• Archives of Pharmacal Research
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• 제24권2호
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• pp.136-143
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• 2001

Fumonisins are specific inhibitors of ceramide synthase in sphingolipid metabolism. An alteration in sphingolipid metabolism as a result of fumonisin exposure is related to cell death (Yoo et al., 1992). The objective of this study was to investigate whether elevated free sphinganine levels are related to the sensitivity of cultured cells to fumonisin exposure. Fumonisin $B_1$ elevated the intracellular free sphinganine concentraions in both LLC-$PK_1$ and Chinese hamster ovary (CHO) cells. However, CHO cells are resistant to fumonisin cytotoxicity at 50${u}m$, while LLC-$PK_1$ cells are sensitive at concentrations greater than 357M. The intracellular concentration of free sphinganine in LLC-$PK_1$ cells treated at 50${u}m$ fumonisin $B_1$ for 72 h was approximately 1450 pmol/mg protein relative to the 37 pmol observed in the control culture. Under the same conditions, the population of apoptotic cells in the 50${u}m$ fumonisin $B_1$-treated culture was approximately 37% of the total compared to 12% in the control. The caspase III-like activity after 72 h in the 50${\mu}$M fumonisin $B_1$-exposed culture Increased to approximately 50 $pmol/mg$ protein/hr compared to 6 $pmol/mg$ protein/hr in the control. L-cycloserine, a serine palmitoyltransferase inhibitory reduced the fumonisin $B_1$-stimulated caspase III-like activity down to the control level. Under the same culture conditions, the intracellular concentration of free sphinganine after-cycloserine plus fumonisin $B_1$ treatment was 140 pmol/mg protein compared to 1450 $pmol/mg$ protein in fumonisin $B_1$ alone. The intracellular concentration of free sphinganine in CHO cells treated with 50${u}m$ fumonisin $B_1$ for 72 h was al)proximately 460 pmol/mg protein, indicating that the mass amount of elevated free sphinganine in the CHO cells was about 32% of that in LLC-$PK_1$ cells. Adding exogenous sphinganine to the CHO cells along with 50${u}m$ fumonisin $B_1$ treatment for 72 h caused both necrosis and apoptosis. In conclusion, the elevated endogenous sphinganine acts as a contributing factor to the fumonisin-induced cell death.

• 대한의생명과학회지
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• 제11권2호
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• pp.135-141
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• 2005

A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element of murine Hoxa-7, has shown to have a toxicity to both prokaryotic and eukaryotic cells when overexpressed. Since Jpk protein harbors a transmembrane domain and a putative endoplasmic reticulum (ER)-retention signal at the N-terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP): Both N-term (Jpk-EGFP) and C-term tagged-Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscopy after staining the cells with ER- and MitoTracker. Since ER stress triggers the ER-stress mediated apoptosis to eliminate the damaged cells, we analyzed the expression pattern of Jpk under ER-stress condition. When MCF7 cells were treated with the ER-stress inducer such as DTT and EGTA, the expression of Jpk was upregulated at the transcriptional level like that of Grp78, a molecular chaperone well known to be overexpressed under ER-stress condition. In the presence of high concentration of ER-sterss inducer (10 mM), about 70 (DTT) to $95\%$ (EGTA) of cells died stronly expressing ($10\~12$ fold) Jpk. Whereas at the low concentration ($0.001\~1.0\;mM$) of the inducer, the expression of Jpk was increased about 2.5 (EGTA) to 5 fold (DTT), which is rather similar to those of ER chaperone protein Grp78. These results altogether indicate that the ER-stress upregulated the expression of Jpk and the excess stress induces the ER-stress induced apoptosis and the concomitant expression of Jpk.

