| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 103 === Abstract
Background:
Renal-cell carcinoma is characterized by a lack of early-warning signs which results in a proportion of patients with metastases and therapeutic inefficiency. 16-hydroxycleroda-3,13-dien-15,16-olide (HCD),was reported to possess various biological functions such as anti-cancer, anti-oxidant, anti-angiogenesis, and anti-inflammation, etc.HCD has been shown to inhibit cancerous cells proliferation including lung, brain, leukemia, colon cells. In the present study, we examined the effects of HCD on tumor regression by using in vitro renal cell carcinoma cell culture.
Materials and methods:
Cell viability was determined by morphological analysis, MTT assays and clonogenic assay. Cell migration was measured by transwell and wound healing method.The levels of mitochondrial membrane potential (examined by JC-1 and Rhodamine123) and intrecellular ROS accumulation (was using Carboxy-H2DCFDA) were determined by flow cytometry. Apoptotic effects were confirmed by acridine orange/ethidium bromide (AO/EB) assay.Cell cycle effects were evaluated by flow cytometry. The related apoptotic caspase signaling molecular, and cell cycle dependent kinases and proteins were examind (CDKs) by immunobloting analysis.
Results:
The anti-proliferative effect of HCD on 786-O and A-498 cells was prominent after 24 h treatment.HCD caused RCC cell death in a concentration- and time-dependent manner.AO/EB assays further confirmed that HCD indeced RCC cells apoptosis. Using flow cytometry, we further confirmed that HCD arrested RCC cells at G2/M phase and increased cell numbers at subG1 phase.By immunoblot analysis,the cell cycle dependent proteins, such as cyclin B1, cyclin D, cyclin E, CDK2 and CDK4 were all down-regulated by HCD treatment.On the other hand, HCD stimulated ROS production and decreased the levels of mitochondrial membrane potential in RCC cells which further activated the intrinsic apoptosic pathways. HCD treatment induced cytochrome c leakage from mitochondrial and reduced the expression of anti-apoptotic protein Bcl-2. Moreover, HCD increased expression of cleaved caspase 3,7,9 and cleaved PARP.
HCD is a multi-kinase drug for RCC cells.It blocked several cellular signaling pathway stimultaneously, therefore,arrested the cell cycle, caused cell death and even inhibit the migration of cancer cells.The HCD-induced signaling pathways include Akt/mTOR/HIF-2α,MEK/ERK, FAK/Paxillin/Vinculin,NF-κB,and HSP70.
In addation,lower concentration of HCD blocked migration in both cell lines without cytotoxicity.Result showed that HCD blocked phosphorylation of NF-κB.These results suggested that HCD may reduce the invasion and migration in RCC cells.
Summary:
The results indicate that HCD exerts cell cycle arrest and caspase-dependent cell death in RCC cells,supporting its potential usage in chemotherapeutics choice for kidney cancer.
Keyword:
16-hydroxycleroda-3,13-dien-15,16-olide (HCD), renal cell carcinoma, apoptosis.
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