• Development and Reproduction
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• v.10 no.3
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• pp.203-209
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• 2006

Ethane 1,2-Dimethane sulfonate(EDS), a toxin which specifically kills Leydig cells(LC), has been widely used to prepare the reversible testosterone(T) depletion rat model. Previous studies including our own clearly demonstrated that the dramatic weight loss of the T-dependent accessory sex organs such as epididymis and seminal vesicle in this 'LC knock-out' rats. These weight loss could be derived from massive and abrupt death of the cells via apoptotic process. The present study was performed to test the effect of EDS administration on the expression of some apoptotic genes in the rat epididymis. Adult male Sprague-Dawley rats($300{\sim}350$ g B.W.) were injected with single dose of EDS(75 mg/kg, i.p.) and sacrificed on Weeks 0, 1, 2, 3, 4, 5, 6 and 7. Tissue weights and the numbers of the epididymal sperm were measured. The transcriptional activities of the bcl-2, bax, Fas and Fas ligand(Fas-L) were evaluated by semi-quantitative RT-PCR. As expected, the weights and the sperm counts of epididymis declined progressively after the EDS treatment during Week 1 and 2. These decrements were discontinued with a gradual return towards normal during Weeks $5{\sim}7$, although the maximal recoveries of the epididymal weights(71%) and sperm count(38%) were subnormal on Week 7. The initial level of bcl-2 transcripts persisted to Week 6 then elevated significantly on Week 7. The level of bax transcripts significantly decreased on Week 6, and no remarkable change was found in the rest of the experimental period. The transcripts for the Fas in epididymis elevated during Weeks $1{\sim}2$, returned to normal on Week 3, and the level persisted to the Week 7. Similarly, the level of Fas-L transcripts elevated during Weeks $1{\sim}3$ and returned to normal after Week 4. Our results demonstrated the transient T depletion by EDS administration could induce the changes in expression of the apoptotic genes in rat epididymis. The activation of Fas and Fas-L in the epididymis of EDS-treated rats might be responsible for the initial apototic process and consequently the tissue damage and the sperm loss. Future studies will attempt to determine the precise molecular mechanism(s) of apoptosis in the rat epididymis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2169-2177
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• 2014

Matrine, a main active component extracted from dry roots of Sophora flavecens, has been reported to exert antitumor effects on A549 human non-small lung cancer cells, but its mechanisms of action remain unclear. To determine effects of matrine on proliferation of A549 cells and assess possible mechanisms, MTT assays were employed to detect cytotoxicity, along with o flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide to analyze cell cycle distribution. Western blotting was performed to determined expression levels of Bax, Bcl-2, VEGF and HDAC1, while a microarray was used to assessed changes of miRNA profiles. In the MTT assay, matrine suppressed growth of human lung cancer cell A549 in a dose- and timedependent manner at doses of 0.25-2.5 mg/ml for 24h, 48h or 72h. Matrine induced cell cycle arrest in G0/G1 phase and decreased the G2/M phase, while down-regulating the expression of Bcl2 protein, leading to a reduction in the Bcl-2/Bax ratio. In addition, matrine down regulated the expression level of VEGF and HDAC1 of A549 cells. Microarray analysis demonstrated that matrine altered the expression level of miRNAs compared with untreated control A549 cells. In conclusion, matrine could inhibit proliferation of A549 cells, providing useful information for understanding anticancer mechanisms.

• Journal of Life Science
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• v.19 no.2
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• pp.157-162
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• 2009

Nitric oxide (NO) is a diffusible, multifunctional and transcellular messenger that has been implicated in numerous physiological and pathological conditions. It has been reported that NO induced apoptosis in tumor cells, macrophage cells and inhibited apoptosis in normal cells, endothelial cells. To examine whether NO could induce apoptosis in MCF-7 cells, cells were treated with SIN-1 (3-morpholinosydnonimine), NO donor. Cell viability did not change in SIN-1 treated cells for 48 h and there was no significantly changes in cell cycle progression or growth pattern by FACS analysis. But p53 protein, an apoptosis-related factor, increased SIN-1 treatment time dependently. Bcl-2, MDM2 and p21 were also accumulated. Bax level did not change. A major role of inhibiting apoptosis by NO in MCF-7 cells, cobalt chloride ($CoCl_2$) was added to cells preincubated with SIN-1. Whereas $CoCl_2$ treated cells underwent apoptosis, for 24 h SIN-1 preincubated cells were not induced apoptosis. Inactivated proteins, MDM2 and bcl-2, by $CoCl_2$ levels also increased in SIN-1 pre-treated cells. These results suggested that SIN-1 blocked p53 by MDM2 activation and inhibited apoptosis by inducing p21 and bcl-2 expression.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.9
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• pp.1257-1264
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• 2016