• International Journal of Oral Biology
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• 제45권1호
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• pp.1-7
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• 2020

Oral lichen planus (OLP) is a chronic inflammatory disease observed in approximately 0.5-2.2% of the population, and it is recognized as a premalignant lesion that can progress into oral squamous cell carcinoma (OSCC). The rate of malignant transformation is approximately 1.09-2.3%, and the risk factors for malignant transformation are age, female, erosive type, and tongue site location. Malignant transformation of OLP is likely related to the low frequency of apoptotic phenomena. Therefore, apoptosis-related genetic factors, like p53, BCL-2, and BAX are reviewed. Increased p53 expression and altered expression of BCL-2 and BAX were observed in OLP patients, and the malignant transformation rate in these patients was relatively higher. The involvement of microRNA (miRNA) in the malignant transformation of OLP is also reviewed. Because autophagy is involved in cell survival and death through the regulation of various cellular processes, autophagy-related genetic factors may function as factors for malignant transformation. In OLP, decreased levels of ATG9B mRNA and a higher expression of IGF1 were observed, suggesting a reduction in cell death and autophagic response. Activated IGF1-PI3K/AKT/mTor cascade may play an important role in a signaling pathway related to the malignant transformation of OLP to OSCC. Recent research has shown that miRNAs, such as miR-199 and miR-122, activate the cascade, increasing the prosurvival and proproliferative signals.

• 생명과학회지
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• 제28권6호
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• pp.718-725
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• 2018

Toll 유사수용체의 TLR4는 세포자연사(apoptosis)와 관련하여 세포의 생존과 증식에 영향을 미치는 것으로 알려져 있다. 본 연구에서는 TLR4의 활성이 부인과 질환 특이적 종양세포의 세포사멸기작에 어떠한 영향을 미치는지 살펴보았다. TLR4의 활성에 의한 세포자연사를 확인하기 위하여 부인암 조직(자궁경부암, 자궁내막암, 난소암)에서 종양세포를 분리하여 초대배양시스템을 구축하였고, lipopolysaccharide (LPS)에 의한 TLR4의 활성유도 과정에서 종양세포의 형태학적 변화를 살펴보았다. 또한, TLR4 매개성 세포사멸 기작을 확인하기 위하여 역전사 중합효소 연쇄반응(RT-PCR)을 통해 유전자 분석을 진행하였다. 연구 결과, 부인암의 초대배양세포에서 세포접촉저지(contact inhibition)현상이 감소되었고, 세포의 배가시간(doubling time)이 단축되어, 종양세포의 성장률 변화를 확인하였다(p<0.05). 자궁근육층(정상조직)의 초대배양세포에서는 민무늬근육 확인 인자인 ITGA5 (an alpha5 integrin marker)의 유전자 발현이 나타났으나, 자궁경부조직의 초대배양세포에서는 발현변화를 확인할 수 없었다. 종양세포의 유전자분석 결과에서 p53과 같은 종양억제인자의 발현이 유의적으로 증가한 반면(p<0.05), 세포사멸 신호기작과 관련하여 TLR4와 Caspase-3의 발현은 감소하였다(Caspase-3, p<0.05). LPS를 처리한 종양세포에서는 LPS 비처리군과 비교 시, TLR4의 발현증가와 함께 Caspase-3의 발현변화가 동반되었다. 이러한 결과들은 TLR4 매개성 apoptosis 유도가 종양세포의 증식억제에 중요한 영향을 미치는 것을 의미하며, TLR4 신호기작을 이용한 종양세포의 새로운 치료적 접근법을 제시할 것으로 기대한다.

• 한국미생물·생명공학회지
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• 제50권3호
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• pp.387-394
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• 2022

Verotoxin-1 (VT-1) or Shiga-like toxin 1 (Stx-1) is produced by enterohemorrhagic Escherichia coli (EHEC) and is an AB5 holotoxin with a strong inhibitor of protein synthesis. VT-1 is a type 2 ribosome-inactivating protein (RIP) that has been shown to have cytotoxic and anticancer potential by inducing necrosis, apoptosis, and cell cycle arrest, making it a promising antitumor candidate. Here, we tested the cytotoxicity of VT-1 on CaCo2 and NCM425 cell lines and the results showed that VT-1 was more potent on CaCo2. Morphological changes were also evaluated on the cellular level and the results showed that VT-1 caused a decrease in viable cell count, altered cell membrane permeability, and an increase in total nuclear intensity. On the other hand, VT-1 displayed a lesser impact on mitochondrial membrane potential (MMP) and cytochrome c release. On the expression of caspases 3 and 9, VT-1 exhibited an insignificant effect on both which alongside the mitochondrial membrane potential (MMP) and cytochrome c results, might indicate that CaCo2 suffered from the necrosis process as a mechanism of cell death after exposure to VT-1.

