Abrin Induces HeLa Cell Apoptosis by Cytochrome с Release and Caspase Activation

( Xiao Ling Qu ),( Liu Ting Qing ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.4

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internudeosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nudear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.

Betulinic Acid Induces Bax/Bak-Independent Cytochrome c Release in Human Nasopharyngeal Carcinoma Cells

Yang Liu,Wenlong Luo 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.5

Betulinic acid (BetA) is an effective and potential anti-cancer chemical derived from plants. BetA can kill a broad range of tumor cell lines, but has no effect on untransformed cells. The chemical also kills melanoma, leukemia, lung, colon, breast, prostate and ovarian cancer cells via induction of apoptosis, which depends on caspase activation. However, no reports are yet available about the effects of BetA on nasopharyngeal carcinoma (NPC), a widely spread malignancy in the world, especially in East Asia. In this study, we first showed that BetA can effectively kill CNE2 cells, a cell line derived from NPC. BetA-induced CNE2 apoptosis was characterized by typical apoptosis hallmarks: caspase activation, DNA fragmentation, and cytochrome c release. Overexpression of Bcl-2 and Bcl-xL could partially prevent apoptosis caused by BetA. Moreover, Bax was not activated during the induction of apoptosis. Bax/Bak knockdown and wild-type CNE2 cells showed the same kinetics of cytochrome c release. We then showed that BetA may impair mitochondrial permeability transition pores (mPTPs), which may partially contribute to cytochrome c release. These observations suggest that BetA may serve as a potent and effective anti-cancer agent in NPC treatment. Further exploration of the mechanism of action of BetA could yield novel break-throughs in anti-cancer drug discovery.

Abrin Induces HeLa Cell Apoptosis by Cytochrome c Release and Caspase Activation

Qu, Xiaoling,Qing, Liuting Korean Society for Biochemistry and Molecular Biol 2004 Journal of biochemistry and molecular biology Vol.37 No.4

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.

Potentiation of Ceramide-Induced Apoptosis by p27kip1 Overexpression

김해종,Kyung Chul Ghil,Moo Sung Kim,Seong Hyun Yeo,천영진,김미영 대한약학회 2005 Archives of Pharmacal Research Vol.28 No.1

The cyclin-dependent kinase inhibitor p27kip1 (p27) has been implicated in the regulation of cell cycle and apoptosis. Recently, we have demonstrated that ceramide induces apoptotic cell death associated with increase in the level of p27 in HL-60 cells. In the present study, we showed that overexpression of p27 increases ceramide-induced apoptotic cell death in HL-60 cells. Furthermore, overexpression of p27 accelerated DNA fragmentation, PARP cleavage and cytochrome c release induced by ceramide. In addition, ceramide induced Bax expression independent of p27. These findings indicate that enhanced effect on apoptosis by p27 is associated with mitochondrial signaling which involves cytochrome c release.

Potentiation of Ceramide-Induced Apoptosis by $p27^{kip1}$ Overexpression

Kim Hae Jong,Ghil Kyung Chul,Kim Moo Sung,Yeo Seong Hyun,Chun Young Jin,Kim Mie Young The Pharmaceutical Society of Korea 2005 Archives of Pharmacal Research Vol.28 No.1

The cyclin-dependent kinase inhibitor$p27^{kip1}$(p27) has been implicated in the regulation of cell cycle and apoptosis. Recently, we have demonstrated that ceramide induces apoptotic cell death associated with increase in the level of p27 in HL-60 cells. In the present study, we showed that overexpression of p27 increases ceramide-induced apoptotic cell death in HL-60 cells. Furthermore, overexpression of p27 accelerated DNA fragmentation, PARP cleavage and cytochrome c release induced by ceramide. In addition, ceramide induced Sax expression independent of p27. These findings indicate that enhanced effect on apoptosis by p27 is associated with mitochondrial signaling which involves cytochrome c release.

