WTAP-induced N6-methyladenosine of PD-L1 blocked T-cell-mediated antitumor activity under hypoxia in colorectal cancer.

N6-Methyladenosine (m6A) is a important process regulating gene expression post-transcriptionally. Programmed death ligand 1 (PD-L1) is a major immune inhibitive checkpoint that facilitates immune evasion and is expressed in tumor cells. In this research we discovered that Wilms' tumor 1-associated protein (WTAP) degradation caused by ubiquitin-mediated cleavage in cancer cells (colorectal cancer, CRC) under hypoxia was inhibited by Pumilio homolog 1 (PUM1) directly bound to WTAP. WTAP enhanced PD-L1 expression in a way that was m6A-dependent. m6A "reader," Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) identified methylated PD-L1 transcripts and subsequently fixed its mRNA. Additionally, we found that T-cell proliferation and its cancer cell-killing effects were prevented by overexpression of WTAP in vitro and in vivo. Overexpression prevented T cells from proliferating and killing CRC by maintaining the expression of PD-L1. Further evidence supporting the WTAP-PD-L1 regulatory axis was found in human CRC and organoid tissues. Tumors with high WTAP levels appeared more responsive to anti-PD1 immunotherapy, when analyzing samples from patients undergoing treatment. Overall, our findings demonstrated a novel PD-L1 regulatory mechanism by WTAP-induced mRNA epigenetic regulation and the possible application of targeting WTAP as immunotherapy for tumor hypoxia.

Up-regulation of PUM1 by miR-218-5p promotes colorectal tumor-initiating cell properties and tumorigenesis by regulating the PI3K/AKT axis.

Background: Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death worldwide. Advanced stage CRC, during the recent past, had a dismal prognosis and only a few available treatments. Pumilio homologous protein 1 (PUM1) is reportedly aberrant in human malignancies, including CRC. However, the role of PUM1 in the regulation of tumor-initiating cells (T-ICs) remains unknown. Methods: The levels of messenger RNAs (mRNAs) were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunoblot analyses. Statistical analyses were performed to determine the associations between the levels of PUM1 and tumor features and patient outcomes. Whether PUM1 is a downstream target of miR-218-5p was verified by bioinformatics target gene prediction and qRT-PCR. Results: Herein, it was found that T-ICs, chemoresistance, and recurrent CRC samples all manifest increased PUM1 expression. Functional investigations have shown that PUM1 increased the self-renewal, tumorigenicity, malignant proliferation, and chemoresistance of colorectal cells. PUM1 activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway biochemically. Furthermore, it was discovered that miR-218-5p specifically targets T-ICs' PUM1 3'-untranslated region (3'-UTR). More importantly, the PUM1/PI3K/AKT axis regulates CRC cells' responses to treatment with cetuximab, and PUM1 overexpression increased cetuximab resistance. More evidence points to the possibility that low PUM1 may predict cetuximab benefits in CRC patients after analysis of the patient cohort, patient-derived tumor organoids, and patient-derived xenografts (PDXs). Conclusions: Taken together, the result of this work points to the critical function of the miR-218-5p/PUM1/PI3K/AKT regulatory circuit in regulating T-ICs characteristics and thus suggests possible therapeutic targets for CRC.

Mesenchymal stem cells improve intestinal integrity during severe acute pancreatitis.

Severe acute pancreatitis (SAP) is an acute inflammatory disease of the pancreas that involves various distant tissues and organs. This study aimed to investigate post-tissue injury repair by mesenchymal stem cells (MSCs) in a rat model of SAP. A total of 54 pathogen-free adult male SD rats were randomly assigned to the groups SAP, SAP + MSCs and sham-operated (SO). SAP was induced by 4% sodium taurocholate, and MSCs were injected via the dorsal penile vein 1 h later. The amylase activity, and tumor necrosis factor (TNF)-α and diamine oxidase (DAO) levels were measured with an enzyme-linked immunosorbent assay (ELISA), while the expression of aquaporin (AQP)-1 was evaluated by immunohistochemical staining. The pathological score of intestinal tissues was also compared among groups. Marked improvement in intestinal necrosis, villi shedding and infiltration of inflammatory cells was observed in the SAP + MSCs group compared to the SAP and SO groups. Amylase, TNF-α, and DAO levels were significantly increased in the SAP + MSCs group. The intestinal expression of AQP-1 was increased at 12 and 24 h post-MSC transplantation compared to the SO group. Rats of the SAP + MSCs group displayed higher pathological scores compared to the SAP group at all time points. Overall, these data showed that MSCs can inhibit systemic inflammation and reduce TNF-α release in a rat model of SAP-induced intestinal injury, suggesting that MSCs exert protective effects on the intestinal barrier during SAP.

