PD-L1 blockade in mitigating severe acute pancreatitis induced pancreatic damage through modulation of immune cell apoptosis.

Acute pancreatitis (AP) is a prevalent gastrointestinal disorder. The immune response plays a crucial role in AP progression. However, the impact of immune regulatory checkpoint PD-L1 on severe acute pancreatitis (SAP) remains uncertain. Hence, this study aimed to examine the influence of PD-L1 on SAP. We assessed PD-L1 expression in neutrophils and monocytes obtained from SAP patients. We induced SAP in C57BL/6J mice, PD-L1 gene-deficient mice, and PD-L1 humanized mice using intraperitoneal injections of cerulein plus lipopolysaccharide. Prior to the initial cerulein injection, a PD-L1 inhibitor was administered. Pancreatic tissues were collected for morphological and immunohistochemical evaluation, and serum levels of amylase, lipase, and cytokines were measured. Flow cytometry analysis was performed using peripheral blood cells. The expression of PD-L1 in neutrophils and monocytes was significantly higher in SAP patients compared to healthy individuals. Likewise, the expression of PD-L1 in inflammatory cells in the peripheral blood of SAP-induced C57BL/6J mice was notably higher than in the control group. In mice with PD-L1 deficiency, SAP model exhibited lower pancreatic pathology scores, amylase, lipase, and cytokine levels compared to wild-type mice. PD-L1 deletion resulted in reduced neutrophil apoptosis, leading to an earlier peak in neutrophil apoptosis. Furthermore, it decreased early monocyte apoptosis and diminished the peak of T lymphocyte apoptosis. Within the SAP model, administration of a PD-L1 inhibitor reduced pancreatic pathology scores, amylase, lipase, and cytokine levels in both C57BL/6J mice and PD-L1 humanized mice. These findings suggest that inhibiting PD-L1 expression can alleviate the severity of SAP.

Mismatch repair protein deficiency and its implications on distant metastasis in colorectal cancer: A comprehensive analysis.

BACKGROUND: While previous studies have indicated variability in distant metastatic potential among different mismatch repair (MMR) states in colorectal cancer (CRC), their findings remain inconclusive, especially considering potential differences across various ethnic backgrounds. Furthermore, the gene regulatory networks and the underlying mechanisms responsible for these variances in metastatic potential across MMR states have yet to be elucidated. METHODS: We collected 2058 consecutive primary CRC samples from the South West of China and assessed the expression of MMR proteins (MLH1, MSH2, MSH6, and PMS2) using immunohistochemistry. To explore the inconsistencies between different MMR statuses and recurrence, we performed a meta-analysis. To delve deeper, we employed Weighted Gene Co-expression Network Analysis (WGCNA), ClueGo, and iRegulon, pinpointing gene expression networks and key regulatory molecules linked to metastasis and recurrence in CRC. Lastly, both univariate and multivariate Cox regression analyses were applied to determine the impact of core regulatory molecules on metastasis. RESULTS: Of the samples, 8.2% displayed deficient MMR (dMMR), with losses of MLH1 and PSM2 observed in 40.8% and 63.9%, respectively. A unique 24.3% isolated loss of PMS2 without concurrent metastasis was identified, a result that diverges from established literature. Additionally, our meta-analysis further solidifies the reduced recurrence likelihood in dMMR CRC samples compared to proficient MMR (pMMR). Two gene expression networks tied to distant metastasis and recurrence were identified, with a majority of metastasis-related genes located on chromosomes 8 and 18. An IRF1 positive feedback loop was discerned in the metastasis-related network, and IRF1 was identified as a predictive marker for both recurrence-free and distant metastasis-free survival across multiple datasets. CONCLUSION: Geographical and ethnic factors might influence peculiarities in MMR protein loss. Our findings also highlight new gene expression networks and crucial regulatory molecules in CRC metastasis, enhancing our comprehension of the mechanisms driving distant metastasis.

RNF6 enhances radioresistance in colorectal cancer via activating the Wnt pathway.