CIC-3 chloride channel blockade protects mouse photoreceptorderived 661W cells against ischemia-reperfusion-induced injury in vitro

Shi Wei Huang,Yuan Yin,Ya Juan Zheng,Ya Ru Dong 대한독성 유전단백체 학회 2015 Molecular & cellular toxicology Vol.11 No.1

Exposure to ischemia/reperfusion leads to the development and progression of retinal degenerative diseases. However, the exact mechanisms are not fully understood. In this article, the role of CIC-3 chloride channel in OGD-R (oxygen-glucose deprivation followed by reperfusion)-induced retinal damage was examined. Mouse photoreceptor-derived 661W cells were treated with the CIC-3 antisense oligonucleotide before exposure to OGD-R. Cell viability, mitochondrial membrane potential, cytochrome-c level, DNA fragmentation, caspase activity and protein expression were detected. Pretreatment of 661W cells with CIC- 3 antisense oligonucleotide significantly decreased OGD-R-mediated toxicity. In addition, apoptosis-related biochemical indicators showed that pre-incubation of CIC-3 antisense oligonucleotide would elevate the mitochondrial membrane potential, decrease the release of cytochrome-c as well as formation of DNA fragmentation, and inhibit activities of caspase-3 and caspase- 9 in exogenous OGD-R-treated 661W cells. Moreover, treatment with CIC-3 antisense oligonucleotide changed the expression of apoptosis-related protein. These results suggest that CIC-3 chloride channel mediates OGD-R-induced apoptosis, at least partially through mitochondrial membrane potential pathway and increasing the levels of proapoptotic molecules in 661W cells. CIC-3 chloride channel blockade may provide a new therapeutic approach for preventing ischemia/reperfusion- induced retinal neural damage.

4-Hydroxynonenal Induces Endothelial Apoptosis through Mitochondrial Depolarization

Dae Yeon Kang(강대연),Ji Young Lee(이지영),Min-Sun Kim(김민선),Chul Hong Kim(김철홍),Hyung Keun Kim(김형근),Sun Mi Lee(이선미),Young Mi Kwon(권영미),Jaewon Lee(이재원),Hyung Suk Baik(백형석),Byung Pal Yu(유병팔),Hae Young Chung(정해 한국생명과학회 2008 생명과학회지 Vol.18 No.11

4-Hydroxynonenal (4-HNE)는 세포내 레독스의 균형을 깨뜨려 혈관 기능 손상을 일으킨다. 본 연구자들은 HNE의 축적이 야기하는 혈관 기능 손상기전을 더 잘 이해하기 위하여 혈관 내피 세포의 미토콘드리아 세포사 메커니즘을 규명하였다. HNE를 처리한 세포에서는 미토콘드리아 막전위 소실과 그에 따른 cytochrome C의 방출이 유도되었으며, Bax의 증가 및 Bcl-2의 감소가 관찰되었다. ROS 제거제인 NAC와 peroxynitrite 제거제인 페니실라민은 HNE가 유도하는 ROS 생성을 차단하여 cytochrome C 방출과 세포사를 억제하였다. 세포에 HNE와 zVAD-fmk(caspase 저해제)를 같이 처리하면 HNE가 유도하는 세포사를 억제하지 못하는데 이는 HNE에 의한 세포사가 caspase에 비의존적 단계일 가능성을 시사하였다. 위의 결과들은 HNE가 유도하는 혈관 내피 세포의 세포사 매커니즘은 미토콘드리아 막전위의 탈분극에 의해 촉발되며 이는 혈관계 항상성의 악화와 노화에 의해 수반되는 혈관 기능 손상을 유도할 것으로 사료된다. The 4-Hydroxynonenal (HNE) affects vascular dysfunctions probably through the interruption of the cellular redox balance. To better understand vascular abnormalities resulting from the accumulation of HNE, we delineated mechanism by which mitochondrial apoptosis occurs in the YPEN-1 endothelial cells. HNE treatment led to the loss of mitochondrial membrane potential (δΨm), resulting in the release of cytochrome c. Data showed decreased Bcl-2 and increased Bax protein levels in HNE-treated cells. NAC, a reactive oxygen species (ROS) scavenger, and penicillamine, the peroxynitrite scavenger, blocked HNE-mediated ROS generation, thereby thwarting the cytochrome c release and apoptosis. The treatment of the cells with zVAD-fmk, a broad range caspase inhibitor did not suppress HNE-induced apoptosis, suggesting that the apoptosis might be the possibility of caspase-independent process. Our findings delineate the underlying mechanism of the HNE induced endothelial apoptosis by triggering depolarization of mitochondria membrane potential that can lead to the deterioration of vasculature homeostasis and subsequent vascular dysfunction with aging.