Extracts from Artemisia annua $Linn\acute{e}$ (AAE) have various functions (anti-malaria, anti-virus, and anti-oxidant). However, the mechanism of the effects of AAE is not well known. Thus, we determined the apoptotic effects of AAE in AGS human gastric carcinoma cells. In this study, we suggested that AAE may exert cancer cell apoptosis through the Akt/mammalian target of rapamycin (mTOR)/glycogen synthase kinase (GSK)-$3{\beta}$ signal pathway and mitochondria-mediated apoptotic proteins. Activation by Akt phosphorylation resulted in cell proliferation through phosphorylation of tuberous sclerosis complex 2 (TSC2), mTOR, and GSK-$3{\beta}$. Thus, de-phosphorylation of Akt inhibited cell proliferation and induced apoptosis through inhibition of Akt, mTOR, phosphorylation of GSK-$3{\beta}$ at serine9, and control of Bcl-2 family members. Inhibition of GSK-$3{\beta}$ attenuated loss of mitochondrial membrane potential and release of cytochrome C. Bax and pro-apoptotic proteins were activated by their translocation into mitochondria from the cytosol. Translocation of Bax induced outer membrane transmission and generated apoptosis through cytochrome C release and caspase activity. We also measured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase assay, Hoechst 33342 staining, Annexin V-PI staining, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining, and Western blotting. Accordingly, our study showed that AAE treatment to AGS cells resulted in inhibition of Akt, TSC2, GSK-$3{\beta}$-phosphorylated, Bim, Bcl-2, and pro-caspase 3 as well as activation of Bax and Bak expression. These results indicate that AAE induced apoptosis via a mitochondrial event through regulation of the Akt/mTOR/GSK-$3{\beta}$ signaling pathways.

• Clinical and Experimental Pediatrics
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• v.50 no.7
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• pp.686-693
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• 2007

Purpose : Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), is a potent inhibitor of inosine-monophosphate dehydrogenase (IMPDH), a new immunosuppressive drug used. It was reported that MPA protected neurons after excitotoxic injury, induced apoptosis in microglial cells. However, the effects of MPA on hypoxic-ischemic (HI) brain injury has not been yet evaluated. Therefore, we examined whether MPA could be neuroprotective in perinatal HI brain injury using Rice-Vannucci model (in vivo) and in rat brain cortical cell culture induced by hypoxia (in vitro). Methods : Cortical cells were cultured using a 18-day-pregnant Sprague-Dawley (SD) rats and incubated in 1% $O_2$ incubator for hypoxia. MPA ($10{\mu}g/mL$) before or after a HI insult was treated. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2 hours of hypoxic exposure (8% $O_2$). MPA (10 mg/kg) before or after a HI insult were administrated intraperitoneally. Apoptosis was measured using western blot and real-time PCR for Bcl-2, Bax, caspase-3. Results : H&E stain revealed increased brain volume in the MPA-treated group in vivo animal model of neonatal HI brain injury. Western blot and real-time PCR showed the expression of caspase-3 and Bax/Bcl-2 were decreased in the MPA-treated group In in vitro and in vivo model of perinatal HI brain injury, Conclusion : These results may suggest that the administration of MPA before HI insult could significantly protect against perinatal HI brain injury via anti-apoptotic mechanisms, which offers the possibility of MPA application for the treatment of neonatal HI encephalopathy.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.741-746
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• 2015

Purpose: To investigate the influence of exogenous p53 upregulated modulator of apoptosis (PUMA) expression on cell proliferation and apoptosis in human non-small cell lung cancer A549 cells and transplanted tumor cell growth in nude mice. Materials and Methods: A549 cells were divided into the following groups: control, non-carrier (NC), PUMA (transfected with pCEP4-(HA) 2-PUMA plasmid), DDP ($10{\mu}g/mL$ cisplatin treatment) and PUMA+DDP (transfected with pCEP4-(HA)2-PUMA plasmid and $10{\mu}g/mL$ cisplatin treatment). The MTT method was used to detect the cell survival rate. Cell apoptosis rates were measured by flow cytometry, and PUMA, Bax and Bcl-2 protein expression levels were measured by Western blotting. Results: Compared to the control group, the PUMA, DDP and PUMA+DDP groups all had significantly decreased A549 cell proliferation (p<0.01), with the largest reduction in the PUMA+DDP group. Conversely, the apoptosis rates of the three groups were significantly increased (P<0.01), and the PUMA and DDP treatments were synergistic. Moreover, Bax protein levels significantly increased (p<0.01), while Bcl-2 protein levels significantly decreased (p<0.01). Finally, both the volume and the weights of transplanted tumors were significantly reduced (p<0.01), and the inhibition ratio of the PUMA+DDP group was significantly higher than in the single DDP or PUMA groups. Conclusions: Exogenous PUMA effectively inhibited lung cancer A549 cell proliferation and transplanted tumor growth by increasing Bax protein levels and reducing Bcl-2 protein levels.