PURPOSE: RNF6 is verified to promote the malignant growth of colorectal cancer (CRC) and its level is linked to prognosis in CRC patients. Radioresistance is a key factor influencing prognosis in CRC. This study aimed to uncover the potential regulation of ring finger protein 6 (RNF6) in CRC radioresistance. METHODS: RNF6 levels in radioresistant and non-radioresistant CRC patients were detected. In vitro and in vivo regulatory effects of RNF6 on radioresistant CRC cell lines and nude mice bearing radioresistant CRC were examined, respectively. The involvement of Wnt pathway in CRC radioresistance was explored by Western blot. RESULTS: RNF6 was highly expressed in radioresistant CRC species than that of non-radioresistant ones. Identically, RNF6 was upregulated in radioresistant CRC cells compared to parental cells. SW1116 cells overexpressing RNF6 were more tolerant to radiotherapy, and similar results were obtained in nude mice bearing radioresistant CRC with overexpression of RNF6. Moreover, the Wnt pathway was activated during RNF6-induced radioresistance improvement in CRC. CONCLUSIONS: RNF6 enhances radioresistance of CRC through activating the Wnt pathway.

Knockdown of PPARδ Induces VEGFA-Mediated Angiogenesis via Interaction With ERO1A in Human Colorectal Cancer.

Angiogenesis is an important mechanism underlying the development and metastasis of colorectal cancer (CRC) and has emerged as a therapeutic target for metastatic CRC (mCRC). Our recent studies found that Peroxisome proliferator-activated receptor ß/δ/D (PPARδ) regulates vascular endothelial growth factor A(VEGFA) secretion and the sensitivity to bevacizumab in CRC. However, its exact effect and underlying mechanisms remain unidentified. In this study, we showed that PPARδ expression was inversely associated with the microvascular density in human CRC tissues. Knockdown of PPARδ enhanced VEGFA expression in HCT116 cells and HUVEC angiogenesis in vitro; these phenomena were replicated in the experimental in vivo studies. By tandem mass tag (TMT)-labeling proteomics and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, endoplasmic reticulum oxidoreductase 1 alpha (ERO1A) was screened and predicted as a target gene of PPARδ. This was verified by exploring the effect of coregulation of PPARδ and ERO1A on the VEGFA expression in HCT116 cells. The results revealed that PPARδ induced VEGFA by interacting with ERO1A. In conclusion, our results suggest that knockdown of PPARδ can promote CRC angiogenesis by upregulating VEGFA through ERO1A. This pathway may be a potential target for mCRC treatment.

Expression profile, molecular functions, and prognostic significance of miRNAs in primary colorectal cancer stem cells.

MicroRNAs (miRNAs) are known to drive the pathogenesis of colorectal cancer (CRC) via the regulation of cancer stem cells (CSCs). We studied the miRNA expression profile of primary CSCs isolated from patients with CRC (pCRCSCs). Compared to pCRCSC-derived differentiated cells, 98 differentially expressed miRNAs were identified in pCRCSCs. Target genes encoding pCRCSC-related miRNAs were identified using a combination of miRNA target databases and miRNA-mRNA regulatory networks from the same patient. The pCRCSC-related miRNA target genes were associated with pathways contributing to malignant phenotypes, including I-kappa B kinase/NF-kappa B signaling, signal transduction by p53 class mediator, Ras signaling, and cGMP-PKG signaling. The pCRCSC-related miRNA expression signature was independently associated with poor overall survival in both the training and validation cohorts. We have thus identified several pCRCSC-related miRNAs with oncogenic potential that could serve as prognostic biomarkers for CRC.

The bromodomain and extra-terminal domain inhibitor JQ1 synergistically sensitizes human colorectal cancer cells to topoisomerase I inhibitors through repression of Mre11-mediated DNA repair pathway.

Camptothecin (CPT) and its derivatives, irinotecan and topotecan are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs. Mechanistically, they induce DNA double-strand breaks (DSBs). Although CPT is an effective chemotherapeutic agent used in the management of advanced colorectal cancer, there exist associated side effects. Herein, we aimed to establish novel drug combinations that can effectively aid in managing the CPT-related side effects. Besides, bromodomain and extra-terminal domain (BET) inhibitors have proved as promising drugs that target epigenetic mechanisms in various cancers, they alter DNA repair processes, hence are a potential candidate for CPT synthetic lethality. A novel BET inhibitor JQ1 synergized with CPT, exerted antiproliferative effects. Through cell cycle analyses and apoptosis assays, we revealed that a combination of CPT and JQ1 induces subG1-phase arrest and enhances cell apoptosis. This combination increased the intensity of γ-H2AX staining, a specific marker of DSBs. Moreover, colorectal cancer cells highly expressing Top1 showed greater sensitivity to JQ1, which was lowered through the lentiviral shRNA-mediated knockdown of Top1. JQ1, combined with CPT, impeded the recruitment of the Mre11-mediated MRN complex. Finally, JQ1 enhanced the in vivo sensitivity of tumors to CPT without inducing toxicity. These results demonstrate that a combination of BET inhibitor with Top1 inhibitor is safe and exerts positive chemotherapeutic effects in colorectal cancer.

miR-124 Intensified Oxaliplatin-Based Chemotherapy by Targeting CAPN2 in Colorectal Cancer.