Sex- and Tissue-related Expression of Two Types of P450 Aromatase mRNA in the Protandrous Black Porgy, Acanthopagrus schlegeli, during Sex Reversal: Expression Profiles Following Exogenous Hormone Administration

Tae Sun Min,Kwang Wook An,길경석,최철영 한국통합생물학회 2009 Animal cells and systems Vol.13 No.4

Cytochrome P450 aromatase (P450arom) catalyzes the conversion of androgens to estrogens and plays an important role in reproduction and development in vertebrates. We investigated the expression patterns of ovarian P450arom (P450aromA) and brain P450arom (P450aromB) mRNA during sex change in black porgy. Maturity was divided into seven stages from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary, and mature ovary). P450aromA expression was significantly higher in the ovarian portion of mostly-ovarian stage fish, and P450aromB expression was highest in the brain of black porgy with mostly-ovarian gonads. Histology showed that testicular tissues were disintegrated with the development of ovarian tissue associated with an increase in the expression of the two P450arom mRNAs during sex change. Interestingly, among various tissues, P450aromA was only expressed in the ovary, and P450aromB was only expressed in the brain. To understand the role of gonadotropin-releasing hormone (GnRH) and estradiol (E2), we injected exogenous hormone (GnRH analogue [GnRHa] and E2) into immature black porgy. In the GnRHa group, expression of the two P450arom genes decreased 12 h after injection, and expression of the two P450arom genes were significantly higher at 6 d after E2 injection. These results provide useful baseline knowledge on the mechanism of natural sex change in black porgy. Cytochrome P450 aromatase (P450arom) catalyzes the conversion of androgens to estrogens and plays an important role in reproduction and development in vertebrates. We investigated the expression patterns of ovarian P450arom (P450aromA) and brain P450arom (P450aromB) mRNA during sex change in black porgy. Maturity was divided into seven stages from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary, and mature ovary). P450aromA expression was significantly higher in the ovarian portion of mostly-ovarian stage fish, and P450aromB expression was highest in the brain of black porgy with mostly-ovarian gonads. Histology showed that testicular tissues were disintegrated with the development of ovarian tissue associated with an increase in the expression of the two P450arom mRNAs during sex change. Interestingly, among various tissues, P450aromA was only expressed in the ovary, and P450aromB was only expressed in the brain. To understand the role of gonadotropin-releasing hormone (GnRH) and estradiol (E2), we injected exogenous hormone (GnRH analogue [GnRHa] and E2) into immature black porgy. In the GnRHa group, expression of the two P450arom genes decreased 12 h after injection, and expression of the two P450arom genes were significantly higher at 6 d after E2 injection. These results provide useful baseline knowledge on the mechanism of natural sex change in black porgy.

Release of Cytochrome c from Isolated Mitochondria by Etoposide

Park, Jung-Hee,Kim, Tae-Hyoung Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.5

The efficacy of chemotherapeutic agents on tumor cells has been shown to be modulated by tumor suppressor gene p53 and its target genes such as Bcl-2 family members (Bax, Noxa, and PUMA). However, various chemotherapeutic agents can induce cell death in tumor cells that do not express the functional p53, suggesting that some chemotherapeutic agents may induce cell death in a p53-independent pathway. Here we showed that etoposide can induce the similar degree of cell death in p53-deficient HCT 116 cells, whereas 5'-FU-mediated cell death is strongly dependent on the existence of functional p53 in HCT 116 cells. Further, we provide the evidence that etoposide can induce the cytochrome c release from isolated mitochondria, and etoposide-induced cytochrome c release is not accompanied with the large amplitude swelling of mitochondria. These data suggest that etoposide can directly induce the mitochondrial dysfunction irrespective of p53 status, and it may, at least in part, account for the p53-independent pathway in cell death induced by chemotherapeutic agents.

Differential Efflux of Mitochondrial Endonuclease G by hNoxa and tBid

Seo, Young-Woo,Park, Sun-Young,Yun, Cheol-Won,Kim, Tae-Hyoung Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.5

The Bcl-2 family of proteins regulates mitochondrial functions during cell death by modulating the efflux of death-promoting proteins such as cytochrome c and endonuclease G. Upon the binding of death ligands to their receptors, caspase-8 cleaves Bid, a BH3-only protein, into tBid that causes the mitochondrial damages resulting in the release of cytochrome c and endonuclease G. Also, another BH3-only protein, hNoxa, has been shown to induce the efflux of cytochrome c from the mitochondria. Whether the efflux proteins from the mitochondria in response to tBid or hNoxa are the same or different, however, has not been addressed. We have demonstrated that endonuclease G activities are not detectable among the proteins released from isolated mitochondria by hNoxa but are detectable in that by tBid. These results suggest that the efflux of proteins from the mitochondria are differentially modulated by tBid and hNoxa.