"Fast track" rehabilitation after gastric cancer resection: experience with 80 consecutive cases.

BACKGROUND: To evaluate the safety, efficacy and outcomes of fast-track rehabilitation applied to gastric cancer proximal, distal and total gastrectomy. METHODS: Eighty consecutive patients undergoing gastric cancer resection performed by a single surgeon, received perioperative multimodal rehabilitation. Demographic and operative data, gastrointestinal function, postoperative hospital stays, surgical and general complications and mortality were assessed prospectively. RESULTS: Of the 80 patients (mean age 56.3 years), 10 (12.5%) received proximal subtotal gastrectomy (Billroth I), 38 (47.5%) received distal (Billroth II), and 32 (40%) received total gastrectomy (Roux-en-Y). Mean operative time was 104.9 minutes and intraoperative blood loss was 281.9 ml. Time to first flatus was 2.8 ± 0.5 postoperative days. Patients were discharged at a mean of 5.3 ± 2.2 postoperative days; 30-day readmission rate was 3.8%. In-hospital mortality was 0%; general and surgical complications were both 5%. CONCLUSIONS: Fast-track multimodal rehabilitation is feasible and safe in patients undergoing gastric cancer resection and may reduce time to first flatus and postoperative hospital stays.

[Correlation of methylation of CpG island in cystathionine beta synthase promoter and clinicopathological features in colorectal cancer].

OBJECTIVE: To explore the association between methylation of cystathionine-beta-synthase (CBS) promoter and clinicopathological features in colorectal cancer. METHODS: Bisulfate sequencing PCR, real-time RT-PCR, and immunohistochemistry were used to investigate the methylation of CpG island in CBS promoter of 95 sporadic colorectal cancers. Software SPSS PASW Statistics was used to analyze the data of the hypermethylation levels in the malignant tissues and the correlation with pathological parameters and clinical outcome. RESULTS: Methylation levels in tumor tissue of patients [(64.9 ± 14.3)%]with colorectal cancer were significantly higher than that in normal tissues[(27.5 ± 13.1)%, P < 0.001]. The CBS mRNA levels in the hypomethylation group (7.22 ± 1.91) were significantly higher than that in the hypermethylation group (2.78 ± 1.12, P < 0.01). Univariate analysis showed that age, pT stage, pN stage, liver metastases, pTNM stage, and CBS hypermethylation level significantly correlated with the survival and recurrence rates of colorectal cancer patients (All P < 0.05). Multivariate analysis showed that CBS hypermethylation level and liver metastasis were independent factors significantly correlated with the recurrence rate and overall survival of the patients (All P < 0.05). CONCLUSIONS: Our study indicates that methylation of CpG island in CBS promoter is correlated with the occurrence and progression of colorectal cancer and plays a role in its tumorigenesis. It might serve as a useful marker for early diagnosis, targeted therapy and prediction of prognosis in colorectal cancer.

Protective effect of transplanted bone marrow-derived mesenchymal stem cells on pancreatitis-associated lung injury in rats.

Severe acute pancreatitis (SAP) is initiated by the premature activation of digestive enzymes within the pancreatic acinar cells, leading to self-digestion and inflammatory responses in pancreatic ductal cells, thus giving rise to systemic inflammatory response syndrome (SIRS). The most common and serious SIRS is pancreatitis-associated lung injury, and inflammatory mediators play an important role in its pathogenesis. Bone marrow-derived mesenchymal stem cells (MSCs) are differentiated into alveolar endothelial cells to replace the damaged alveolar endothelial cells and inhibit inflammatory response in the injured lung tissues. In this study, we aimed to investigate the therapeutic effect of bone marrow-derived MSCs in rats with pancreatitis-associated lung injury. Experimental SAP was induced by a retrograde injection of 5% sodium taurocholate into the biliopancreatic duct of 75 male Sprague-Dawley rats, which were divided into the SAP group (n=25), the MSC group (n=25) and the sham-operated group (n=25) to explore the pathology and function of lung tissues and the regulation of inflammatory mediators. Pulmonary edema was estimated by measuring water content in the lung tissues. Pulmonary myeloperoxidase (MPO) activity was detected using spectrophotometry. Serum amylase was detected using the Automatic Biochemistry Analyzer. Tumor necrosis factor-α (TNF-α) and substance P (SP) mRNA levels were determined by quantitative reverse transcriptase-polymerase chain reaction. Our results showed that serum amylase activity was significantly decreased in the MSC group compared to the SAP group. Pulmonary edema was significantly diminished (p<0.05) in the MSC group compared to the SAP group. Typical acute lung injury was observed in the SAP group, and the pathological changes were mild in the MSC group. The expression of TNF-α and SP mRNA in lung tissue was diminished in the MSC group compared to the SAP group. In conclusion, MSC transplantation attenuates pulmonary edema and inflammation, and reduces the mRNA expression of TNF-α and SP in pancreatitis-associated lung injury.

