• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.756-766
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• 2005

The signaling mechanism underlying aburatubolactam C-induced FasL upregulation was investigated in human Jurkat T cells. After treatment with aburatubolactam C, the src-family PTKs $p56^{lck}\;and\;p59^{fyn}$, and MAP kinases ERK2 and JNK1, were activated prior to FasL upregulation; Both $p56^{lck}\;and\;p59^{fyn}$ were directly activated 2.4- and 2.2-fold, respectively, in vitro by aburatubolactam C. The aburatubolactam C-induced cellular changes, including the activation of ERK2 and INK1, and FasL upregulation, were completely prevented by the PTK inhibitor genistein. The activation of protein kinase C (PKC$\delta,\;\epsilon\;and\;\mu$ was also induced following aburatubolactam C treatment. Although the activation of $p56^{lck}$ and tyrosine phosphorylation of the cellular proteins were not blocked by the PKC inhibitor GFl09203X, the activation of ERK2 was completely abrogated, along with a detectably enhanced JNK1 activation; FasL upregulation, and apoptosis. However, the FasL upregulation and apoptosis were significantly inhibited by the PKC activator PMA, with a remarkable increase in the ERK2 activation. The cytotoxic effect of aburatubolactam C was reduced in the presence of the anti-Fas neutralizing antibody ZB-4. Although ectopic expression of Bcl-2 failed to completely block the cytotoxicity of aburatubolactam C, it was clearly suppressed. The c-Fos mRNA expression was upregulated in a biphasic manner, where the second phasic expression overlapped with the FasL upregulation. Accordingly, these results demonstrate that aburatubolactam C-induced apoptosis is exerted, at least in part, by FasL upregulation dictated by activation of the PTK ($p56^{lck}\;and\;p59^{fyn}$) /JNKI pathway, which is negatively affected by the concurrent activation of the PKC/ERK2 pathway proximal to PTK activation.

• Molecules and Cells
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• v.25 no.3
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• pp.390-396
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• 2008

Calreticulin (CRT) is one of the major $Ca^{2+}$ binding chaperone proteins of the endoplasmic reticulum (ER) and an unusual luminal ER protein. Postnatally elevated expression of CRT leads to impaired development of the cardiac conductive system and may be responsible for the pathology of complete heart block. In this study, the molecular mechanisms that affect $Ca^{2+}$-dependent signal cascades were investigated using CRT-overexpressing cardiomyocytes. In particular, we asked whether calreticulin plays a critical role in the activation of $Ca^{2+}$-dependent apoptosis. In the cells overexpressing CRT, the intracellular calcium concentration was significantly increased and the activity of PKC and level of SECAR2a mRNA were reduced. Phosphorylation of Akt and ERKs decreased compared to control. In addition the activity of the anti-apoptotic factor, Bcl-2, was decreased and the activities of pro-apoptotic factor, Bax, p53 and caspase 8 were increased, leading to a dramatic augmentation of caspase 3 activity. Our results suggest that enhanced CRT expression in mature cardiomyocytes disrupts intracellular calcium regulation, leading to calcium-dependent apoptosis.

• Journal of Acupuncture Research
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• v.21 no.2
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• pp.41-55
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• 2004

Objective : To investigate the possible molecular mechanism (s) of melittin as a candidate of anti-cancer drug, we examined the effects of the compound on the growth of human lung carcinoma cell line A549. Methods : Growth inhibitory study, flow cytometry analysis, SDS-polyacrylamide gel electrophoresis and Western blot analysis, RT-PCR and in vitro caspases activity assay were performed. Results : Melittin treatment declined the cell viability of A549 cells in a concentration-dependent manner, which was associated with induction of apoptotic cell death. Melittin treatment down-regulated the levels of Bcl-XS/L mRNA and protein expression of A549 cells, an anti-apoptotic gene, however, the those of Bax, a pro-apoptotic gene, were up-regulated. Melittin induced the proteolytic cleavage and activation of caspase-3 and caspase-9 protease in a dose-dependent manner without alteration of inhibitor of apoptosis proteins family and Akt expression. Western blot analysis and RT-PCR data revealed that the levels of tumor suppressor p53 and cyclin-dependent kinase inhibitor p21 were also remained unchanged. Conclusions : Taken together, these findings suggest that melittin-induced inhibition of human lung cancer cell growth is associated with the induction of apoptotic cell death via regulation of several major growth regulatory gene products, and melittin may have therapeutic potential in human lung cancer.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.307-311
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG ($33.1{\pm}9.6$ vs $21.7{\pm}8.3%$). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP-treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The mRNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.3
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• pp.283-289
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• 2015

