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Clitocine potentiates TRAIL-mediated apoptosis in human colon cancer cells by promoting Mcl-1 degradation

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Abstract

Among anti-cancer candidate drugs, TRAIL might be the most specific agent against cancer cells due to its low toxicity to normal cells. Unfortunately, cancer cells usually develop drug resistance to TRAIL, which is a major obstacle for its clinical application. One promising strategy is co-administrating with sensitizer to overcome cancer cells resistance to TRAIL. Clitocine, a natural amino nucleoside purified from wild mushroom, is recently demonstrated that can induce apoptosis in multidrug-resistant human cancer cells by targeting Mcl-1. In the present study,we found that pretreatment with clitocine dramatically enhances TRAIL lethality in its resistant human colon cancer cells by inducing apoptosis. More importantly, combination of clitocine and TRAIL also effectively inhibits xenograft growth and induces tumor cells apoptosis in athymic mice. The disruption of the binding between Mcl-1 and Bak as well as mitochondrial translocation of Bax mediated by clitocine are identified as the key underlying mechanisms, which leading to mitochondrial membrane permeabilization. Enforced exogenous Mcl-1 can effectively attenuate clitocine/TRAIL-induced apoptosis by suppressing the activation of intrinsic apoptotic pathway. Furthermore, clitocine regulates Mcl-1 expression at the posttranslational level as no obvious change is observed on mRNA level and proteasome inhibitor MG132 almost blocks the Mcl-1 suppression by clitocine. In fact, more ubiquitinated Mcl-1 was detected under clitocine treatment. Our findings indicate that clitocine is potentially an effective adjuvant agent in TRAIL-based cancer therapy.

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Acknowledgments

This work was supported in part by National Natural Science Foundation of China (No. 31570811), Natural Science Foundation of Zhejiang Province (LY15C020001) and Si-Yuan Foundation.

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    Authors and Affiliations

    1. Zhejiang University, Research Centre of Siyuan Natural Pharmacy and Biotoxicology, College of Life Sciences, Zijinggang Campus, Hangzhou, 310058, People’s Republic of China

      Jian-guo Sun, Xue-li Zeng, Jun Xiang, Xia Li, Ping Wu & Fei-yan Liu

    2. Zhejiang University, Joint Centre of Zhejiang University and The Chinese University of Hong Kong on Natural Products and Toxicology Research, Zijinggang Campus, Hangzhou, People’s Republic of China

      Jian-guo Sun, Ping Wu, Kwok Pui Fung & Fei-yan Liu

    3. The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, People’s Republic of China

      Feng Ruan

    4. School of Biomedical Sciences (SBS), The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People’s Republic of China

      Kwok Pui Fung

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    Jian-guo Sun, Feng Ruan, and Xue-li Zeng have contributed equally to this work.

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    10495_2016_1273_MOESM1_ESM.tif

    Figure S1. HPLC analysis for clitocine. HP1100 C8 column (Hypersil ODS 20×250 mm) was used in this assay with a mobile phase of Methanol/water (95:5 (v/v)) at a flow rate of 8ml/min.Supplementary material 1 (TIFF 1942 kb)

    10495_2016_1273_MOESM2_ESM.tif

    Figure S2. The cytotoxicity of TRAIL and clitocine on six human colon cancer cell lines. A, human colon cells SW620, SW480, LS411N, Caco-2, HCT116 and RKO were treated with indicated concentrations of TRAIL for 24h and then analyzed by MTT assay to determine the sensitivities of different cancer cell lines to TRAIL. B, Human colon cancer cells were treated with indicated concentrations of clitocine for 48 h and subject to MTT assay. Data are presented as mean ± SD, n=3. Supplementary material 2 (TIFF 602 kb)

    10495_2016_1273_MOESM3_ESM.tif

    Figure S3. The intrinsic apoptotic pathway is indispensable for sensitization activity of Clitocine on TRAIL in human colon cancer cells. LS411N and SW620 cells were pre-treated with vehicle, 20 μM caspase 8 or 9 inhibitor for 2h followed by 0.2 μM clitocine treatment for 24h. Then, cells were incubated with 100 ng/ml TRAIL for another 12h. After treatment, LS411N and SW620 cells were subjected to Annexin V-FITC & PI staining. Flow cytometry assay was performed to detect the percentage of apoptotic cells. Data are presented as mean ± SD, n=3. ** P < 0.01 vs. respective control. Supplementary material 3 (TIFF 466 kb)

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    Sun, Jg., Ruan, F., Zeng, Xl. et al. Clitocine potentiates TRAIL-mediated apoptosis in human colon cancer cells by promoting Mcl-1 degradation. Apoptosis 21, 1144–1157 (2016). https://doi.org/10.1007/s10495-016-1273-y

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