 cells to VCR, we compared the cytotoxicity of VCR between KBv200 and their parental sensitive KB cells. IC₅₀ of VCR to KB and KBv200 cells was 69.8 nmol/L and 3577.2 nmol/L, respectively (Figures 2A and Figures 2B). The resistance of KBv200 cells to VCR was approximately 51.2-fold higher than that of the parental sensitive KB cells in our experimental system. The cells were incubated with 10 μmol/L of VRP and a full range of concentrations of VCR. At the concentration of 10 μmol/L, VRP lowered the IC₅₀ of VCR to 189.6 nmol/L in KBv200 cells (Figure 2B). This gave 18.9-fold reversal of MDR. In the sensitive KB cells, however, the IC₅₀ of VCR was 65.4 nmol/L in the presence of VRP at the concentration of 10 μmol/L (Figure 2A). These results suggested VRP was very effective on reversing MDR in vitro.

Figure 2 VRP enhanced cytotoxicity of VCR to KB cells (A) and KBv200 cells (B).

VRP did not enhance the sensitivity of anti-Fas (CH-11) to P-gp positive KBv200 cells

CH-11 induced a similar cytotoxicity to both P-gp positive KBv200 cells and their parental P-gp negative KB cells. The IC₅₀ was 39.3±3.6 (ng/mL) and 38.5±4.2 (ng/mL) for KBv200 and KB cells, respectively (P>0.05). Furthermore, 10 μmol/L of VRP did not significantly enhance the sensitivity of KBv200 cells to anti-Fas (CH-11, Figure 3).

Figure 3 VRP did not enhance cytotoxicity of anti-Fas to KB cells (A) and KBv200 cells (B).

KBv200 cells did not show resistance to anti-Fas (CH-11)-mediated apoptosis and VRP did not sensitize KBv200 cells to anti-Fas (CH-11)-mediated apoptosis

To examine whether P-gp positive cells failed to undergo apoptosis induced by anti-Fas/Fas, apoptosis was assessed by Hoechst 33258 staining. Treatment with 20 ng/mL anti-Fas (CH-11) resulted in significant apoptosis in parental KB cells, as well as in drug-resistant KBv200 cells. Condensed and fragmented nuclei were found, and normal cells showed an even distribution of the stain throughout the nucleus with flocculated chromatin. The apoptotic rate was 39.0% and 40.5% in KB and KBv200 cells respectively after treatment with 20 ng/mL CH-11 for 24 h. Anti-Fas (CH-11)/Fas induced similar apoptosis in P-gp positive KBv200 cells and parental sensitive KB cells. Moreover, treatment with CH-11 (20 ng/mL) resulted in similar cell apoptosis with or without VRP (10 μmol/L). The results are shown in Figure 4 and Table 1.

Table 1 Effect of VRP on the apoptotic rate in cells treated with CH-11 (mean±SD).

	Control	VRP (10 mmol/L)	CH-11 (20 ng/mL)	VRP+CH-11
KB	3.5±1.3	3.0±0.8	39.0±6.1	40.3±6.8
KBv200	2.8±1.0	3.5±1.3	40.5±3.4	41.3±3.4

Figure 4 Cell morphology and DNA fragmentation.

VRP did not sensitize activation of caspase-8 and -3 in KBv200 cells

The induction of apoptosis by anti-Fas was assessed by Western blot. VRP did not induce apoptosis alone. Treatment with 20 ng/mL anti-Fas (CH-11) resulted in significantly reduced levels of caspase-8 and -3 in KBv200 cells. KBv200 cells did not demonstrate greater CH-11 mediated-caspase-8 and -3 activation in the presence of 10 μmol/L VRP (Figure 5). These results were in agreement with the apoptosis assessed by Hoechst 33258 staining.

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.

DISCUSSION

Following the introduction of more effective drugs and the development of better-designed chemotherapy strategies, the treatment of cancer has improved. However, the development of multidrug resistance (MDR) is still a problem and remains an obstacle to successful chemotherapy. Many cellular changes have been associated with the development of MDR, including expression of ATP-binding cassette (ABC) transport proteins, multidrug resistance-associated protein (MRP1), and P-glycoprotein. Resistance mediated by these proteins is associated with altered drug transport, resulting in decreased intracellular drug accumulation[21-25].

P-gp has been demonstrated to efflux a wide range of structurally and functionally diverse compounds[22,24]. Recently it has been proposed that in addition to mediating drug resistance through drug transport, P-glycoprotein may also play a role in resistance to apoptosis[26]. Resistance to apoptosis is an important factor in drug resistance, ultimately determining the cellular response to drug treatment. This supports the suggestion that P-glycoprotein can protect cells against apoptotic stimuli that function in a caspase-dependent manner by transporting a key caspase or adaptor molecules out of cells or by inhibiting caspase activition through decreasing intracellular ATP or altering intracellular pH[27,28]. On the other hand, P-gp-expressing cells are not resistant to caspase-independent cell death mediated by pore-forming proteins and granzyme B as well as by other factors[10,29,30].

However, P-gp does not interfere with the formation of DISC since equivalent amounts of pro-caspase-8 are associated with the Fas-receptor[31]. Cullen et al[19,27], showed that MDR mediated by MRP and P-gp does not correlate with resistance to Fas-mediated cell death or to caspase-3 activation. Sikora et al[32], also found that there is no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells.

Our results tended to exclude that the multidrug transporter P-gp played an important role in the inhibition of apoptosis by inhibiting activation of caspase-8 and -3. In fact, anti-Fas/Fas induces similar cytotoxicity and apoptotic rate in P-gp overexpressing KBv200 cells as in their parental sensitive KB cells. In addition, the P-gp inhibitor VRP strongly reverses the resistance to VCR and VCR-induced apoptosis in KBv200 cells. But verapamil does not increase the cytotoxicity and the rate of apoptosis induced by anti-Fas antibody in the same cells. KBv200 cells do not exhibit greater caspase-8 and -3 degradation in the presence of 10 μmol/L VRP.

Notarbartolo et al[33], and Campone et al[34], have shown that P-gp-expressing HL-60 cells are resistant to caspase-3-dependent apoptosis due to the presence of XIAP in these cells. This suggests that the novel family of IAPs may have a responsibility in such a process.

In conclusion, overexpression of P-gp in KBv200 cells does not influence the apoptotic pathway leading to activation of caspase-8 which in turn activates caspase-3.

ACKNOWLEDGMENTS

We thank Professor Liu YS (Chinese Academy of Medical Sciences, Beijng, China) for providing KB and KBv200 cell lines.