Colchicine-induced apoptosis in human normal liver L-02 cells by mitochondrial mediated pathways
New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, Jiangsu, China. Toxicology in Vitro
(Impact Factor: 2.9).
02/2012; 26(5):649-55. DOI: 10.1016/j.tiv.2012.01.024
Colchicine is an alkaloid that has been widely used to treat gout. It also has a curative effect on cancer. Although many studies have shown that its effect on cell apoptosis was mediated by the activation of caspase-3, the pathways involved in the process remained obscure. Here we show some evidence regarding the missing information using human normal liver cells L-02 in our study. The effect of colchicine on apoptosis in L-02 cells and the apoptosis-associated signaling pathways were determined using different tests including cell viability assay, Annexin V and propidium idodide binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential assay, caspase activity assay and Western blot analysis. We found that colchicine-induced a dose-dependent drop of cell viability in L-02 cells; early apoptosis happened when cells were treated with 0.1μM of colchicine. The colchicine-induced loss of mitochondrial membrane potential, activation of caspase-3 and 9, up-regulation of Bax and down-regulation of Bcl-2 showed an evidence for the colchicine activity on apoptosis, at least, by acting via the intrinsic apoptotic pathway.
Available from: Malgorzata Czyz
- "At this concentration it was also not highly effective against clonogenic cells. It has been shown recently that colchicine exerted anti-proliferative effect in hepatocellular carcinoma cell lines , but the significance of this finding is diminished by observation that it also reduced viability in normal liver cells . Given these results, colchicine is probably not a good drug candidate for the treatment of melanoma. "
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ABSTRACT: Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study.
We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed.
Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC.
Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.
PLoS ONE 03/2014; 9(3):e90783. DOI:10.1371/journal.pone.0090783 · 3.23 Impact Factor
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ABSTRACT: Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early phase of apoptosis induced by both apoptosis inducing agents. The analysis of expression of the apoptosis related genes in MonoMac6 cultures treated with puromycin and exposed to PEMF performed in reverse transcription of polymerase chain reaction (PCR) assay has shown changes in mRNA of genes engaged in intrinsic apoptotic pathway and pathway with AIF abundance. The most influenced was expression of gene belonging to pro-apoptotic family of Bcl-2 and AIF agent. Examination of immunoblots developed with anti-AIF antibody showed that cytosol content of AIF protein was diminished after puromycin and PEMF treatment of MonoMac6 cells. The obtained results indicate that PEMF affects induction of apoptosis in MonoMac6 cells stimulated to death with inducing agents to a different extent. Main finding of the current results is that, PEMF stimulation of MonoMac6 cells simultaneously treated with puromycin caused changes in the Bcl-family genes expression as well as in caspase independent pathway of apoptosis inducing factor (AIF).
Journal of physiology and pharmacology: an official journal of the Polish Physiological Society 10/2012; 63(5):537-45. · 2.39 Impact Factor
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ABSTRACT: Nitrogen-containing bioactive alkaloids of plant origin play a significant role in human health and medicine. Several semisynthetic antimitotic alkaloids are successful in anticancer drug development. Gloriosa superba biosynthesizes substantial quantities of colchicine, a bioactive molecule for gout treatment. Colchicine also has antimitotic activity, preventing growth of cancer cells by interacting with microtubules, which could lead to the design of better cancer therapeutics. Further, several colchicine semisynthetics are less toxic than colchicine. Research is being conducted on effective, less toxic colchicine semisynthetic formulations with potential drug delivery strategies directly targeting multiple solid cancers. This article reviews the dynamic state of anticancer drug development from colchicine semisynthetics and natural colchicine production and briefly discusses colchicine biosynthesis.
Current Medicinal Chemistry 12/2012; 20(7). DOI:10.2174/092986713805219073 · 3.85 Impact Factor
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