Studies for anti-tumor activities and reversing p-glycoprotein mediated multidrug resistance induced by novel quinoline derivatives

Abstract

Cancer is a major health threaten worldwide. P-glycoprotein mediated multidrug resistance is one of the main obstacles for successful cancer chemotherapy. Quinoline core has been shown to possess a promising role in the development of novel anti-cancer agents. The development of novel anti-cancer drugs with high efficacy, low toxicity and minimal anti-cancer drug resistance is thus essential and still a great challenge. In the study of the anti-cancer effect of a novel synthesized quinoline compound 91b1, cytotoxic effect on the four cancer cell lines (A549, AGS, KYSE150, and KYSE450) and one non-tumor cell line NE3 was determined by MTS cytotoxicity assay compared with the widely used chemotherapeutic drug CDDP. The MTS50 values of compound 91b1 were 15.38 μg/mL, 4.28 μg/mL, 4.17 μg/mL, 1.83 μg/mL, and 2.17 μg/mL for A549, AGS, KYSE150, KYSE450, and NE3 cells respectively. The MTS50 values of CDDP were 6.23 μg/mL, 13.00 μg/mL, 13.2μg/mL, 6.83 μg/mL, and 1.17 μg/mL for A549, AGS, KYSE150, KYSE450, and NE3 cells respectively. The compound 91b1 showed stronger cytotoxicity to cancer cells and less toxicity to non-tumor cells compared with CDDP. cDNA microarray analysis on the compound 91b1 treated (9.5μg/mL) KYSE150 cells compared with the parental cells identified lumican as one of the most significantly downregulated targets. By qPCR analysis, lumican was shown to be suppressed in mRNA with compound 91b1 treatment in a dose-dependent manner. Cell proliferation assay was performed on four cancer cell lines (A549, AGS, KYSE150, and KYSE450), which all showed an obvious reduction in proliferation rate after treated with compound 91b1 (10 μg/mL). Cell cycle analysis was performed on A549 and KYSE450 cells to identify the effect of compound 91b1 on cancer cell growth. Cancer cells were observed to be arrest at G2/M phase and decreased at G0/G1 phase after compound 91b1 (10 μg/mL) treatment. Wound healing assay for A549, KYSE150, AGS, KYSE70, and KYSE510 cell lines showed that compound 91b1 suppressed cancer cell migration in vitro. What's more, compound 91b1 inhibited solid tumor (KYSE150 and KYSE450) growth in nude mice xenograft model using KYSE450 with the reduction of tumor volume by 51.06 % compared with the vehicle control group after 25 days' treatment). The compound 91b1 showed mild liver damage with no significant changes being observed on liver functional parameters including levels of ALT, AST, TP, and Tbile in animal serum after treatment and was relatively safe than doxorubicin at therapeutic dose in the mouse model. These results implied that the compound 91b1 could effectively induce anti-cancer activity both in vitro and in vivo on the studied tumors. In the study of the functions of lumican in cancer cells, up-regulation of lumican was detected in cancer cell lines (KYSE30, KYSE70, KYSE150, KYSE510, and SLMT1) and tumor samples isolated from cancer patients with esophageal carcinoma. The trans-well matrigel invasion assay demonstrated that lumican protein enhanced the migration ability of KYSE150 cells. Cell proliferation assay on four cancer cell lines (A549, AGS, KYSE150, and KYSE450) showed that protein lumican (250 ng/mL) increased cancer cell proliferation rate. Wound healing test of A549, AGS, and KYSE150 cell lines showed that protein lumican enhanced cancer cell invasion. Subcutaneous injection of lumican transfected NIH 3T3 cells (NIH 3T3/Lum) generated a subcutaneous mass in athymic nude mice. Bio-Plex cell signaling assay was performed on KYSE150 cells treated with either compound 91b1 or human recombined lumican protein in order to identify the most associated pathway by which compound 91b1 suppressed cancer cell progression and tumor development via down-regulating lumican expression. The phosphorylated level of Erk1/2 revealed consistent trend of increase or decrease after treatment with either compound 91b1 or protein lumican respectively (phosphorylated level of Erk1/2 was increased after compound 91b1 treatment and decreased after reclumican treatment; while compound 91b1 down-regulated lumican expression in KYSE150 cells). The overall results suggested that protein lumican promoted cancer cell proliferation, invasion and migration in esophageal carcinoma cells KYSE150, by enhancing tumor development. It is inferred that compound 91b1 induced anti-cancer effect through Erk1/2 signaling pathway by downregulating lumican expression. In the study of reversal effect of p-glycoprotein modulated multi-drug resistance (MDR) of compound 160a, DOX-resistance cancer cells of lung cancer (A549) and esophageal cancer (KYSE150) were established from parental cancer cells and cultured in 1.0 μg/mL doxorubicin in medium to maintain its resistance to doxorubicin. P-glycoprotein was predicted to be the possible interaction target of compound 160a by molecular docking analysis using Similarity Ensemble Approach (SEA). The synergistic effect of compound 160a combined with doxorubicin was determined based on cytotoxicity (A549/DOX and KYSE150/DOX cell lines) and cell proliferation (A549/DOX, KYSE150, LCC6/MDR, and MX100 cell lines) tests and analyzed by Compusyn program with CI value being smaller than 1. The above results demonstrated that the compound 160a showed synergistic effect to enhance the cytotoxicity and inhibition of cell proliferation induced by doxorubicin against the DOX-resistance cancer cell lines. The effect of compound 160a on p-glycoprotein was also tested by multi-drug resistance assay using calcein AM as the substrate of p-glycoprotein, and detected by flow cytometry and confocal microscopy. It was indicated that the compound 160a increased the intracellular calcein AM concentration by inhibiting the MDR effect induced by p-glycoprotein. Doxorubicin accumulation test also offered consistent results that 160a exhibited greater potency by increasing DOX accumulation against DOX-resistance cancer cells (LCC6/MDR, KYSE150/DOX, and A549/DOX cells) than verapamil, a positive control. The duration of reversal MDR effect test also showed that the compound 160a could persist the MDR effect for 1 hour. The overall results of this part showed that the novel quinoline compound 160a possessed synergistic effect when combined with doxorubicin against DOX-resistance cancer cells. The molecular mechanism of the synergistic effect of the compound 160a was explained by being able to reverse the multi-drug resistance modulated by the p-glycoprotein based drug-efflux effect. To conclude, the results of the overall work provided the possible novel therapeutic directions for cancer therapy at molecular level. The findings of the anti-cancer actions of the novel quinoline compound 91b1 and the mechanisms associated with lumican offered a new therapeutic target. The results about the MDR reversal effect of the novel quinoline compound 160a showed the great potential of 160a to overcome the chemoresistance in cancer treatment in future.