Propolis Inhibits UVA-Induced Apoptosis of Human Keratinocyte HaCaT Cells by Scavenging ROS.

Propolis is a resinous material collected by honeybees from several plant sources. This research aimed at showing its protective effect against UVA-induced apoptosis of human keratinocyte HaCaT cells. Using Hoechst staining, it was demonstrated that propolis (5 and 10 µg/mL) significantly inhibited the apoptosis of HaCaT cells induced by UVA-irradiation. Propolis also showed the protective effect against loss of mitochondrial membrane potential induced by UVA-irradiaiton in HaCaT cells. Propolis also inhibited the expression of activated caspase-3 induced by UVA-irradiation. To investigate the role of ROS in UVA-induced apoptosis and protection by propolis, the generation of ROS was determined in cells. The results showed that the generation of ROS was markedly reduced in cells pretreated with propolis. Consequently, propolis protected human keratinocyte HaCaT cells against UVA-induced apoptosis, which might be related to the reduction of ROS generation by UVA-irradiation.

Erratum to "Propolis Inhibits Neurite Outgrowth in Differentiating SH-SY5Y Human Neuroblastoma Cells" [Toxicol. Res. 32 (2016) 239-243].

[This corrects the article on p. 239 in vol. 32, PMID: 27437091.].

Propolis Inhibits Neurite Outgrowth in Differentiating SH-SY5Y Human Neuroblastoma Cells.

Propolis is a multicomponent, active, complex resinous substance collected by honeybees from a variety of plant sources. We have studied the effect of propolis on neurite outgrowth of SH-SY5Y human neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Propolis, at a concentration of 3 µg/mL, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells treated with propolis (0.3~3 µg/mL) for 48 hr was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 0.3 to 3 µg/mL propolis resulted in decreased level of transglutaminase and 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The results indicate that propolis is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells.

Cadmium inhibits neurite outgrowth in differentiating human SH-SY5Y neuroblastoma cells.

Cadmium, a highly ubiquitous heavy metal, is well known to induce neurotoxicity. However, the underlying mechanism of cadmium-mediated neurotoxicity remains unclear. We have studied cadmium inhibition of neurite outgrowth using human SH-SY5Y neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Cadmium, at a concentration of 3 µmol/L, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells 48 hours after cadmium treatment (1-3 µmol/L cadmium) was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 1 to 3 µmol/L cadmium resulted in decreased level of cross-reactivities with 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The reactive oxygen species (ROS) scavenger, NAC (N-acetyl-l-cysteine), recovered the expression of GAP-43 in cadmium-treated cells. The results indicate that cadmium is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells and that this effect might result from ROS generation by cadmium.

Cadmium inhibits the differentiation of 3T3-L1 preadipocyte through the C/EBPα and PPARγ pathways.

Cadmium, a well-known toxic heavy metal, affects cellular physiology by disturbing cellular signaling pathways. We investigate the effect of cadmium on cellular differentiation using 3T3-L1 preadipocyte cell lines as an in vitro model. Here, it was shown that cadmium (3 µM) significantly decreased both glycerol-3-phosphate dehydrogenase (GPDH) activity and lipid accumulation of differentiating 3T3-L1 cells in a dose-dependent manner. In addition, inhibitory action of cadmium on differentiating 3T3-L1 cells was effective only at the initial stage of 3T3-L1 preadipocyte differentiation. Western blot analysis revealed that cadmium suppressed the expression of CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activator receptor gamma (PPARγ) proteins, key transcriptional activators for adipogenesis, in a dose-dependent manner. These results suggest that the inhibitory effects of cadmium on 3T3-L1 preadipocyte differentiation, as indicated by a decrease in GPDH activity and lipid accumulation, a marker of adipogenesis, appeared to be mediated through the downregulated expression of C/EBPα and PPARγ proteins.

Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli using two-step ultra-rapid real-time PCR.

