title: 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. EC50values 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.
authors.affiliation: College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-ku, Seoul, 151-742, South Korea.
authors.forename: Parthasarathy
authors.initials: P
authors.lastname: Arumugam
authors.affiliation: College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-ku, Seoul, 151-742, South Korea.
authors.forename: Annie
authors.initials: A
authors.lastname: Samson
authors.affiliation: College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-ku, Seoul, 151-742, South Korea.
authors.forename: Jieun
authors.initials: J
authors.lastname: Ki
authors.affiliation: College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-ku, Seoul, 151-742, South Korea. jmsong@snu.ac.kr.
authors.forename: Joon Myong
authors.initials: JM
authors.lastname: Song
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002166:Camptothecin / Q000494:pharmacology*; D045744:Cell Line, Tumor; D051152:Clusterin / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D063154:Mitochondrial Dynamics / Q000187:drug effects / Q000235:genetics*; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects / Q000235:genetics; D000083162:Mitochondrial Permeability Transition Pore; D009336:Necrosis / Q000235:genetics*; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; C495242:CLU protein, human; D051152:Clusterin; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D002166:Camptothecin
keywords: Clusterin; Mitochondrial permeability transition pore; Necrosis; Topoisomerase inhibitor-1; Total internal reflection fluorescence microscope