| Multidrug resistance(MDR) is a major obstacle to curative treatment of leukemia. Researches on the mechanisms of drug resistance have been performed, but recently, more and more studies are focused on Glucosylceramide synthase(GCS) which is highly associated with MDR. It is known that GCS is a glycosyltransferase in sphingolipid metabolism. It transfers a glucose reside from UDP-glucose to ceramide(Cer) for glucosylceramide(Glc Cer) thus eliminating cellular levels of Cer and providing synthetic precursor for senior glycosphingolipid. As a second messenger molecule in sphingolipid signal transduction, Cer is involved in the regulation of various physiological processes such as cell growth、differentiation、aging and cell death. Reduced levels of Cer which inhibit cell differentiation and apoptosis are thus considered to be one of the mechanisms of GCS induced MDR.Previous studies on GCS-induced drug resistance in tumors are focused on its impact on membrane proteins functioning as drug efflux pumps. P170 glycoprotein(P-glycoprotein, P-gp) coded by multidrug resistance gene 1(MDR1) is the most studied molecule related to MDR. When the drugs enter the cell, P-gp binds to drug molecules and then strengthens the intercellular transfer of diverse chemotherapeutic agents from the inside to the outside of cells driven by the ATP-binding cassette transporter and keeps drugs at a low level. It is considered that the overexpression of GCS, which increases the efflux of chemotherapeutic agents through upregulating P-gp, is one of the mechanisms of MDR in various tumors. However, even if anti-cancer agents reach their sites of action by passing drug efflux systems, some cells still exhibit resistance via inhibition of pro-apoptosis signaling pathway, suggesting that dysregulation of apoptosis also plays a very important role in MDR generation.Bcl-2 is a tumor-specific gene family. It regulates cell apoptosis on the mitochondria membrane. The family is composed of three groups related by structure and function:(1)the BH3 proteins like Bid that sense cellular stress and activate;(2)the executioner proteins like Bax that permeabilize the mitochondrial outer membrane and then activate the effector caspases of apoptosis;(3)the anti-apoptotic members like Bcl-2 that inhibit both the BH3 and the executioner proteins. Bcl-2 impedes the apoptotic process primarily by inhibiting the release of cytochrome c from mitochondria. Large amounts of data show that the increase of Bcl-2 or the Bax protein reduction is probably responsible for the non-sensitivity of tumors to chemotherapeutics. In contrast, upregulation of Bax or elimination of Bcl-2 may sensitize tumor cells to these agents.Previous studies done in our lab demonstrated that GCS and Bcl-2 were co-overexpressed in K562/A02 leukemia drug-resistant cell line compared with its sensitive counterpart, K562. While, there was no difference in Bax expression between K562 and K562/A02 cells. GCS knockdown led to remarkable reduction in Bcl-2 expression, but did not apparently influence the expression level of Bax. In addition, the MDR of K562/A02 cell line was highly associated with ERK signaling pathway that regulated the influence of GCS on P-gp expression. Based on the fact that the ERK signaling pathway was closely related to cell apoptosis, ERK induced signaling transduction was supposed to be a common pathway after a variety of stimuli. Studies also found that the activation of ERK pathway was a necessary prerequisite for mitochondrial depolarization, the release of cytochrome C, caspase 3 activation and cell apoptosis. Herein, we explored the molecular mechanisms of GCS upregulating Bcl-2 expression and investigated whether ERK signaling was associated with the inhibition of cell apoptosis that caused by GCS, in the hope of conquering MDR of tumors.OBJECTIVE: To explore the effect of GCS on the expression level of apoptosis-related gene Bcl-2 in the K562/A02 leukemia multidrug-resistant cell line, in the hope of understanding the mechanisms of GCS-induced MDR in leukemia.METHODS: The expressions of Bcl-2 m RNA and protein, as well as the expressions of members of MAPK family were measured by real-time PCR and western blotting in K562/A02 cells after GCS was knockdown by si RNA transfection; the effect of treatments on apoptosis was examined by Flow Cytometry(FCM); the IC50 value of AMD for K562/A02 cells was determined by CCK-8 kit and the level of Cer was examined by normal phase high performance liquid chromatography coupled atmospheric pressure chemical ionization-mass spectrometry(LC/MS). After being incubated with 20 μM U0126 for 24 h, the IC50 value of ADM for K562/A02 cells was determined by CCK-8; FCM was used to observe the apoptosis of cells exposed to 10 μg/ml ADM for 24 h in the absence or presence of U0126(20μM); real-time PCR and western blotting were used to measure the effects of U0126 or Cer on Bcl-2 m RNA and protein, as well as the activation of ERK signaling pathway.RESULTS: Knockdown of GCS caused by GCS si RNA increased Cer level, suppressed the expression levels of Bcl-2 m RNA and protein, enhanced ADM-induced cell apoptosis, and sensitized K562/A02 cells to ADM. Western blotting showed that GCS knockdown led to remarkable reduction in phosphorylated ERK, but did not apparently influence total ERK or other MAPK kinases expressions even their phosphorylated forms. U0126 treatment enhanced apoptosis and increased the cell sensitivity to ADM in K562/A02 cells. Bcl-2 m RNA and protein and p-ERK expressions were suppressed by U0126 in a dose- and time-dependent manner. The phosphorylation of ERK, as well as Bcl-2 expression was decreased upon C6-Cer exposure.CONCLUSIONS: GCS promoted anti-apoptotic Bcl-2 expression via the ERK signaling pathway so as to decrease the sensitivity of K562/A02 cells to ADM and induce multidrug resistance of leukemia. The decrease of Cer,as a substrate of enzymatic reaction catalyzed by GCS, was upstream signaling of ERK activation when GCS was overexpressed. |
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