Antiproliferative and Cytotoxic Effects of Resveratrol in Mitochondria-Mediated Apoptosis in Rat B103 Neuroblastoma Cells

Resveratrol, a natural compound, has been shown to possess anti-cancer, anti-aging, anti-inflammatory, anti-microbial, and neuroprotective activities. In this study, we examined the antiproliferative and cytotoxicity properties of resveratrol in Rat B103 neuroblastoma cells; although it's molecular mechanisms for the biological effects are not fully defined. Here, we examined the cellular cytotoxicity of resveratrol by cell viability assay, antiproliferation by BrdU assay, DNA fragmentation by DNA ladder assay, activation of caspases and Bcl-2 family proteins were detected by western blot analyses. The results of our investigation suggest that resveratrol increased cellular cytotoxicity of Rat B103 neuroblastoma cells in a dose-and time-dependent manner with IC50 of 17.86 microM at 48 h. On the other hand, incubation of neuroblastoma cells with resveratrol resulted in S-phase cell cycle arrests which dose-dependently and significantly reduced BrdU positive cells through the downregulation of cyclin D1 protein. In addition, resveratrol dose-dependently and significantly downregulated the expression of anti-apoptotic protein includes Bcl-2, Bcl-xL and Mcl-1 and also activates cleavage caspase-9 and-3 via the downregulation of procaspase-9 and -3 in a dose-dependent manner which indicates that involvement of intrinsic mitochondria-mediated apoptotic pathway. In conclusion, resveratrol increases cellular cytotoxicity and inhibits the proliferation of B103 neuroblastoma cells by inducing mitochondria-mediated intrinsic caspase dependent pathway which suggests this natural compound could be used as therapeutic purposes for neuroblastoma malignancies.

A Mechanism for the Up-regulation of the IL-8 Gene Expression in Keratinocytes by All-trans Retinoic Acid / 대한피부과학회지

BACKGROUND: Retinoic acid (RA) has been reported to induce the up-regulation of inflammatory cytokines such as IL-1, TNF-alpha and IL-8 in dermal fibroblasts and keratinocytes. There is no evidence to support a direct interaction between the RA-mediated transcriptional machinery and IL-8 gene transcription. OBJECTIVE: The aim of this study is to clarify the mechanism of the up-regulation of IL-8 in keratinocytes by RA. METHODS: The IL-1, IL-8, TNF-alpha and MCP-1 mRNA expressions in HaCaT cells stimulated by RA were measured by quantitative RT-PCR. The effects of a NF-kappaB inhibitor and IL-1 receptor antagonist (ra) on the IL-8 mRNA expression were measured by quantitative RT-PCR. Electrophoretic motility shift assay (EMSA) was conducted on the RA-stimulated HaCaT cells that were or were not treated with NF-kappaB inhibitor to measure the NF-kappaB binding activity in each group. The phospho-IkappaB activity in the HaCaT cells after stimulation with RA was also measured by Western blotting. RESULTS: An up-regulation of the IL-8 gene expression by RA was demonstrated in the HaCaT cells. The inhibition assay revealed the involvement of the NF-kappaB binding site of the IL-8 gene in the RA-enhanced promoter activity. EMSA demonstrated that RA enhanced the formation of the DNA-NF-kappaB complex. There was no evidence to support IL-1 as an intermediate stimulus between the RA-mediated transcriptional machinery and IL-8 gene transcription. Western blot analysis revealed increased phospho-IkappaB activity in the HaCaT cells after stimulation with RA. CONCLUSION: Our result suggested that the IL-8 gene expression of HaCaT cells after RA stimulation is caused by the activation of IKK and the dissociation of IkappaB from NF-kappaB and the transcription of NF-kappaB in the nucleus.

The Effect of Pemphigus Sera on Apoptosis of Keratinocytes / 대한피부과학회지

BACKGROUND: Pemphigus is an autoimmune blistering disease of skin and mucous membrane characterized by loss of adhesion between keratinocytes, a process known as acantholysis. The target molecules of autoantibodies are desmogleins which belong to cadherin family proteins of desmosome. Independently from the autoantibodies involved, the question whether apoptosis plays a role in the pathogenesis of pemphigus has not been addressed. OBJECTIVES: The purpose of this study was to determine if pemphigus sera induce apoptosis in HaCaT cells and to elucidate the mechanism of apoptosis induced by pemphigus sera. METHODS: We treated pemphigus sera on HaCaT cells for 48h and investigated whether apoptosis was induced or not through FACS analysis and TUNEL staining. In addition, we performed western blot analysis to detect cleavage of PARP, cytosolic cytochrome c and activation of caspase-8, 3, which play a major role in apoptosis. Soluble Fas ligand protein level in pemphigus sera was compared with the level in normal sera by ELISA. Pemphigus sera from which IgG was eliminated, were treated on HaCaT cells to elucidate the role of IgG in pemphigus sera-induced apoptosis. RESULTS: Using FACS analysis with Annexin-V and PI staining, we have shown that pemphigus sera induced apoptosis in HaCaT cells. TUNEL staining assay also confirmed the apoptosis were induced in HaCaT cells after treatment of pemphigus sera. Western blot analysis showed activation of caspase-3, release of cytochrome c, cleavage of PARP and decrease of procaspase-8 which is proform of active caspase-8 in HaCaT cells treated with pemphigus sera. We could not detect increased Fas ligand protein level in pemphigus sera compared with the level in normal sera. Moreover, since the removal of IgG obliterated the apoptosis inducing effects of pemphigus sera in HaCaT cells, IgG might be involved in the activation of caspase-8 and caspase-3. CONCLUSION: We concluded that treatment of pemphigus serum induces the apoptosis of HaCaT cells through, at least in part, activation of caspase-8, caspase-3 and cytochrome c related mechanism, and that IgG in the pemphigus serum rather than Fas ligand is involved in the apoptosis. These results suggest that apoptosis may play an important role in the pathomechanism of pemphigus lesion.