Abstract
BackgroundDiscovering how to regulate mitochondrial function to reduce cancer growth holds great potential for future cancer therapy development. Here we explore the effects of cryptotanshinone (CPT), a natural product derived from Salvia miltiorrhiza, on mitochondria of osteosarcoma (OS) both in vitro and in vivo, and further elucidate the underlying molecular mechanisms.MethodsCytotoxicity in the CPT treated OS cells was analyzed by flow cytometry, CCK8, TUNEL assay and colony formation assays. Flow cytometric analysis was performed to evaluate the effect of CPT on cell cycle of OS cells. Mitochondrial morphology was examined by staining with the mitochondrial membrane potential -sensitive fluorochrome, MitoTracker Red (CMXRos). Immunoblotting, confocal-immunofluorescence staining, co-immunoprecipitation were used to examine the expression and interaction between CPT-mediated Drp1 and Bax. Finally, the synergistic effect of CPT on OS cells was validated using a mouse xenograft tumor model.ResultsIn this study, we found CPT treatment induced S-phase arrest, apoptosis, and mitochondrial fragmentation in OS cells. CPT also effectively activated caspase-dependent apoptosis, which could be blocked by pan-caspase inhibitor Z-VAD-FMK. Moreover, we herein provide evidence that treatment with CPT resulted in mitochondrial fragmentation, which is mediated by dynamin-related protein 1 (Drp1), a key mediator of mitochondrial fission. Pursuing this observation, downregulation of Drp1 via silencing RNA could abrogate the induction of apoptosis and mitochondrial fragmentation induced by CPT. Finally, we demonstrate that CPT induced Drp1, which interacted directly with Bcl-2-associated X protein (Bax), which contributed to driving Bax translocation from the cytosol to the mitochondria.ConclusionsOur findings offer insight into the crosstalk between mitochondrial fragmentation and inhibition of osteosarcoma cell growth in response to CPT.
Highlights
Osteosarcoma is the most commonly occurring form of malignant bone tumor
These results indicate that CPT-induced dynamin-related protein 1 (Drp1) upregulation results in mitochondrial fission, which contributes to OS cell death
The results were expressed as the means ± standard deviation (SD) from three independent experiments. **P < 0.01, significantly different compared with control caspase-8, and cleaved caspase-9 as well as higher expressions of Bak, Bcl-2-associated X protein (Bax) and Bak; whereas the level of Bcl-2 was decreased in comparison with the vehicle control group (Fig. 7a)
Summary
Osteosarcoma is the most commonly occurring form of malignant bone tumor. Osteosarcoma can affect people of all ages, it most often occurs in children and teens who are still growing, which indicates genetic and molecular alterations that disrupt osteoblast differentiation are important factors in the etiology of the disease. Treat ments of osteosarcoma include surgery, radiation, chemotherapy, or a combination of radiotherapy and chemotherapy; such therapies are often negatively charac terized by toxicity and side effects. Considering the long-term and short-term toxicities of chemotherapeutic agents commonly used to treat osteosarcoma, a more promising approach targets the development of effective, and nontoxic therapeutic strategies, using active constitutive agents extracted from natural sources. We explore the effects of cryptotanshinone (CPT), a natural product derived from Salvia miltiorrhiza, on mitochondria of osteosarcoma (OS) both in vitro and in vivo, and further elucidate the underlying molecular mechanisms
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