| Clostridium difficile is a Gram-positive normal bacteria that locating in intestinal tract of human and animal. It is the main cause of antibiotic-associated diarrhea and pseudomembranous colitis in case of the imbalance of intestinal flora, especially in the context of the abuse of antibiotics. In the past decade, the morbidity and mortality of C. difficile infection has increased significantly due to the emergence of hypervirulent strains. As the major virulence factors of C. difficile with similar action mechanism, the pro-apoptotic and pro-inflammation properties of toxin A(Tcd A) and toxin B(Tcd B) have been widely reported. The prior work of our group have found that recombinant Tcd B(r Tcd B) can induce immunogenicity cell death(ICD) in CT26 tumor cell. ICD is a new cell death modality that dying cells will stimulate immune responses against dead-cell antigens. The immunogenic characteristics of ICD are mainly mediated by damage-associated molecular patterns(DAMPs), which include changes in the composition of the cell surface as well as the release of soluble mediators. Up to date, only several clinically successful anticancer agents can induce ICD. Moreover, ER stress and ROS play import role in regulating the immunogenicity of dying cancer cells and the pathways involved in the emission of several vital DAMPs. Although great progress has been achieved in understanding the pathologic mechanism of Tcd A and Tcd B, many debates existing in mechanisms of cell death induced by them, and whether the reactive oxygen species(ROS) and endoplasmic reticulum stress(ER stress) play roles during which is still unknown. This study mainly checked the induction of ROS and ER stress in r Tcd B intoxicated cells and their roles in cell death induced by Tcd B.The results showed that dose-independent ROS increase was induced in r Tcd B intoxicated CT26 cells. The dynamic change of ROS level was found within 24 hours. The three pharmacological repression agents of ROS, N-acetyl-L-cysteine(NAC, the working concentration is 5m M), diphenyleneiodonium chloride(DPI, the working concentration is 5μM) or antimycin A(Anti.A, the working concentration is 5μM) not only significantly decreased PS exposure and the percent of sub-G1 population, but also significantly inhibited the down-regulation of Bcl-2 protein and the upregulation of phosphorylated STAT3 protein, which suggests that ROS involves in apoptosis caused by r Tcd B by regulation of Bcl-2 and STAT3 proteins in CT26 cells. ROS was induced by r Tcd B in HCT-8 cells, and NAC protects r Tcd B intoxicated HCT-8 cells by acting on the expression of Bcl-2 proteins, the mitochondrial membrane potential and the turnover of LC3 B protein.The PERK and IRE1 branch of unfolded protein response(UPR) was activated by r Tcd B. However, the mutant r Tcd B losing glucosyltransferase activity also evoked them, which means ER stress induced by r Tcd B is glucosyltransferase independent. Beside, it is mutant r Tcd B but not r Tcd B induced the upregulation of CHOP protein(a key marker of ER stress related apoptosis), which suggests the expression of CHOP protein is controlled by glucosyltransferase. As a selectively inhibitor of de-phosphorylation of e IF2α, salubrinal not only obviously alleviated cytotoxic and cytopathic effect of r Tcd B, but also decreased LDH release, PS exposure, the percent of sub-G1 population and the activation of caspase 9 induced by r Tcd B. These data sufficiently confirmed that salubrinal is an effective protectant for r Tcd B intoxicated cells. Further study found that salubrinal delayed the process of glucosylation of Rac1 in r Tcd B intoxicated CT26 cells, and it also inhibited the ratio of LC3B-I/LC3B-II after 36 hours treatment. In summary, ER stress induced by r Tcd B is not involved in cell death caused by r Tcd B.The results of animal experiments showed that after challenge with live CT26 cells, the mice inoculated with cells treated with r Tcd B, r Tcd B(500 ng/m L) and TUCDA, or r Tcd B(500 ng/m L) and 4-PBA have no tumor development, suggests that ER stress is not involved in r Tcd B induced ICD. Tumor development were found in mice inoculated with cells treated with r Tcd B and DPI, or r Tcd B and Anti.A. No tumor development were found in the mice inoculated with cells treated with r Tcd B and NAC. The reason may lies in that NAC playing its role after production of ROS, while DPI and Anti.A inhibiting the production of ROS from source. Further study found that massive supernatant secretion of HSP70, HSP90 and HMGB1 proteins induced by r Tcd B were significantly inhibited by DPI and Anti.A, but not NAC. Besides, the obviously increased surface-exposed CRT in dying r Tcd B treated cells was decreased by DPI and Anti.A but not NAC. However, NAC, DPI and Anti.A have no effect on the release of ATP induced by r Tcd B, but they significantly inhibited the accumulation of LC3B-II protein in CT26 cells. All together, these data suggests that ROS involving in r Tcd B induced ICD by control location of some key DAMPs and autophagy caused by r Tcd B.In a word, this study confirms the induction of ROS and ER stress in r Tcd B intoxicated CT26 cells, and ROS play important role in apoptosis and ICD induced by r Tcd B, but ER stress is not involved in the two processes. |
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