| Chemotherapy is the most effective treatment for patients who suffer from cancers. The effectiveness of chemotherapy, however, is seriously limited by multidrug resistance (MDR). To explore the mechanisms of MDR will help to improve the the efficiency of chemotherapy. Factors that affect drug sensitivity include the pharmacokinetic profile of the drug, evasion of apoptosis, MAPK signaling pathway, alterations in the drug target and processing of drug-induced damage.JWA is a novel structurally microtubule associated protein (MAP), not only related to differentiation and/or apoptosis inducers (e.g. TPA, ATRA, 4HPR, and As2O3) mediated leukemia cells differentiation and apoptosis, but actively responsive to stress stimulations (e.g. heat shock, oxidative stress). However, the underlying molecular mechanism of JWA gene and involved signal pathways in the regulation of MDR is still unknown.In this study, we chose VP16 as the apoptosis inducing chemotherapeutic agent, to explore whether JWA is involved in MDR in cancer cells and the underlying molecular mechanisms in human choriocarcinoma cell line JAR and its chemoresistant cell line JAR/VP16 as cell culture models.1. JWA is required for VP16 induced apoptosisThe Hoechst 33258 staining assay and Annexin V-PE flow cytometry assays showed that VP16 induced apoptosis in both JAR and JAR/VP16 tumor cells in a dose-dependent manner. However, the two cell lines indicated differential sensitivity to VP16 exposure. For JAR cells, treatment with 1×10-3 g/L VP16 for 48 h induced about 90 % apoptosis; however, induced only 16% apoptosis in JAR/VP16 cells. Western blot analysis showed enhanced JWA expressions in JAR cells. These data suggest the increased JWA expression may be involved in VP16 induced apoptosis.To evaluate whether increased JWA expression is required for VP16 induced apoptosis in both JAR and JAR/VP16 cells, we established stable JWA deficient JAR cells. Interestingly, these JWA deficient cells showed significant resistance to VP16 treatment when compared with their vector controlled cells. These results indicating that JWA protein may act as a pro-apoptotic molecule in the process of apoptosis induced by VP16.2. JWA through mitochondrial pathway enhances VP16 induced apoptosisHow does JWA play its pro-apoptotic role in VP16 triggered cell apoptosis? Genetic and biochemical evidences indicate that apoptosis proceeds in two major cell death pathways: an intrinsic pathway that involves mitochondrial membrane permeabilization and release of several apoptogenic factors, followed by Caspase-9 activation; or an extrinsic apoptotic signaling pathway that occurs via Caspase-8 activation. To determine which Caspases linked signal pathways were involved in VP16 triggered and JWA dependent cell apoptosis, studies examined expressions of Caspase-8 and Caspase-9 and their potential cleavages. Data showed that VP16 treatment (1×10-3 g/L for 48 hr) significantly triggered Caspase-9 but not Caspase-8 cleavage, this phenomenon was obviously attenuated in JWA deficient cells. Suggesting that JWA exerted its pro-apoptotic effect mostly through the intrinsic pathway in VP16 triggered cell apoptosis. To testify whether Caspase-9 activation regulated by JWA may be due to the changes of mitochondrial membrane potential (Δψm), we further performed FACS analysis to detect the loss ofΔψm. Interestingly, results revealed that siJWA-JAR cells treatment with VP16(1×10-3g/L for 48 hr) were still maintained theirΔψm in contrast of markedly losingΔψm in control cells. It was suggested that loss of mitochondria membrane potential in the presence of JWA expression may be linked to Caspase-9 activation.3.Molecular mechanisms underlying the effect of JWA on VP16 induced apoptosisMAPK signal pathways to be responsible for mitochondria mediated cell apoptosis. To further investigate the role of JWA on VP16 induced apoptosiswe tested whether the role of JWA was associated with MAPK activations. We observed the effects of JWA on VP16 (0, 1×10-3 g/L for 48 hr) induced expression or phosphorylation of MAPK molecules. The results showed the VP16 activated phosphorylations of P38, JNK and transcription factor c-Jun were almost completely blocked by knock-down JWA expression; although the total expressions (unphosphorylation level) of these signal molecules were unaffected in VP16 treated JAR/VP16 and siJWA-JAR cells. Data indicating that the role of JWA on VP16 induced JAR cell apoptosis was realized via MAPK signal pathway.4. JWA is required for the down-regulation of JNK on P-glycoproteinIt is believed that inhibition of P-glycoprotein function or inhibition of its expression may reverse P-glycoprotein-mediated MDR phenotype and improve the effectiveness of chemotherapy. Researches found that cancer cells overexpressing P-glycoprotein had minor endogenous JNK activity. The reduction of P-glycoprotein in JAR and C1-JAR cells treated with VP16 was clearly seen as p-JNK and p-Jun were highly expressed. We then treated cells with 20umol/L SP600125 for 48 h, a small molecular inhibitor of JNK to examine whether the down-regulation of P-glycoprotein is required for JNK activity. Data showed that SP600125 not only inhibited the basic activity of JNK but also inhibited transcription factor c-Jun activity in the presence of JWA. Furthermore, SP600125 remarkably abolished the down-regulatory effect of JNK on P-glycoprotein accompanied with JWA. These results indicated a role for JNK in mediating the effect of JWA on P-glycoprotein.In summary, the results presented here indicate that (1) VP16 induced apoptosis in JAR and JAR/VP16 cells. (2) JWA is required for VP16 induced activation of P38 and JNK which further closely linked to changes of mitochondrial membrane potential and the activation of Caspase-9; these cascade events play a central role for VP16 inducing apoptosis in JAR cells. JWA may exert its pro-apoptotic effect on VP16 induced apoptosis through the following pathway: VP16→JWA→MAPK family(P38,JNK)→mitochondrial membrane potential→Caspase-9→apoptosis. (3) JWA might be required for the down-regulatory of JNK on P-glycoprotein. JWA may regulate P-glycoprotein through the following pathway: JWA→JNK→c-Jun→ P-glycoprotein. |
PDF Full Text Request