LPA protects intestinal epithelial cells from  apoptosis by inhibiting the mitochondrial pathway

We previously showed ( Gastroenterology 123: 206–216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through Gi-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.

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Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochromec

AJP Cell Physiology ◽

10.1152/ajpcell.00351.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. C1290-C1297 ◽

Cited By ~ 46

Author(s):

Qing Yuan ◽

Ramesh M. Ray ◽

Leonard R. Johnson

Keyword(s):

Cytochrome C ◽

Mammalian Cells ◽

Caspase 3 ◽

Intestinal Epithelial ◽

Caspase 9 ◽

Cytochrome C Release ◽

Polyamine Synthesis ◽

Antiapoptotic Proteins ◽

Induced Apoptosis ◽

Rate Limiting

C1297, 2002; 10.1152/ajpcell.00351.2001.We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome cfrom mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with α-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-xL and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.

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Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b-dependent mechanism

AJP Cell Physiology ◽

10.1152/ajpcell.00335.2019 ◽

2020 ◽

Vol 318 (4) ◽

pp. C732-C739

Author(s):

Fangyi Liu ◽

Xiao Wang ◽

Hua Geng ◽

Heng-Fu Bu ◽

Peng Wang ◽

...

Keyword(s):

Epithelial Cells ◽

Protein Expression ◽

Intestinal Epithelial Cells ◽

Specific Inhibitor ◽

In Silico Analysis ◽

Interferon Γ ◽

Luciferase Assay ◽

Epithelial Cell Line ◽

Dependent Manner ◽

Intestinal Epithelial

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3′-untranslated region (UTR) contains a putative binding site for miR-92b. Luciferase assay showed that Ifnγ inhibited Sirt6 3′-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3′-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b-mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation.

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Identification of apoptotic genes mediating TGF-β/Smad3-induced cell death in intestinal epithelial cells using a genomic approach

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00437.2005 ◽

2007 ◽

Vol 292 (1) ◽

pp. G28-G38 ◽

Cited By ~ 18

Author(s):

Yanna Cao ◽

Lu Chen ◽

Weili Zhang ◽

Yan Liu ◽

Harry T. Papaconstantinou ◽

...

Keyword(s):

Caspase 3 ◽

Target Genes ◽

Transforming Growth Factor ◽

Intestinal Epithelial ◽

Cytochrome C Release ◽

Apoptotic Pathway ◽

Genomic Approach ◽

Apoptotic Genes ◽

Induced Apoptosis

Transforming growth factor (TGF)-β-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-β inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-β-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-β-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-β/Smad3. Total RNA samples were analyzed using Affymetrix oligonucleotide microarrays. We found that TGF-β regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-β activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-β induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-β-induced apoptosis in RIE-1/Smad3 cells.

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Effect of Berberine from Coptis chinensis on Apoptosis of Intestinal Epithelial Cells in a Mouse Model of Ulcerative Colitis: Role of Endoplasmic Reticulum Stress

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/3784671 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Shen Yan ◽

Liu Yingchao ◽

Wang Zhangliu ◽

Ruan Xianli ◽

Li Si ◽

...

Keyword(s):

Ulcerative Colitis ◽

Endoplasmic Reticulum ◽

Epithelial Cells ◽

Endoplasmic Reticulum Stress ◽

Caspase 3 ◽

Intestinal Epithelial Cells ◽

High Dose ◽

Intestinal Epithelial ◽

Model Group ◽

Caspase 12

The purpose of this study was to verify the effect of berberine (BBR) on endoplasmic reticulum stress (ERS) and apoptosis of intestinal epithelial cells (IECs) in mice with ulcerative colitis (UC). BALB/c mice were randomly divided into five groups as follows: blank control, model, and low-, medium-, and high-dose BBR. A dextran sodium sulfate- (DSS-) induced model of UC was prepared, and the low-, medium-, and high-dose BBR groups were simultaneously gavaged with a BBR suspension for 7 d. Disease activity index (DAI) was assessed, and tissue damage index (TDI) was assessed from colon samples after the last administration. TUNEL assays were used to detect apoptosis of IECs. Immunohistochemistry and/or real-time PCR were applied to determine the expression of GRP78, caspase-12, and caspase-3. In all BBR treatment groups, clinical symptoms of colitis and histopathological damage were significantly reduced. The high-dose BBR group exhibited particularly pronounced decrease (p<0.01) in both DAI (0.48 ± 0.36) and TDI (1.62 ± 0.64) relative to the model group (1.50 ± 0.65 and 3.88 ± 0.04, respectively). In colon tissues of the model group, the number of apoptotic IECs was significantly increased; the expression of GRP78, caspase-12, and caspase-3 proteins was significantly increased; and the expression of the GRP78 mRNA was upregulated. In low-, medium-, and high-dose BBR groups, the number of apoptotic IECs was significantly reduced. Moreover, GRP78 and caspase-3 expression levels were significantly decreased in the medium- and high-dose BBR groups, caspase-12 expression was significantly decreased in the high-dose BBR group, and the GRP78 mRNA expression level was significantly decreased in the high-dose BBR group. BBR can effectively reduce the rate of IEC apoptosis in UC mice and alleviate the inflammatory response in the colon. The underlying mechanism seems to involve ERS modulation and inhibition of ERS-mediated activation of the caspase-12/caspase-3 apoptosis signaling pathway.

