Study On The Multidrug-resistant Effect Of Acridine Derivative LTW02 And Its Mechanism In Tumor Cells

Objective:To evaluate the anticancer effect and anti-multidrug resistant effect of LTW02 in vitro, a series of novel acridine derivatives,and explore the possible anticancer mechanism.Methods:The drug resistance fold of K562 /ADM was detected by MTT in two human leukemia cells K562(human leukemia cells) and K562/ADM(adriamycin-resistant human leukemia cells). The anti-proliferative effects of LTW02 was assessed by MTT assay. The function of P-gp were assessed with rhodamine accumulation. Apoptotic morphology was observed by HCS with Hoechst33342 staining and cell cycle of K562/ADM was measured by FCM with PI staining. We also researched mitochondrial membrane potential(MMP) and the situation of Autophagy by HCS with RH123/Hoechst33342. The situation of autophagy were assessed by FCM with MDC and Lyso-Tracker Red fluorescent staining respectively.The protein expression of P-gp, the members of Caspase and Bcl-2, cytochrome c, LC3,BECN1 and other relative proteins were assessed by western blot, and analyzed their relationships.Results:MTT assay results showed that the IC50(48 h) of doxorubicin was(1.62±0.10) μg/ml and(178.14±7.72) μg/ml in K562 and K562/ADM cells respectively. The drug resistance fold of K562/ADM was 110.MTT assay demonstrated that LTW02 exerted an excellent anti-proliferative effects in dose- and timedependent manner in K562 and K562/ADM cells, the IC50(48 h) of LTW02 was(15.09±0.19) μM and(14.96±0.26) μM, respectively.Rhodamine accumulation assay demonstrated that LTW02 increased the Rh123 accumulation of K562/ADM cells in a dose-dependent manner. Western Blot results further showed that LTW02down-regulated the expressing of P-gp in K562/ADM cells.Hoechst33342 staining and cell cycle analysis both confirmed that LTW02 induced cell apoptosis in dose-dependent manner in resistant human leukemia cells K562/ADM.After treatment with LTW02, wefound that the tumor cells appeared typical apoptotic bodies and condensed or fragmented nucleus. PI staining assay showed that LTW02 has little effect on the cell cycle of K562/ADM. But there was a apoptosis peak in the DNA histogram in dose-dependent manner.To further investigated the mechanism of apoptosis induced by LTW02, the MMP was measured with Rhl23/Hoechst33342 double staining by HCS. The results indicated that LTW02 induce the MMP loss and destroy membrane integrity of K562/ADM cells in dose-manner. Furthermore, the accumulation of Cyto-c in cytosol was observed and the active forms of procaspase-9 and procaspase-3 were detected by Western Blot. We also found LTW02 down-regulated the expression of the antiapoptotic protein Bcl-2 and up-regulated the expression of the proapoptotic protein Bax. Furthermore, we also found LTW02up-regulated the expression of p53.MDC staining and Lyso-Tracker Red staining both confirmed that LTW02 induced cell autophagy in dose-dependent manner in resistant human leukemia cells K562/ADM. Western Blot results further showed that the activity of autophagy related protein Beclin 1 significantly increased, and the transformation MAP LC3β from typeⅠto typeⅡalso increased in K562/ADM cells after after treatment with LTW02. We also found LTW02 down-regulated the expression of mTOR.Conclusions:LTW02 exerted potent cytotoxicity against multi-drug resistant human leukemia cell line K562/ADM.LTW02 plays its anti-multidrug resistance role through inhibiting the expression and function of P-gp, and inducing apoptosis and autophagy in K562/ADM cells.