A rapid detection method for Enterohemorrhagic Escherichia coli (EHEC), which has the virulent stx2 gene, was developed using a two-step, ultra-rapid real-time (URRT) PCR. URRT PCR was designed to detect the stx2 gene using a microchip-based, real-time PCR system, GenSpector TMC-1000, which only has a 6 microl total reaction volume with an extremely short denaturation step and combined annealing/extension step (1 and 3 s, respectively) for each cycle. Specific primers for the stx2 gene were designed to amplify a 100 bp region known for genetic stability among the various EHEC strains. Using the URRT PCR method, stx2 gene could be detected in 7 min 8 s including melting point (Tm) analysis. The detection limit for the stx2 gene for URRT-PCR was estimated to be 3 c.f.u./PCR with the amplification product having a consistent Tm of 85.2 +/- 0.4 degrees C. This method was tested for the various applications relevant to the different EHEC strains and was useful for the rapid detection of stx2-carrying EHEC strains.

Propolis inhibits the proliferation of human leukaemia HL-60 cells by inducing apoptosis through the mitochondrial pathway.

Propolis, a natural product derived from plant resins collected by honeybees, has been reported to exert a wide spectrum of biological functions. This research aimed at investigating the effect of propolis on the proliferation of human leukaemia HL-60 cells and whether propolis might induce apoptosis in HL-60 cells. The results showed dose- and time-dependent decreases in the proliferation of HL-60 cells treated with propolis (above 3 microg mL(-1) of propolis). Further studies revealed that the anti-proliferative effects of propolis were caused by inducing apoptosis. Agarose electrophoresis of genomic DNA of HL-60 cells treated with propolis showed the ladder pattern typical for apoptotic cells. Propolis induced the activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase in HL-60 cells. Propolis also induced the release of cytochrome c from mitochondria to cytosol. Taken together, these findings demonstrate that the inhibitory effect of propolis on HL-60 cell proliferation is caused by inducing apoptosis through the mitochondrial pathway.

Long-term cultured skeletal muscle-derived neural precursor cells and their neurogenic potentials.

For cell-based therapy, it is necessary to obtain sufficient cell quantities for cell transplantation to the diseased or injured site. However, a given tissue has only a limited number of stem cells, making it necessary to expand stem cell source through long-term culture. In this study, we evaluated whether our recently described skeletal muscle-derived neural precursor (SMNP) cells can be cultured long-term without alteration of their neural precursor characteristics. Our results showed that SMNP cells can be cultured over approximately 16 months, but their growth rate and neurogenic potential gradually decrease in a culture time-dependent manner. Importantly, approximately 120-day cultured SMNP cells retain their self-renewal ability, neural precursor characteristics, and high neurogenic potential. These results suggest that SMNP cells may be an appropriate cell source for regenerative or reparative therapies against neuronal disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits neurite outgrowth in differentiating human SH-SY5Y neuroblastoma cells.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental pollutant, is known to induce neurodevelopmental and neurobehavioral deficits. However, the underlying mechanism of TCDD-mediated neurotoxicity has remained unclear. We have studied TCDD inhibition of neurite outgrowth using human SH-SY5Y neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). TCDD, at concentrations of 3 nM or 5 nM, had no significant effect on the viability of either undifferentiating or differentiated SH-SY5Y cells. However, differentiating SH-SY5Y cells exhibited a distinct decrease of neurite outgrowth 48 h after TCDD treatment in a dose-dependent manner. TCDD treatment 12h or 24h after RA stimulation did not elicit a significant inhibition of neurite outgrowth, whereas TCDD cotreatment with RA or TCDD treatment at 6h after RA stimulation significantly inhibited neurite outgrowth. Western blot analysis of cell extracts of RA-stimulated differentiating SH-SY5Y cells showed increased level of cross reactivities with tissue glutaminase (TGase) antibody compared to control extracts, in a time-dependent manner. By contrast, treatment of differentiating SH-SY5Y cells with 1-5 nM TCDD resulted in decreased level of cross-reactivities with TGase antibody in a dose-dependent manner. The results indicate that TCDD is able to inhibit neurite outgrowth by differentiating SH-SY5Y cells and that this effect might result from reduced levels of TGase.

