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

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.