• 동의생리병리학회지
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• 제18권6호
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• pp.1652-1660
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• 2004

Apoptosis of vascular smooth muscle cells(VSMCs) is essential in atherogenesis, being a factor that modulates its early progression rather than a terminal event in the course of the disease. Various stimuli, including oxide lipoproteins, altered hemodynamic stress and free radical, can induced VSMCs apoptosis in vitro. The protective effects of Sophorae Radix (SR) on apoptotic cell death induced by H₂O₂ were investigated in VSMCs. The viability of VSMCs was markedly decreased by H₂O₂. Sophorae Radix protected the H202-induced apoptotic death of VSMCs, which was characterized as nuclear fragmentation and increase of sub-G0/G1 fraction .. Sophorae Radix decreased the activation of caspase-3 like protease induced by H₂O₂ and recovered control level from H202-induced PARP, Bak, Bcl-XL and mitochondrial membrane potential. These results suggest that Sophorae Radix protected VSMCs apoptotic death induced by H₂O₂ via inactivation of caspase-3 and modulation of mitochondrial function. Also, the expression profile of proteins by using two-dimensional (2-D) gel electrophoresis was screened. Future investigations will need to explore the use of an anti atherosclerotic therapy of Sophorae Radix, which relies on inhibition of the proapoptotic activation of the vascular smooth muscle cells.

• Journal of Microbiology and Biotechnology
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• 제31권2호
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• pp.189-196
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• 2021

Periplanetasin-2 from cockroach exhibits broad-spectrum antimicrobial activity. The underlying antibacterial mechanisms rely on the stimulation of reactive oxygen species overproduction to induce apoptotic cell death. A promising strategy to increase the bioavailability of periplanetasin-2 involves reducing the dose through combination therapy with other antibacterials that show synergistic effects. Thus, the synergistic antibacterial activity of periplanetasin-2 with conventional antibacterial agents and its mechanisms was examined against Escherichia coli in this study. Among the agents tested, the combinations of periplanetasin-2 with vancomycin and chloramphenicol exhibited synergistic effects. Periplanetasin-2 in combination with vancomycin and chloramphenicol demonstrated antibacterial activity through the intracellular oxidative stress response. The combination with vancomycin resulted in the enhancement of bacterial apoptosis-like death, whereas the combination with chloramphenicol enhanced oxidative stress damage. These synergistic interactions of periplanetasin-2 can help broaden the spectrum of conventional antibiotics. The combination of antimicrobial peptides and conventional antibiotics is proposed as a novel perspective on treatments to combat severe bacterial infection.

• 한국식품영양과학회지
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• 제32권3호
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• pp.437-443
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• 2003

선행연구에서 라이코펜이 HT-29세포의 증식을 억제하는 것을 관찰하였기 때문에 본 연구는 그 기전을 연구하기 위하여 수행되 었다. 라이코펜이 HT-29 세포의 사멸을 유도하는지 조사하기 위해서 여러 농도의 라이코펜이 포함된 배지에서 세포를 4일 동안 배양하였다. 라이코펜 농도의 증가에 따라 사멸되는 세포에서 나타나는 특징의 하나인 DNA fragmentation이 증가하는 것을 관찰하였다. Western blot을 수행하여 얻은 결과에 의하면 라이코펜이 IGF-IR, IRS-1, PI3K, Akt와 같은 IGF-IR pathway에 속하는 단백질의 수준을 감소시켰다. IGF-IR의 인산화를 유도하기 위해서 라이코펜이 포함된 배지에서 세포를 배양하고 IGF-I을 첨가하여 0, 5, 10, 60분간 배양한 다음 IGF-IR antibody를 이용하여 immunoprecipitation을 수행하였다. 라이코펜은 IGF-I에 의한 IGF-IR, IRS-1의 인산화와 IGF-IR와 PI3K의 결합을 감소하고 인산화된 Akt를 감소시켰다. 이와 같은 IGF-IR signaling의 억제는 이 대장암세포에 존재하는 IGF-II의 autocrine loop을 억제하는 원인이 될 수 있어, 라이코펜의 암세포증식을 억제하는 기전 중의 하나가 될 수 있다. 라이코펜은 토마토와 그 가공품에 많이 존재하는 물질로 자연적인 식사를 통해 많이 섭취할 수 있는 물질이다. 라이코펜의 항암 기전을 밝혀냄으로써 미래 암예방과 치료를 위한 중요한 기능성 영양소를 생산할 수 있는 기초를 마련해줄 수 있을 것으로 기대된다.

