Abstract
BackgroundHarmine is a beta-carboline alkaloid from the plant Peganum harmala. Previous studies found that harmine inhibited metastasis of B16F-10 melanoma cells. This study aims to elucidate the role of harmine in apoptosis of B16F-10 cells.MethodsB16F-10 melanoma cells were treated in the presence and absence of harmine in vitro. Morphological changes, cell cycle and expression of various pro and anti- apoptotic genes were analyzed for the study of apoptosis.ResultsMorphological observation and DNA laddering assay showed that harmine treated cells displayed marked apoptotic characteristics, such as nuclear fragmentation, appearance of apoptotic bodies and DNA laddering fragment. TUNEL assay and flow cytometric analysis also confirmed apoptosis. Furthermore, RT-PCR analysis showed that harmine induced apoptosis in B16F-10 melanoma cells by up-regulating Bax and activating Caspase-3, 9 and p53 and down-regulating Bcl-2. Harmine also up-regulated Caspase-8 and Bid, indicating that harmine affected both extrinsic and intrinsic pathways of apoptosis. This study also showed inhibitory effects of harmine on some transcription factors and pro- inflammatory cytokines that protect cell from apoptosis.ConclusionHarmine activates both intrinsic and extrinsic pathways of apoptosis and regulates some transcription factors and pro-inflammatory cytokines.
Highlights
Harmine is a beta-carboline alkaloid from the plant Peganum harmala
Apoptosis involves a sequence of specific morphological changes in a dying cell: condensation of the cytoplasm and nuclear chromatin, followed by breakage of cells into membrane bound apoptotic bodies containing a variety of cytoplasmic organelles and nuclear fragments, which are engulfed by neighboring cells and macrophages [2]
Harmine up to 2 μg/mL, was not directly cytotoxic to B16F10 melanoma cells and concentrations of 0.5, 1 and 2 μg/mL were used for further experiments
Summary
Harmine is a beta-carboline alkaloid from the plant Peganum harmala. Previous studies found that harmine inhibited metastasis of B16F-10 melanoma cells. This study aims to elucidate the role of harmine in apoptosis of B16F-10 cells. Apoptosis pathways can generally be divided into signaling via the death receptors (extrinsic) or the mitochondria (intrinsic) pathways. Both pathways lead to activation of the members of highly selective proteases referred to as ‘Caspases’ [3]. P53 is a nuclear transcription factor that accumulates in response to cellular stress, including DNA damage and oncogene activation. This triggers transcriptional trans activation of p53 target genes such as p21, p27, Bax, leading to cell cycle arrest, senescence and/or apoptosis [7]. The p53 tumour-suppressor protein can intervene at every major step in apoptotic pathways as a key regulator of apoptosis and carcinogenesis [8]
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