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Knockdown of clusterin alters mitochondrial dynamics, facilitates necrosis in camptothecin-induced cancer stem cells

Abstract

The existence of a well-established drug resistance mechanism in cancer stem cells (CSC) complicates the cancer treatment. Clusterin (CLU) plays a key role in maintaining the integrity of endoplasmic reticulum (ER) during drug-induced stress. Hence, silencing the CLU could significantly reduce the inherent drug resistance mechanism of CSC. The combination of drug-induced cytotoxicity, as well as the suppression of drug resistance in CSC, could circumvent the recurrence capability of the tumor. In the present study, camptothecin (CPT)-induced apoptosis and necrosis in CSC with and without siCLU treatment were simultaneously measured using Qdot-based total internal reflection fluorescence microscope (TIRF). In addition, to elucidate the mechanism of CPT-induced cytotoxicity in CLU-suppressed CSC, expression of Bcl-2, Bax, Bak, and PARP and mitochondrial permeability transition pore (MPTP) were studied. EC50 values of CPT-induced apoptosis and necrosis were significantly reduced (p < 0.01) in CLU-suppressed MCF-7 and CSC. Significantly increased MPTP (p < 0.001) and cytosolic Ca2+ (p < 0.001) were observed in CPT-treated CLU-suppressed CSC as compared to the normal CSC. Elevated expression of Bax, Bak, and cleaved PARP and reduced expression of Bcl-2 and cytosolic ATP were observed in CPT-treated CLU-suppressed CSC. Observed results indicate that silencing the expression of CLU could improve the anticancer efficacy of CPT at 128.4-nM concentration by equally inducing necrotic signals along with apoptosis. Furthermore, the developed high content TIRF assay based on the CLU-suppressed CSC could be an ideal and beneficial tool for rapidly analyzing the cytotoxicity of anti-cancer agents.

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2015R1A2A1A05001842 and 2016R1A4A1010796). This work was also supported by the Agency for Defense Development through Chemical and Biological Defense Research Center. We are grateful to the Research Institute of Pharmaceutical Sciences at Seoul National University for providing experimental equipment. The authors declare no competing financial interests.

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Correspondence to Joon Myong Song.

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Arumugam, P., Samson, A., Ki, J. et al. Knockdown of clusterin alters mitochondrial dynamics, facilitates necrosis in camptothecin-induced cancer stem cells. Cell Biol Toxicol 33, 307–321 (2017). https://doi.org/10.1007/s10565-016-9378-1

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  • DOI: https://doi.org/10.1007/s10565-016-9378-1

Keywords

  • Clusterin
  • Topoisomerase inhibitor-1
  • Necrosis
  • Mitochondrial permeability transition pore
  • Total internal reflection fluorescence microscope