Our previous study demonstrated that miR-124 was downregulated in colorectal cancer (CRC) compared with normal mucosa, and the downregulated expression of miR-124 was an independent prognostic factor in CRC patients. However, the function of miR-124 in CRC patients treated with chemotherapy is currently unclear. The aim of this study was to determine the miR-124 expression and its regulative role in oxaliplatin (L-OHP)-based chemotherapy of CRC patients. We observed that low miR-124 expression was correlated with worse overall survival (OS) in the 220 patients who received postoperative chemotherapy of 5-fluorouracil [5-FU]+leucovorin+L-OHP (FOLFOX) or capecitabine+L-OHP (XELOX). miR-124 overexpression promoted L-OHP-induced, but not 5-FU-induced, cytotoxicity and apoptosis in HT29 and SW480 cells. CAPN2 was a direct target of miR-124, and its protein expression was reduced by forced expression of miR-124. miR-124 inhibited tumorigenesis and promoted OS of mice bearing xenograft tumors, especially upon L-OHP treatment. miR-124 also promoted L-OHP-induced apoptosis and restrained CAPN2 protein expression in xenograft tumors. Our results suggest that miR-124 could be considered as both a predictor of L-OHP-based chemotherapy for personalized treatment and a therapeutic target for CRC.

The expression of SOX9, Tiam1, and PTEN is correlated with angiogenesis and prognosis in gastric cancer.

BACKGROUND: With a high rate of metastasis and recurrence, gastric cancer (GC), a common malignant tumor, often has a poor prognosis. GC tissues have abnormal expressions of the Sry-related HMG-box family of transcription factors (SOX9), T-lymphoma invasion and metastasis-inducing factor 1 (Tiam1), and phosphatase and tensin homolog deleted on chromosome ten (PTEN). Meanwhile, vascular endothelial growth factor (VEGF) have been reported to play an important role in tumor angiogenesis. This study aimed to analyze the correlation of SOX9, Tiam1, and PTEN with angiogenesis and prognosis in GC. METHODS: A total of 90 patients who underwent GC surgery at our hospital between January 2017 and October 2018 were enrolled. The expressions of SOX9, Tiam1, PTEN, and VEGF in GC tissues and adjacent normal tissues were detected by immunohistochemistry and the differences were analyzed. Spearman's correlation coefficient was applied to analyze the relationship between the expression levels of SOX9, Tiam1, PTEN, and VEGF. The patients were followed-up. RESULTS: The positive expression rates of SOX9, Tiam1, PTEN, and VEGF in GC tissues were 75.56%, 61.11%, 52.22%, and 48.89%, respectively, compared with 6.67%, 4.44%, 97.78%, and 2.22%, respectively, in the adjacent normal tissues (P<0.05). Spearman's correlation analysis showed that the expression of SOX9 (r=0.349, P=0.001) and Tiam1 (Tiam1: r=0.370, P=0.000) in GC tissues was positively correlated with VEGF expression; however, PTEN was negatively correlated with VEGF (r=-0.311, P=0.000). There were no significant differences in SOX9, Tiam1, or PTEN expression in GC tissues from patients of different genders or ages (P>0.05). When the tumor was low differentiated, with lymph node metastasis or high TNM staging, the positive expression rates of SOX9 and Tiam1 were significantly increased (P<0.05), while the positive expression rate of PTEN was significantly decreased (P<0.05). The log-rank test results showed that the three-year survival rates of the groups with positive SOX9 (54.41%) and Tiam1 expression (49.09%) were significantly lower than those of the groups with negative SOX9 and Tiam1 expression (77.27% and 77.13%, respectively; P<0.05). The 3-year survival rate in the PTEN positive expression group was significantly higher than that of the PTEN negative expression group (76.60% vs. 41.80%; P<0.05). CONCLUSIONS: SOX9 and Tiam1 are highly expressed, in GC tissues while there is a low expression of PTEN. The expression levels of SOX9, Tiam1, and PTEN are all linearly correlated with VEGF expression, and they have important effects on angiogenesis and is closely related to the three-year survival rate of patients with GC.