This study surveys the improvement characteristics in old-aged muscular mitochondria by bio-active materials coated fabric (BMCF). To observe the effects, the fabric (10 and 30%) was worn to old-aged rat then the oxygen consumption efficiency and copy numbers of mitochondria, and mRNA expression of apoptosis- and mitophagy-related genes were verified. By wearing the BMCF, the oxidative respiration significantly increased when using the 30% materials coated fabric. The mitochondrial DNA copy number significantly decreased and subsequently recovered in a dose-dependent manner. The respiratory control ratio to mitochondrial DNA copy number showed a dose-dependent increment. As times passed, Bax, caspase 9, PGC-$1{\alpha}$ and ${\beta}$-actin increased, and Bcl-2 decreased in a dose-dependent manner. However, the BMCF can be seen to have had no effect on Fas receptor. PINK1 expression did not change considerably and was inclined to decrease in control group, but the expression was down-regulated then subsequently increased with the use of the BMCF in a dose-dependent manner. Caspase 3 increased and subsequently decreased in a dose-dependent manner. These results suggest that the BMCF invigorates mitophagy and improves mitochondrial oxidative respiration in skeletal muscle, and in early stage of apoptosis induced by the BMCF is not related to extrinsic death-receptor mediated but mitochondria-mediated signaling pathway.

• Nutrition Research and Practice
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• v.15 no.3
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• pp.294-308
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• 2021

BACKGROUD/OBJECTIVES: Allomyrina dichotoma larva (ADL), one of the many edible insects recognized as future food resources, has a range of pharmacological activities. In a previous study, an ADL extract (ADLE) reduced the hepatic insulin resistance of high-fat diet (HFD)-induced diabetic mice. On the other hand, the associated molecular mechanisms underlying pancreatic beta-cell dysfunction remain unclear. This study examined the effects of ADLE on palmitate-induced lipotoxicity in a beta cell line of a rat origin, INS-1 cells. MATERIALS/METHODS: ADLE was administered to high-fat diet treated mice. The expression of apoptosis-related molecules was measured by Western blotting, and reactive oxidative stress generation and nitric oxide production were measured by DCH-DA fluorescence and a Griess assay, respectively. RESULTS: The administration of ADLE to HFD-induced diabetic mice reduced the hyperplasia, 4-hydroxynonenal levels, and the number of apoptotic cells while improving the insulin levels compared to the HFD group. Treatment of INS-1 cells with palmitate reduced insulin secretion, which was attenuated by the ADLE treatment. Furthermore, the ADLE treatment prevented palmitate-induced cell death in INS-1 cells and isolated islets by reducing the apoptotic signaling molecules, including cleaved caspase-3 and PARP, and the Bax/Bcl2 ratio. ADLE also reduced the levels of reactive oxygen species generation, lipid accumulation, and nitrite production in palmitate-treated INS-1 cells while increasing the ATP levels. This effect corresponded to the decreased expression of inducible nitric oxide synthase (iNOS) mRNA and protein. CONCLUSIONS: ADLE helps prevent lipotoxic beta-cell death in INS-1 cells and HFD-diabetic mice, suggesting that ADLE can be used to prevent or treat beta-cell damage in glucose intolerance during the development of diabetes.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.