cytochrome-c-t and 10-hydroxycamptothecin

Neuroblastoma (NB), the most common extracranial solid tumor in childhood, remains one of the most challenging types of cancer to treat. Therefore, the search for novel effective drugs for its treatment is essential. The present study used 10-hydroxycamptothecin (HCPT), which is a naturally occurring alkaloid anticancer agent extracted from the Chinese tree, Camptotheca acuminata, and has a strong anticancer activity in vitro and in vivo. HCPT is able to induce apoptosis in cells of various tumor types. However, few studies have been conducted on its efficacy in NB, and its apoptosis-inducing mechanism has not been elucidated. In the present study, the in vitro effects of HCPT on apoptosis in the human NB cell line, SMS-KCNR, and its underlying molecular mechanisms were investigated. Cell proliferation was measured by an MTT assay and apoptosis was measured using DAPI staining and flow cytometric analysis. In addition, western blot analysis was used to evaluate the apoptosis-associated signaling pathways. HCPT was observed to markedly inhibit cell proliferation and induce apoptosis in SMS-KCNR cells at a relatively low concentration (2.5-20 nM). DAPI staining revealed typical apoptotic feature, namely apoptotic body formation. The flow cytometric analysis revealed that the number of apoptotic cells increased from 20.89% (for 2.5 nM) to 97.66% (for 20 nM) following HCPT treatment for 48 h. Western blot analysis revealed that p53, cytoplasmic cytochrome c, cleaved caspase-3 and poly ADP-ribose polymerase (PARP) proteins were significantly upregulated, while the mitochondrial cytochrome c and pro-caspase-3 proteins were downregulated. However, the B-cell lymphoma 2 and Bcl-2-associated X proteins were unaffected. The results indicated that HCPT may inhibit proliferation and induce apoptosis in the SMS-KCNR cells. The possible mechanism of apoptosis induction is the p53-mediated mitochondrial apoptotic signaling pathway, which promotes cytochrome c release and induces apoptosis by activating caspase-3 and PARP. Our study provides experimental evidence for HCPT as a potent therapeutic drug in NB treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Camptothecin; Caspase 3; Cell Proliferation; Cytochromes c; Humans; Neuroblastoma; Tumor Suppressor Protein p53

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bombyx; Camptothecin; Caspase 3; Cell Line; Cytochromes c; Endoplasmic Reticulum; Insect Proteins; Mitochondrial Membranes; Permeability; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53