Deletion Of XIAP reduces the severity of acute pancreatitis via regulation of cell death and nuclear factor-κB activity.

Severe acute pancreatitis (SAP) still remains a clinical challenge, not only for its high mortality but the uncontrolled inflammatory progression from acute pancreatitis (AP) to SAP. Cell death, including apoptosis and necrosis are critical pathology of AP, since the severity of pancreatitis correlates directly with necrosis and inversely with apoptosis Therefore, regulation of cell death from necrosis to apoptosis may have practicably therapeutic value. X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the inhibitor of apoptosis proteins (IAP) family, but its function in AP remains unclear. In the present study, we investigated the potential role of XIAP in regulation of cell death and inflammation during acute pancreatitis. The in vivo pancreatitis model was induced by the administration of cerulein with or without lipopolysaccharide (LPS) or by the administration of l-arginine in wild-type or XIAP-deficient mice, and ex vivo model was induced by the administration of cerulein+LPS in AR42J cell line following XIAP inhibition. The severity of acute pancreatitis was determined by serum amylase activity and histological grading. XIAP deletion on cell apoptosis, necrosis and inflammatory response were examined. Caspases activities, nuclear factor-κB (NF-κB) activation and receptor-interacting protein kinase1 (RIP1) degradation were assessed by western blot. Deletion of XIAP resulted in the reduction of amylase activity, decrease of NF-κB activation and less release of TNF-α and IL-6, together with increased caspases activities and RIP1 degradation, leading to enhanced apoptosis and reduced necrosis in pancreatic acinar cells and ameliorated the severity of acute pancreatitis. Our results indicate that deletion of XIAP switches cell death away from necrosis to apoptosis and decreases the inflammatory response, effectively attenuating the severity of AP/SAP. The critical role of XIAP in cell death and inflammation suggests that inhibition of XIAP represents a potential therapeutic strategy for the treatment of acute pancreatitis.

Effects of Tocilizumab on Experimental Severe Acute Pancreatitis and Associated Acute Lung Injury.

OBJECTIVE: To examine the therapeutic effects of tocilizumab, an antibody against interleukin-6 receptor, on experimental severe acute pancreatitis and associated acute lung injury. The optimal dose of tocilizumab and the activation of interleukin-6 inflammatory signaling were also investigated. DESIGN: Randomized experiment. SETTING: Research laboratory at a university hospital. SUBJECT: Experimental severe acute pancreatitis in rats. INTERVENTIONS: Severe acute pancreatitis was induced by retrograde injection of sodium taurocholate (50 mg/kg) into the biliopancreatic duct. In dose-study, rats were administered with different doses of tocilizumab (1, 2, 4, 8, and 16 mg/kg) through the tail vein after severe acute pancreatitis induction. In safety-study, rats without severe acute pancreatitis induction were treated with high doses of tocilizumab (8, 16, 32, and 64 mg/kg). Serum and tissue samples of rats in time-study were collected for biomolecular and histologic evaluations at different time points (2, 6, 12, 18, and 24 hr). MEASUREMENTS AND MAIN RESULTS: 1) Under the administration of tocilizumab, histopathological scores of pancreas and lung were decreased, and severity parameters related to severe acute pancreatitis and associated lung injury, including serum amylase, C-reactive protein, lung surfactant protein level, and myeloperoxidase activity, were all significant alleviated in rat models. 2) Dose-study demonstrated that 2 mg/kg tocilizumab was the optimal treatment dose. 3) Basing on multi-organ pathologic evaluation, physiological and biochemical data, no adverse effect and toxicity of tocilizumab were observed in safety-study. 4) Pancreatic nuclear factor-κB and signal transducer and activator of transcription 3 were deactivated, and the serum chemokine (C-X-C motif) ligand 1 was down-regulated after tocilizumab administration. CONCLUSIONS: Our study demonstrated tocilizumab, as a marketed drug commonly used for immune-mediated diseases, was safe and effective for the treatment of experimental severe acute pancreatitis and associated acute lung injury. Our findings provide experimental evidences for potential clinical application of tocilizumab in severe acute pancreatitis and associated complications.