• Tuberculosis and Respiratory Diseases
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• v.45 no.5
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• pp.962-974
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• 1998

Background: Alteration of p53 tumor suppressor genes is most frequently identified in human neoplasms, including lung carcinoma. It is well known that bcl-2 oncoprotein protects cells from apoptosis. Recent studies have demonstrated that bcl-2 expression is associated with favorable prognosis for patients with non-small cell lung carcinoma. However, the precise biologic role of bcl-2 in the development of these tumors is still obscure. p53 and bcl-2 have important regulatory influence in the apoptotic pathway and thus their relationship is of interest in tumorigenesis, especially lung cancer. Purpose: The author investigated to know the prognostic significance of the expression of p53 and bcl-2 in radically resected non-small cell lung cancer. Method: 84 cases of formalin-fixed paraffin-embedded blocks from resected primary non-small cell lung cancer from 1980 to 1994 at Hanyang University Hospital were available for both clinical follow-up and immunohistochemical staining using monoclonal antibodies for p53 and bcl-2. Results : The histologic classification of the tumor was based on WHO criteria., and the specimens included 45 squamous cell carcinomas(53.6%), 28 adeonocarcinomas(33.3%) and 11 large cell carcinomas(13.1 %). p53 immunoreactivity was noted in 47 cases of 84 cases(56.0%). bcl-2 immunoreactivity was noted in 15 cases of 84 cases(17.9%). The mean survival duration was $64.23{\pm}10.73$ months in bcl-2 positive group and $35.28{\pm}4$. 39 months in bcl-2 negative group. The bcl-2 expression was significantly correlated with survival in radically resected non-small cell lung cancer patients(p=0.03). The mean survival duration was $34.71{\pm}6.12$ months in p53 positive group and $45.35{\pm}6.30$ months in p53 negative group(p=0.21). The p53 expression was not predictive for survival. There was no correlation between combination of the different status of p53 and bcl-2 expression in our study. Conclusions : The interaction and the regulation of new biologic markers, such as those involved in the apoptotic pathway, are complex. bcl-2 overexpression is a good prognostic factor in non-small cell lung cancer and p53 expression is not significantly associated with the prognostic factor in non-small cell lung cancer.

• Radiation Oncology Journal
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• v.18 no.4
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• pp.329-336
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• 2000

Backgrounds : The purpose of this study was to identify drugs that can enhance radioresponse of murine fepatocarcinorna. Methods : CSH/HeJ mice bearing 8 mm tumors of murine fepatocarcinorna, HCa-1, were treated with 25 Gy radiation and one of the following drugs: 5-Fu, 150 mghg; adriamycin, 8 mg/kg; cisplatin, 6 mg/kg; paclitaxel, 40 mg/kg; and gemcitabine, 50 mg/kg. Tumor response to the treatment was determined by tumor growth delay assay and by enhancement factor. Apoptotic level was assessed in tissue sections. Expression of regulating molecules was analyzed by western blotting for p53, Bcl-2, Bax, Bcl-XL, Bcl-XS, and p21$^{WAF1/CIP1}$. Results :Among the drugs tested, only gemcitabine enhanced the antitumor effect of radiation, with enhancement factor of 1.6. Induction of apoptosis by a combination of gemcitabine and radiation was shown as only additive level. In analysis of radiation-induced expression of regulating molecules, the most significant change by combining gemcitabine was activation of p21$^{WAF1/CIP1}$ Conclusion :Gemcitabine is the first drug showing an enhancement of radioresponse in murine hepatocarcinoma, when combined with radiation. The key element of enhancement is thought to be p21$^{WAF1/CIP1}$.

• Archives of Pharmacal Research
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• v.29 no.12
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• pp.1140-1146
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• 2006

Ceramide analogs are potential chemotherapeutic agents. We report that a ceramide analog induces apoptosis in human prostate cancer cells. The ceramide analog induced cell death through an apoptotic mechanism, which was demonstrated by DNA fragmentation, the cleavage of poly ADP ribose polymerase (PARP), and a loss of membrane asymmetry. Treating the cells with ceramide analog resulted in the release of various proapoptotic mitochondrial proteins including cytochrome c and Smac/DIBLO into the cytosol, and a decrease in the mitochondrial membrane potential. In addition, the ceramide analog decreased the phospho-Akt and phospho-Bad levels. The expression of the antiapoptotic Bcl-2 decreased slightly with increasing Bax to Bcl-2 ratio. These results suggest that the ceramide analog induces apoptosis by regulating multiple signaling pathways that involve the mitochondrial pathway.

• Journal of Life Science
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• v.17 no.6 s.86
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• pp.766-771
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• 2007

The co-infection with hepatitis B virus (HBV) and hepatitis C Virus (HCV) is associated with a more severe liver disease and increased frequency in the development of hepatocellular carcinoma com-pared to those with single infection. Here, we demonstrated that HBV X protein (HBx) and HCV Core cooperatively up-regulated the level of p53 in human hepatoma HepG2 cells. The elevated p53 subsequently stimulated the expression of proapoptotic Bax whereas it repressed the expression of antiapoptotic Bcl2. These effects, however, were not observed in p53-negative Hep3B cells. Consistently to their cooperative regulation of apoptotic effectors, HBx and HCV Core additively stimulated cisplatin-mediated apoptotic cell death of HepG2 but not of Hep3B cells. These results may help to explain the development of a more severe liver disease in patients co-infection with HBV and HCV as well as some contradictory results on the roles of HBx and Core in apoptosis.