Involvement of Blimp-1 and AP-1 dysregulation in the 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated suppression of the IgM response by B cells.

B cell differentiation and humoral immune responses are markedly suppressed by the persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The suppression of humoral immune responses by TCDD occurs by direct actions on the B cell and involves activation of the aryl hydrocarbon receptor. Transcriptional regulation of paired box gene 5 (Pax5), an important regulator of B cell differentiation, is altered by TCDD in concordance with the suppression of B cell differentiation and humoral immunoglobulin M response. We hypothesized that TCDD treatment leads to dysregulation of Pax5 transcription by interfering with the basic B cell differentiation mechanisms and aimed to determine the effects of TCDD on upstream regulators of Pax5. A critical regulator of B cell differentiation, B lymphocyte-induced maturation protein-1 (Blimp-1) acts as a transcriptional repressor of Pax5. In lipopolysaccharide (LPS)-activated murine B cell lymphoma, CH12.LX, Blimp-1 messenger RNA, and DNA-binding activity within the Pax5 promoter were suppressed by TCDD. Furthermore, LPS activation of CH12.LX cells upregulated DNA-binding activity of activator protein 1 (AP-1) at three responsive element-like motifs within the Blimp-1 promoter. TCDD treatment of LPS-activated CH12.LX cells suppressed AP-1 binding to these motifs between 24 and 72 h, in concordance with the suppression of Blimp-1 by TCDD. A more comprehensive analysis at 72 h demonstrated that the suppression of AP-1 binding within the Blimp-1 promoter by TCDD was concentration dependent. In summary, our findings link the TCDD-mediated suppression of Blimp-1 through AP-1 to the dysregulation of Pax5, which ultimately leads to the suppression of B cell differentiation and humoral immune responses.

Apoptosis Induction by Menadione in Human Promyelocytic Leukemia HL-60 Cells.

Cell death induced by menadione (vitamin K-3,2-methyl-1,4-naphthoquinone) has been investigated in human promyelocytic leukemia HL-60 cells. Menadione was found to induce both apoptosis and necrosis in HL-60 cells. Low concentration (1~50 µM) of menadione induced apoptotic cell death, which was demonstrated by typical DNA ladder patterns on agarose gel electrophoresis and flow cytometry analysis. In contrast, a high concentration of menadione (100 µM) induced necrotic cell death, which was demonstrated by DNA smear pattern in agarose gel electrophoresis. Necrotic cell death was accompanied with a great reduction of cell viability. Menadione activated caspase-3, as evidenced by both increased protease activity and proteolytic cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP) into 85 kDa cleavage product. Caspase-3 activity was maximum at 50 µM of menadione, and very low at 100 µM of menadione. Taken together, our results showed that menadi-one induced mixed types of cell death, apoptosis at low concentrations and necrosis at high concentrations in HL-60 cells.

Activity of protein kinase C modulates the apoptosis induced by polychlorinated biphenyls in human leukemic HL-60 cells.

Polychlorinated biphenyls (PCBs) induce apoptotic cell death of HL-60 cells. In the present study, we examined the possible involvement of protein kinase C (PKC) in PCB-induced apoptosis of HL-60 cells. Treatment of cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, suppressed DNA fragmentation induced by PCBs in HL-60 cells. Treatment with another active phorbol ester, phorbol-12,13-dibutyrate (PDBu), also suppressed PCB-induced DNA fragmentation, whereas 4alpha-phorbol-12,13-didecanoate (4alphaPDD), an inactive phorbol ester, did not affect PCB-induced apoptosis of HL-60 cell. Moreover, 1-oleoyl-2-acetyl-sn-glycerol (OAG), an activator of PKC that is not a phorbol ester, also suppressed PCB-induced DNA fragmentation. However, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, increased DNA fragmentation induced by PCBs. These results demonstrate that the activation of PKC is responsible for the suppression of PCB-induced apoptosis of HL-60 cells. Furthermore, inhibition of PKC promotes DNA fragmentation of HL-60 cells treated with PCBs, thereby suggesting the involvement of PKC activity in PCB-induced apoptosis of HL-60 cells.