Realgar-mediated growth inhibition on HaCaT human keratinocytes is associated with induction of apoptosis
School of Chinese Medicine, Faculty of Science, Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, PR China. International Journal of Molecular Medicine
(Impact Factor: 2.09).
09/2009; 24(2):189-96. DOI: 10.3892/ijmm_00000222
Traditional Chinese medicine has long been used to treat a variety of ailments including skin diseases. Our previous study has revealed the ethanolic extract of realgar, a common ingredient used in psoriasis treatment in Chinese medicine, to possess potent anti-proliferative action on cultured HaCaT cells of human keratinocyte origin. In the present study, the mechanisms of action of the observed growth inhibitory action of realgar were investigated. Several bioassay methods were employed to elucidate whether cellular apoptosis is involved in the realgar-induced growth inhibition of the skin cells. Morphologically, nuclear condensation and DNA fragmentation were observed when HaCaT cells were exposed to the realgar extract. DNA fragmentation induced by the treatment of realgar was also evident as detected by gel electrophoresis and the TUNEL method. Cell cycle analysis by propidium iodide (PI) staining demonstrated the appearance of sub-G1 peak and cell cycle arrest at the G1 phase upon realgar treatment. Quantitative analysis by annexin V-PI staining revealed that the realgar-induced apoptotic event was dose-dependent. Furthermore, realgar was able to activate caspase-3 expression when examined by Western blot analysis. Our experimental data unambiguously confirm that induction of cellular apoptosis is mainly responsible for the observed growth inhibition brought about by realgar on the HaCaT keratinocytes, and this finding helps place the traditional use of this mineral for psoriasis treatment on a scientific footing.
Available from: PubMed Central
- "Compared to As2O3, As4S4 is generally well tolerated with moderate side effects and possesses the biologic property of less toxical and adverse reaction . Although recent studies revealed that the therapeutic action of As4S4 is closely associated with the induction of cellular apoptosis , , , , the definitive molecular mechanism of action of As4S4 in APL therapy still remains unknown. In the present study, As4S4 was further confirmed to inhibit the growth of RA-resistant NB4-R1 cells in a time- and dose-dependent manner. "
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ABSTRACT: Tetra-arsenic tetra-sulfide (As4S4) is an arsenic compound with anti-tumor activity, especially in acute promyelocytic leukemia (APL) that are resistant to retinoic acid (RA). Although recent studies revealed that the therapeutic action of As4S4 is closely associated with the induction of cellular apoptosis, the exact molecular mechanism of action of As4S4 in RA-resistant APL remains to be clarified. In this study, we found that As4S4-induced apoptosis was accompanied by reduced mRNA and protein expression of SET gene in RA-resistant NB4-R1 cells. Moreover, RNAi knockdown of SET gene further promoted As4S4-induced apoptosis, while SET over-expression inhibited it, suggesting that As4S4 induces apoptosis through the reduction of SET protein in NB4-R1 cells. We also demonstrated that the knockdown of SET gene resulted in the upregulation of protein phosphatase 2 (PP2A) expression and the downregulation of promyelocytic leukemia and retinoic acid receptor α fusion gene (PML-RARα) expression, which were enhanced by As4S4 treatments. By contrast, over-expression of SET gene resulted in PP2A downregulation and PML-RARα upregulation, which were abolished by As4S4 pretreatment. Since PP2A is a pro-apoptotic factor and PMLRARα is an anti-apoptotic factor, our results suggest that As4S4-induced apoptosis in NB4-R1 cells is through the downregulation of SET protein expression, which in turn increases PP2A and reduces PML-RARα expressions to lead to cell apoptosis.
Available from: ncbi.nlm.nih.gov
- "MTT assays demonstrated that realgar nanoparticles, purified realgar, and traditional realgar inhibited the growth of C6 cells in a dose- and time-dependent manner (Figure 4). These findings are consistent with those of previous studies showing that realgar significantly suppressed the proliferation of tumor cells, including HaCaT, SiHa, and NB4-R1 cells, in a dose-dependent manner.6–8 Realgar nanoparticles showed the greatest anti-proliferative effect, followed by purified realgar and then traditional realgar (P < 0.05 for each). "
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ABSTRACT: Our objective was to prepare a new nano-sized realgar particle and characterize its anti-tumor effect on tumor cells.
Nanoparticles were prepared by coprecipitation and were detected by transmission electron microscopy, scanning electron microscopy, energy dispersive spectrometry (EDS), and dynamic light scattering. An anti-proliferative effect of realgar nanoparticles on rat glioma (C6) cells was determined by the MTT assay. Cell cycle and apoptosis rates were observed by flow cytometry. Apoptosis-related gene expression was detected by immunofluorescence staining.
Realgar nanoparticles were successfully prepared. The particles were spherical, with an average diameter of approximately 80 nm, and contained arsenic and sulfur elements. Realgar nanoparticles inhibited C6 cell proliferation and induced apoptosis in a dose- and time-dependent manner. Treatment of C6 cells with realgar nanoparticles significantly increased the proportions of cells in S and G2/M phases, decreased the proportion of cells in G0/G1 phase, downregulated Bcl-2 expression, and substantially upregulated Bax expression.
Realgar nanoparticles significantly inhibited C6 glioma cell proliferation and promoted cell apoptosis by inducing the upregulation of Bax and downregulation of Bcl-2 expression. Realgar nanoparticles are a promising in vitro anti-cancer strategy and may be applicable for human cancer therapy studies.
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ABSTRACT: We carried out live imaging of PC12 cells expressing SCAT3, a caspase-3 cleavage peptide sequence linking two fluorescent proteins, ECFP and Venus, which function respectively as the donor and acceptor for FRET. Live imaging of SCAT3-expressing cells was performed from 60 to 300 min after exposure to sodium arsenite (NaAsO(2): 0, 1, 5, or 10 μM) was initiated. We then measured the emission ratio of ECFP to Venus to monitor the activity of caspase-3 and found that the ratio was temporally and dose-dependently increased by NaAsO(2). The mean ECFP/Venus emission ratio between 200 and 300 min after exposure to NaAsO(2) at a dose of 5 or 10 μM, but not at 1 μM, was significantly higher than that in the control group. We showed by other methods that NaAsO(2) significantly increased the amount and activity of mature caspase-3 and the amount of nucleosomes generated from DNA fragmentation, and decreased cell viability. However, methods other than live imaging required a longer time and higher doses of NaAsO(2) than did live imaging to detect significant effects. This result suggests that live imaging using SCAT3 is a useful method for the screening of chemical toxicities and for improving the efficiency of toxicity evaluation.
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