Role of bone marrow-derived mesenchymal stem cells in a rat model of severe acute pancreatitis.

AIM: To investigate the role and potential mechanisms of bone marrow mesenchymal stem cells (MSCs) in severe acute peritonitis (SAP). METHODS: Pancreatic acinar cells from Sprague Dawley rats were randomly divided into three groups: non-sodium deoxycholate (SDOC) group (non-SODC group), SDOC group, and a MSCs intervention group (i.e., a co-culture system of MSCs and pancreatic acinar cells + SDOC). The cell survival rate, the concentration of malonaldehyde (MDA), the density of superoxide dismutase (SOD), serum amylase (AMS) secretion rate and lactate dehydrogenase (LDH) leakage rate were detected at various time points. In a separate study, Sprague Dawley rats were randomly divided into either an SAP group or an SAP + MSCs group. Serum AMS, MDA and SOD, interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α levels, intestinal mucosa injury scores and proliferating cells of small intestinal mucosa were measured at various time points after injecting either MSCs or saline into rats. In both studies, the protective effect of MSCs was evaluated. RESULTS: In vitro, The cell survival rate of pancreatic acinar cells and the density of SOD were significantly reduced, and the concentration of MDA, AMS secretion rate and LDH leakage rate were significantly increased in the SDOC group compared with the MSCs intervention group and the Non-SDOC group at each time point. In vivo, Serum AMS, IL-6, TNF-α and MAD level in the SAP + MSCs group were lower than the SAP group; however serum IL-10 level was higher than the SAP group. Serum SOD level was higher than the SAP group at each time point, whereas a significant between-group difference in SOD level was only noted after 24 h. Intestinal mucosa injury scores was significantly reduced and the proliferating cells of small intestinal mucosa became obvious after injecting MSCs. CONCLUSION: MSCs can effectively relieve injury to pancreatic acinar cells and small intestinal epithelium, promote the proliferation of enteric epithelium and repair of the mucosa, attenuate systemic inflammation in rats with SAP.

[Protective effect of N-acetylcysteine on the intestinal barrier dysfunction after radiation injury in rats].

OBJECTIVE: To evaluate the protective effect of N-acetylcysteine (NAC) on the intestinal barrier dysfunction in rats after extensive abdominal radiation with X ray. METHODS: Twenty-four Spraque-Dawley male rats were divided into normal control group (n=8), radiation group (n=8), and radiation+NAC group (300 mg/kg) (n=8). Radiation injury was induced by X ray with a single dose of 10 Gy. NAC was administered from 4 days before irradiation to 3 days after radiation. Three days after radiation, all the rats were euthanized. The terminal ileum was collected for crypt survival assay and ileal villi count. The tissue samples from mesenteric lymph nodes (MLN), spleen, and liver were harvested under sterile conditions for microbiological analysis and ileum samples were harvested for biochemical analysis. The blood levels of D-lactate, endotoxin and diamine oxidase (DAO) and the ileum samples levels of nitric oxide(NO) were also measured. RESULTS: Rats in radiation+NAC group had a higher survival rate of intestinal crypt [(76.84+/-4.82)% vs (49.64+/-5.48)%, P<0.01], higher intestinal villus count [(8.56+/-0.68)/mm vs (4.02+/-0.54)/mm, P<0.01], lower NO concentration [(0.48+/-0.12) mumol/g vs (0.88+/-0.16) mumol/g, P<0.01], lower levels of D-lactate, endotoxin and DAO (P<0.05 or P<0.01), and significantly decreased enteric bacteria cultured from mesenteric lymph nodes and other tissues as compared with the radiation group (P<0.05 or P<0.01). CONCLUSION: NAC protects the small intestine from radiation-induced injury maybe through the inhibition of NO in rats.

Safety and efficacy of Qingre Buyi Decoction in the treatment of acute radiation proctitis: a prospective, randomized and controlled trial.