• Journal of Ginseng Research
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• 제45권1호
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• pp.126-133
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• 2021

Background: 20(S)-protopanaxadiol (20(S)-PPD), one of the aglycone derivatives of major ginsenosides, has been shown to have an anticancer activity toward a variety of cancers. This study was initiated with an attempt to evaluate its anti-cancer activity toward human endometrial cancer by cell and xenograft mouse models. Methods: Human endometrial cancer (HEC)-1A cells were incubated with different 20(S)-PPD concentrations. 20(S)-PPD cytotoxicity was evaluated using MTT assay. Apoptosis was detected using the annexin V binding assay and cell cycle analysis. Cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-9 were assessed using western blotting. HEC-1A cell tumor xenografts in athymic mice were generated by inoculating HEC-1A cells into the flank of BALB/c female mice and explored to validate 20(S)-PPD anti-endometrial cancer toxicity. Results: 20(S)-PPD inhibited HEC-1A cell proliferation in a dose-dependent manner with an IC50 value of 3.5 μM at 24 h. HEC-1A cells morphologically changed after 20(S)-PPD treatment, bearing resemblance to Taxol-treated cells. Annexin V-positive cell percentages were 0%, 10.8%, and 58.1% in HEC-1A cells when treated with 0, 2.5, and 5 μM of 20(S)-PPD, respectively, for 24 h. 20(S)-PPD subcutaneously injected into the HEC-1A cell xenograft-bearing mice three times a week for 17 days manifested tumor growth inhibition by as much as 18% at a dose of 80 mg/kg, which sharply contrasted to controls that showed an approximately 2.4-fold tumor volume increase. These events paralleled caspase-9 activation and PARP cleavage. Conclusion: 20(S)-PPD inhibits endometrial cancer cell proliferation by inducing cell death via a caspase-mediated apoptosis pathway. Therefore, the 20(S)-PPD-like ginsenosides are endowed with ample structural information that could be utilized to develop other ginsenoside-based anticancer agents.

• BMB Reports
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• 제38권4호
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• pp.447-456
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• 2005

TNF-$\alpha$ plays a pivotal role in inflammation processes which are mainly regulated by endothelial cells. While TNF-$\alpha$ induces apoptosis of several cell types like tumor cells, endothelial cells are resistant to TNFa mediated cell death. The cytotoxic effects of TNF-$\alpha$ on most cells are only evident if RNA or protein synthesis is inhibited, suggesting that de novo RNA or protein synthesis protect cells from TNF-$\alpha$ cytotoxicity, presumably by NF-${\kappa}B$ mediated induction of protective genes. However, the cytoprotective genes involved in NF-${\kappa}B$ dependent endothelial cell survival have not been sufficiently identified. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-$\alpha$ inducible genes in human arterial endothelial cells related to cell survival and cell cycle. The TNF-$\alpha$-induced expression of the RNA binding protein $p54^{nrb}$ and the 14-3-3 protein HS1 as shown here for the first time may contribute to the TNF-$\alpha$ mediated cell protection of endothelial cells. These genes have been shown to play pivotal roles in cell survival and cell cycle control in different experimental settings. The concerted expression of these genes together with other genes related to cell protection and cell cycle like DnaJ, $p21^{cip1}$ and the ubiquitin activating enzyme E1 demonstrates the identification of new genes in the context of TNF-$\alpha$ induced gene expression patterns mediating the prosurvival effect of TNF-$\alpha$ in endothelial cells.