Synthesis of a Dual Functional Anti-MDR Tumor Agent PH II-7 with Elucidations of Anti-Tumor Effects and Mechanisms

Ye Su,Tao Cheng,Juanni Liu,Yunhui Hu,Yaohong Tan,Hua Miao,Yingdai Gao,Caiyun Wang,Chunzheng Yang,Yuanfu Xu,Dongsheng Xiong,Jianxiang Wang,Xin Cheng,Yuan Zhou,Yinliang Xie,Arto Urtti

doi:10.1371/journal.pone.0032782

Abstract

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux.

Highlights

Clinical cancer treatments still rely heavily on chemotherapy, especially for metastatic cancers and hematological malignancies
We found a series of PH II-7 responsive genes including cell cycle related genes, apoptosis related genes, multi-drug resistance (MDR) related genes (Fig. 7A), these are put to further screening and validation with real-time polymerase chain reaction (PCR)
Compared to the chemotherapeutic agent ADM, PH II- 7 is especially potent against 5 pairs of human cancer cell lines and their corresponding MDR sublines that are highly resistant to ADM (Fig. 2B)

Summary

Introduction

Clinical cancer treatments still rely heavily on chemotherapy, especially for metastatic cancers and hematological malignancies. Other ABC family members, e.g. ABCC1 [3,4,5], ABCG2 [6,7,8], have since been shown to affect drug efflux. The P-gp represents the most well established mechanism for MDR, with many clinical chemotherapy drugs being its substrates. Other P-gp-targeting anti-MDR strategies include but not limited to: P-gp specific peptides [16] or antibodies [17], downregulation of MDR1 gene with transcriptional repressors [18,19] or siRNAs [20,21], novel agents that are not substrates of P-gp [22,23], or encapsulation of chemo-agents to evade P-gp efflux [24]

Methods

Results

Conclusion