OBJECTIVE: To investigate the efficiency, safety, and possible mechanisms of Qingre Buyi Decoction (QBD) in the treatment of acute radiation proctitis (ARP). METHODS: This study was a single center, prospective, single blind, randomized, and placebo-controlled clinical trial. A total of 60 patients with ARP was equally and randomly distributed into the control group (conventional treatment) and the combination group (conventional treatment plus QBD). The changes of main Chinese medicine clinical symptoms and signs, including stomachache, diarrhea, mucous or bloody stool before and after treatment, and their adverse reactions were observed after the two-week treatment. Also, D-lactate and diamine oxidase (DAO) levels, hepatic and renal function were measured. Cure rates, effective rates, and recurrence rates were compared between the two groups. RESULTS: The blood levels of both DAO and D-lactate were significantly decreased in the combination group as compared with those in the control group (P<0.05 or P<0.01). All main clinical symptoms and signs were alleviated more significantly in the combination group (P<0.01). The main symptom scores also were significantly decreased after treatment in the control group (P<0.01), except those for mucous or bloody stool (P>0.05). Compared to the control group, the improvements of stomachache, diarrhea, defecation dysfunction, and stool blood in the combination group were significantly better (P<0.05 or P<0.01). For the combination group, the curative rate, effective rate, and recurrence rate was 76.67%, 16.67%, and 6.67%, respectively. On the other hand, for the control group, the rate was 53.33%, 16.67%, and 30.00%, respectively. The total curative effect was significantly better in the combination group than in the control group (P<0.05). However, the recurrence rate was similar between the two groups (P>0.05). The hepatic and renal function remained normal in both groups (P>0.05). In addition, no severe adverse event was found in both groups. CONCLUSIONS: Addition of QBD to the conventional treatment can effectively alleviate the damage of intestinal mucosal barrier function and improve all main clinical symptoms and signs of the ARP. The combination of conventional treatment with Chinese herbal medicine QBD is effective and safe for ARP.

[Evaluation of endoscopy-assisted laparoscopic surgery in the treatment of early colon cancer].

OBJECTIVE: To evaluate the endoscopy-assisted laparoscopic surgery in the treatment of early colon carcinoma. METHODS: The data of 55 early colon cancer patients, including 30 male, 25 female with mean age of 54 years(ranged 42 to 68), undergone endoscopy-assisted laparoscopic surgery at the colon were reviewed retrospectively. RESULTS: From March 2002 to December 2007, 55 early colon cancer patients were treated with endoscopy-assisted laparoscopic surgery in our institute. In 53 cases, a laparoscopic and endoscopic cooperative bowel segment resection was performed at first. Of these 53 patients, 11 cases then received laparoscopic and endoscopic cooperative radical anatomical resection according to the result of frozen section. Two cases were transferred to open surgery because of small intestinal inflation after endoscopic location. The mean operative time of cooperation was 90 min (55-240 min), and the mean blood loss was 50 ml(10-200 ml). In 51 cases(92.7%), the time for flatus passage was 2 to 3 days. The mean postoperative hospital stay was 5 d(2-15 d). No postoperative complications were found. Follow-up data were obtained by clinical examination and personal communication via telephone. The median follow-up was 42 months(ranged 3-72). Most of the patients were alive except one case died of myocardial infarction during the follow-up period. None of the patients with early colon cancer treated by the cooperative surgery had relapse or metastasis. Two cases of T1N1Mx underwent adjuvant chemotherapy. CONCLUSIONS: Endoscopy-assisted laparoscopic surgery offers a minimal-invasive and safe therapeutic approach for early colon cancer. The early colon cancer may be a good indication for endoscopy-assisted laparoscopic surgery when the endoscopic mucosal resection is inadequate.

[Clinical efficacy of buzhong yiqi pill combined with imodium in treating post-operational diarrhea in patients of colonic cancer].

OBJECTIVE: To investigate the clinical efficacy of Buzhong Yiqi Pill (BYP) combined with imodium in treating post-operational diarrhea in patients undergoing colonic cancer surgery. METHODS: Eighty patients with diarrhea after colorectal cancer surgery were randomized into two groups equally, the control group were treated with imodium (loperamide hydrochloride) and the treatment group treated by BYP combined with imodium. The therapeutic efficacy was analyzed and evaluated comprehensively depending upon a defecation check table developed from the XU Zhong-fa's 5-item 10-integrable system. RESULTS: After treatment, the improvements of the anal controlling capacity, the defecatory sensation, the frequency of defecation in the treatment group were significantly better than those in the control group (P < 0.01 or P < 0.05). The integral function of defecation in the treatment group was obvionsly improved by the end of treatment when compared with before treatment and the control group (P < 0.01). CONCLUSION: The clinical efficacy of the BYP combined with imodium in treating post-operational diarrhea after colorectal cancer surgery were better than that of imodium alone.