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Pro-apoptotic effect of haem oxygenase-1 in human colorectal carcinoma cells via endoplasmic reticular stress.

ABSTRACT: Several biological effects of haem oxygenase (HO)-1, including anti-inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO-1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO-1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)-3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT-15, LOVO and HT-29 cells in serum-free (SF) conditions with increased HO-1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP-induced apoptosis and HO-1 protein expression in human colon cancer cells. CoPP-induced apoptosis of colon cancer cells was prevented by the addition of the pan-caspase inhibitor, Z-VAD-FMK (VAD), and the Casp-3 inhibitor, Z-DEVD-FMK (DEVD). N-Acetyl cysteine inhibited reactive oxygen species-generated H₂ O₂ -induced cell death with reduced intracellular peroxide production, but did not affect CoPP-induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO-1 expression by those cells, and knockdown of HO-1 protein expression by HO-1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO₄ or biliverdin, induced DNA ladders, and cleavage of Casp-3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R-like ER kinase (PERK), and eukaryotic initiation factor 2? (eIF2?) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM- and CoPP-induced apoptosis. Increased GRP78 level and formation of the HO-1/GRP78 complex were detected in CORM- and CoPP-treated human CRC cells. A pro-apoptotic role of HO-1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.

SUBMITTER: Wu MS

PROVIDER: S-EPMC6653387 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Pro-apoptotic effect of haem oxygenase-1 in human colorectal carcinoma cells via endoplasmic reticular stress.

Wu Ming-Shun MS Chien Chih-Chiang CC Chang Jungshan J Chen Yen-Chou YC

Journal of cellular and molecular medicine 20190614 8

Several biological effects of haem oxygenase (HO)-1, including anti-inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO-1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO-1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)-3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT-15, LOVO and HT-2 ...[more]

PMID: 31199053

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Project description:Sustained endoplasmic reticular stress (ERS) is implicated in aggressive metastasis of cancer cells and increased tumor cell proliferation. Cancer cells activate the unfolded protein response (UPR), which aids in cellular survival and adaptation to harsh conditions. Inhibition of apoptosis, in contrast, is a mechanism adopted by cancer cells with the help of the inhibitor of an apoptosis (IAP) class of proteins such as Survivin to evade cell death and gain a proliferative advantage. In this study, we aimed to reveal the interrelation between ERS and Survivin. We initially verified the expression of Survivin in Winnie (a mouse model of chronic ERS) colon tissues by using immunohistochemistry (IHC) and immunofluorescence (IF) in comparison with wild type Blk6 mice. Additionally, we isolated the goblet cells and determined the expression of Survivin by IF and protein validation. Tunicamycin was utilized at a concentration of 10 µg/mL to induce ERS in the LS174T cell line and the gene expression of the ERS markers was measured. This was followed by determination of inflammatory cytokines. Inhibition of ERS was carried out by 4Phenyl Butyric acid (4PBA) at a concentration of 10 mM to assess whether there was a reciprocation effect. The downstream cell death assays including caspase 3/7, Annexin V, and poly(ADP-ribose) polymerase (PARP) cleavage were evaluated in the presence of ERS and absence of ERS, which was followed by a proliferative assay (EdU click) with and without ERS. Correspondingly, we inhibited Survivin by YM155 at a concentration of 100 nM and observed the succeeding ERS markers and inflammatory markers. We also verified the caspase 3/7 assay. Our results demonstrate that ERS inhibition not only significantly reduced the UPR genes (Grp78, ATF6, PERKandXBP1) along with Survivin but also downregulated the inflammatory markers such as IL8, IL4, and IL6, which suggests a positive correlation between ERS and the inhibition of apoptosis. Furthermore, we provided evidence that ERS inhibition promoted apoptosis in LS174T cells and shortened the proliferation rate. Moreover, Survivin inhibition by YM155 led to a comparable effect as that of ERS inhibition, which includes attenuation of ERS genes and inflammatory markers as well as the promotion of programmed cell death via the caspase 3/7 pathway. Together, our results propose the interrelation between ERS and inhibition of apoptosis assigning a molecular and therapeutic target for cancer treatment.

Dataset Information

Pro-apoptotic effect of haem oxygenase-1 in human colorectal carcinoma cells via endoplasmic reticular stress.

Publications

Pro-apoptotic effect of haem oxygenase-1 in human colorectal carcinoma cells via endoplasmic reticular stress.

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