Berberine, a naturally occurring isoquinoline alkaloid, is present in a number of important medicinal plants. Berberine has a wide range of biochemical and pharmacological effects, including anticancer effects. In this study, we elucidated the mechanism of antiangiogenic activity of berberine using in vivo and in vitro models. In vivo antiangiogenic activity was studied using B16F-10 melanoma cells and induced capillary formation in C57BL/6 mice. Berberine, at 10 mg/kg body weight, showed significant inhibition in tumor-directed capillary formation and in various proangiogenic factors, such as vascular endothelial growth factor (VEGF), and proinflammatory mediators, such as interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNF-α), and granulocyte macrophage colony-stimulating factor (GM-CSF), which are involved in tumor angiogenesis. At the same time, it could also increase antitumor factors, such as IL-2 and tissue-inhibitor metalloproteinase (TIMP) levels in the serum. Berberine could also inhibit endothelial motility, migration, tube formation, and vessel sprouting from rat aortic ring in vitro. Further, berberine inhibited various transcription factors involved in tumor development and angiogenesis, such as NF-ĸB, c-Fos, CREB, and ATF-2. mRNA expression levels of proangiogenic factors, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and hypoxia-inducible factor (HIF), were also downregulated in tumor cells after treatment with berberine. Drastically elevated expressions of HIF and VEGF mRNA by tumor cells under hypoxic conditions were also decreased after treatment with berberine. This result clearly demonstrates that the antiangiogenic activity of berberine is mainly mediated through the inhibition of various proinflammatory and pro-angiogenic factors and the major ones are HIF, VEGF, COX-2, NO, NF-ĸB, and proinflammatory cytokines.
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.
Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli, cabbage, cauliflower, etc. SFN has received a great deal of attention because of its ability to inhibit cell proliferation and induce apoptosis in several tumor cell lines. Previously, we have demonstrated that SFN inhibits the metastasis of B16F-10 melanoma cells in both in vivo and in vitro models. Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high tendency to metastasize. In this study, we investigated the influence of SFN on the induction of apoptosis in B16F-10 melanoma cells, which was evidenced by morphological changes such as membrane blebbing, presence of apoptotic bodies, DNA condensation, and also by nuclear DNA fragmentation. SFN-induced apoptosis was associated with the activation of caspases 3 and 9, Bax, and p53 and the downregulation of Bcl-2, caspase-8, Bid, and NF-kB. Caspase-3 is a most likely candidate to mediate SFN-induced apoptosis. In addition to the caspase-dependent pathway, our results also showed the involvement of proinflammatory cytokines, namely tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-12p40, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the nuclear translocation of factors kappa B (NF-κB) p65, NF-κB p50, NF-κB c-Rel, c-FOS, ATF-2, and CREB-1 in SFN-induced apoptosis. These results raise the possibility that SFN may be a promising candidate for molecular-targeting chemotherapy against melanoma.
The present study demonstrated the potential antimetastatic and antiinvasive effect of berberine using both in vivo mouse lung metastasis and in vitro models. Administration of berberine resulted in significant suppression of B16F-10 melanoma induced tumor nodule formation and enhanced the survival of tumor-bearing mice. Berberine treatment also decreased various biochemical parameters associated with lung metastasis. These inhibitory actions may be due to the significant suppression of several signaling molecules such as ERK1/2, NF-κB, ATF-2 and CREB involved in the transcription signaling pathways for MMP gene expression. It could also inhibit the migration and invasion of highly metastatic murine melanoma cells in a dose-dependent manner in vitro. The results clearly show that berberine could significantly inhibit experimental lung metastasis produced by intravenous injection of B16F-10 melanoma cells and this effect could be linked to the down-regulation of metastasis-related signaling molecules.
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