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Platycodon grandiflorum Induces Apoptosis in SKOV3 Human Ovarian Cancer Cells Through Mitochondrial-Dependent Pathway

The American Journal of Chinese Medicine ◽

10.1142/s0192415x10007919 ◽

2010 ◽

Vol 38 (02) ◽

pp. 373-386 ◽

Cited By ~ 12

Author(s):

Qin Hu ◽

Ruile Pan ◽

Liwei Wang ◽

Bo Peng ◽

Jingtian Tang ◽

...

Keyword(s):

Ovarian Cancer ◽

Cytochrome C ◽

Cancer Cells ◽

Caspase 3 ◽

Ovarian Cancer Cells ◽

Mitochondrial Cytochrome ◽

Cytochrome C Release ◽

Apoptotic Pathway ◽

Platycodon Grandiflorum ◽

Mitochondrial Cytochrome C

Platycodon grandiflorum (Jacq.) A. DC., a Chinese food and medicine, has been used as expectorant traditionally. The present study aimed to investigate the effect of Platycodon grandiflorum extract (PGE) on SKOV3 ovarian cancer cells. 3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to monitor cell numbers, Annexin-V/propidium iodide (PI) staining, RT-PCR and Western blot were used to examine cell apoptosis, caspases activation. Bcl-2 and Bax expressions and mitochondrial cytochrome c release. Our result showed that PGE-induced apoptosis was associated with activation of caspase-3, -8 and -9, down-regulation of Bcl-2, up-regulation of Bax and release of mitochondrial cytochrome c to cytosol. The data indicate that PGE may have anti-tumor effect mainly via caspase-3 and caspase-9 dependent apoptotic pathway.

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JS-K, a Novel Nitric Oxide (NO) Generator, Induces Cytochrome c Release and Caspase Activation in HL-60 Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v104.11.3415.3415 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3415-3415

Author(s):

Paul J. Shami ◽

Vidya Udupi ◽

Margaret Yu ◽

Swati Malaviya ◽

Joseph E. Saavedra ◽

...

Keyword(s):

Cytochrome C ◽

Caspase 3 ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Caspase 8 ◽

Caspase 9 ◽

Cytochrome C Release ◽

Dose Dependent Fashion ◽

Acute Myelogenous ◽

Dose Dependent

Abstract NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity. We have previously shown that JS-K induces apoptosis in HL-60 cells by a caspase dependent mechanism (Molecular Cancer Therapeutics2:409-417,2003). The purpose of this study was to determine the pathway through which JS-K induces apoptosis. Western blot analysis showed that treatment of HL-60 cells with JS-K (0 – 1 μM) for 6 hours results in release of Cytochrome c from mitochondria in a dose dependent fashion. Treatment with JS-K resulted in a dose dependent activation of Caspase 9. Sixteen and 24 hours after exposure to 1 μM JS-K, Caspase 9 activity was induced by 393 ± 93% and 237 ± 13% of control, respectively (p = 0.03 at the 24 hours time point). Treatment with JS-K resulted in a dose dependent activation of Caspase 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 3 activity was 208 ± 3.4 % of control (p = 0.02). Treatment with JS-K also resulted in a dose dependent activation of Caspase 8, but to a lesser extent than Caspase 9 and 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 8 activity was 144 ± 5.3 % of control (p = 0.04). We conclude that JS-K activates the intrinsic pathway of apoptosis in leukemia cells by inducing the release of Cytochrome c from mitochondria. (NO1-CO-12400).

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