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Colchicine induces autophagy and senescence in lung cancer cells at clinically admissible concentration: potential use of colchicine in combination with autophagy inhibitor in cancer therapy

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Tumor Biology

Abstract

Colchicine is a well-known and potent microtubule targeting agent, but the therapeutic value of colchicine against cancer is limited by its toxicity against normal cells. But, there is no report of its cytotoxic potential against lung cancer cell, at clinically permissible or lower concentrations, minimally toxic to non-cancerous cells. Hence, in the present study, we investigated the possible mechanism by which the efficacy of colchicine against lung cancer cells at less toxic dose could be enhanced. Colchicine at clinically admissible concentration of 2.5 nM had no cytotoxic effect and caused no G2/M arrest in A549 cells. However, at this concentration, colchicine strongly hindered the reformation of cold depolymerised interphase and spindle microtubule. Colchicine induced senescence and reactive oxygen species mediated autophagy in A549 cells at this concentration. Autophagy inhibitor 3-methyladenine (3-MA) sensitised the cytotoxicity of colchicine in A549 cells by switching senescence to apoptotic death, and this combination had reduced cytotoxicity to normal lung fibroblast cells (WI38). Together, these findings indicated the possible use of colchicine at clinically relevant dose along with autophagy inhibitor in cancer therapy.

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Acknowledgments

The work was supported by grants from DST, Govt. of India (No. SR/SO/BB-14/2008) and DBT, Government of India (No. BT/ PR12889/AGR/36/624/2009) to GC. Confocal Microscope facility is from University-DBT-IPLS programme, Government of India (No. BT/PR14552/INF/22/123/2010), and FACS instrument facility is developed by grants from National Common Minimum Project, Government of India. SB was a senior research fellow of University Grant Commission. SD is a Senior Research Fellow of CSIR, Government of India. AG was a fellow of DBT-CU-IPLS programmne, Government of India (No. BT/PR14552/INF/22/123/2010).

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Authors and Affiliations

  1. Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB, 700 019, India

    Surela Bhattacharya, Amlan Das, Satabdi Datta, Arnab Ganguli & Gopal Chakrabarti

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  1. Surela Bhattacharya
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Fig. S1

Colchicine inhibited reformation of cold depolymerized interphase microtubule in A549 cells at low concentration Panel a shows normal interphase microtubule in A549 cells before incubating the cells at 4°C whereas panel b shows microtubule network after 3 h of cold depolymerization. Cold media was replaced by warm media in the absence (Panel c) and presence of 2.5 nM (Panel d) of colchicine and incubated for 3 h at 37°C. Microtubules were tagged by anti- α-tubulin antibody, subsequently FITC conjugated secondary antibody (green) and nucleus were probed by DAPI (blue). (GIF 42 kb)

High resolution image (TIF 1787 kb)

Fig. S2

Colchicine suppressed the reassembly of the cold depolymerized mitotic spindle microtubules in A549 cells Cultured A549 cells were incubated with nocodazole for 20h. After removing nocodazole, cells were kept on ice for 30 min in absence (Panel a-c) and presence of 2.5 nM colchicine (Panel g-i) and further incubated at 37°C in absence (Panel d-f) and presence of 2.5 nM colchicine (Panel j-l). (GIF 25 kb)

High resolution image (TIF 1533 kb)

Fig. S3

(a) Effect of colchicine on the ratio of soluble and polymerized tubulin in A549 cells. . A549 cells, treated without or with colchicine (2.5 and 50 nM), were lysed by a hypotonic lysis buffer. The insoluble and soluble tubulin fractions were separated by centrifugation. Western blot was performed using monoclonal antibody against α-tubulin. (b) Effect of colchicine on the ratio of soluble and polymerized tubulin in A549 cells before and after cold treatment. (c) Effect of 3-MA on the microtubule structure of A549 cells. Cultured A549 cells were incubated in absence and presence of 3-MA (5 mM) for 48 h, fixed and stained using antibody against α-tubulin (red) and nucleus with DAPI (blue). (GIF 15 kb)

High resolution image (TIF 1448 kb)

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Bhattacharya, S., Das, A., Datta, S. et al. Colchicine induces autophagy and senescence in lung cancer cells at clinically admissible concentration: potential use of colchicine in combination with autophagy inhibitor in cancer therapy. Tumor Biol. 37, 10653–10664 (2016). https://doi.org/10.1007/s13277-016-4972-7

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