Allosteric inhibition reveals SHP2-mediated tumor immunosuppression in colon cancer by single-cell transcriptomics

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING-TBK1-IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.

Applications of BGUSs inhibition in tumor prevention and chemotherapy / 中国临床药理学与治疗学

Bacterial β-glucuronidases (BGUSs) are an important type of hydrolase produced by gut microbiota and play an important role in tumor development and chemotherapy by deconjugating a large number of endogenous and exogenous glucuronides. In recent years, BGUSs inhibition has emerged as a promising approach to reduce cancer risk and alleviate gastrointestinal toxicity of chemotherapy drugs. However, a growing body of evidence underlines great genetic and structural diversity, functional promiscuity, and varied inhibition propensity of BGUSs, which have posed enormous challenges to identifying BGUSs involved in a specific pathophysiological or pharmacological process and developing effective inhibitors. In this review, we summarize the latest advances in structure and function of BGUSs and review the findings of BGUSs-mediated carcinogen reactivation and deconjugation of chemotherapy drugs in recent years, as well as the discovery of BGUSs inhibitors and preclinical investigation of their applications in cancer prevention and drug therapy. Finally, we discuss the prospects of BGUSs inhibition as a new strategy for tumor prevention and drug therapy.

Overexpression of a leucine transfer RNA gene tL(CAA)K improves the acetic acid tolerance of Saccharomyces cerevisiae / 生物工程学报

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.

Effects of perioperative probiotics administration on patients with colorectal cancer / 中华临床营养杂志

Objective To evaluate the effects of perioperative probiotics administration on patients with colorectal cancer and to explore its possible mechanism.Methods Seventy patients with colorectal cancer who were scheduled to undergo radical colorectomy at Shanghai Sixth People's Hospital between May 2011 and July 2011 were randomly divided with random number table into the control group (n =35) and the treatment group (n =35).The two groups in 5 days preoperatively and 7 days postoperatively were given daily doses of probiotics preparation consisting of two combined live bacteria and placebo,respectively.The structure of intestinal epithelial tight junction was observed by electron microscopy in colorectal tissue specimens collected during the operation.The expression of tight junctional protein was detected using Western blot and real-time RT-PCR technology.Intestinal epithelial permeability was evaluated by Ussing Chamber system.Stool samples and blood samples were collected on the 7th day after operation.The diversity of faecal flora was analyzed by terminal restriction fragment length polymorphism (T-RFLP) technique,and the quantitative detection of specific bacteria was conducted by bacterial culture.Clinical parameters including the first exhaust and defecate time,distension and diarrhea incidence,systemic inflammatory response,and postoperative infective complications were recorded.Results Compared with the control group,the treatment group showed better intestinal epithelial tight junction ultrastructure.The expression of tight junction proteins occludin,claudin-1,and ZO-1 (protein:all P ＜ 0.001 ; mRNA:P =0.005,0.001,0.006) and the transepithelial electrical resistance [(28.3 ±5.2) Ω · cm2 vs.(22.1 ± 4.7) Ω · cm2,P =0.002] were significantly increased,the large molecule permeability [(0.91 ± 0.17) ％ vs.(1.65 ± 0.33) ％,P ＜ 0.001] reduced,the diversity of intestinal flora (P=0.006) increased,the growth of intestinal Bifidobacteria [(143.4 ±35.9) vs.(100.0 ±0.0),P=0.002] and Lactobacilli [(111.3 ± 52.9) vs.(100.0 ± 0.0),P ＜ 0.001] promoted,and the growth of Clostridium perfringens [(66.2 ±23.7) vs.(100.0 ±0.0),P ＜0.001] inhibited in the treatment group.The treatment group also showed shorter postoperative exhaust [(2.5 ± 1.7) d vs.(4.5 ±2.0) d,P ＜0.001] and defecate time [(5.0 ± 1.3) d vs.(6.3 ± 1.1) d,P =0.002],lower incidence of diarrhea (20％ vs.40％,P =0.005) and abdominal distension (35 ％ vs.60％,P =0.021).Conclusion Probiotics used perioperatively in patients with colorectal cancer can effectively enhance the intestinal epithelia barrier function,maintain the homeostasis of gut flora,shorten the postoperative first exhaust and defecate time,reduce the incidence of diarrhea and abdominal distension,and promote the recovery of intestinal function.

Effects of interleukin-10 in epithelial-mesenchymal transition and endoplasmic reticulum stress on intestinal fibrosis / 中华消化杂志

Objective To investigate the effects of interleukin-10 on mice intestinal fibrosis and epithelial-mesenchymal transition(EMT),and the relation between these effects and endoplasmic reticulum stress(ERS).Methods Forty-two IL-10 knockout(IL-10-/-)mice were divided into fibrosis model group(n=18),IL-10 treatment group(n=12)and solvent control group(n =12),another 18 wild-type mice were taken as negative control group.IL-10 and 0.9％ NaCl were intraperitonealy injected in IL-10 treatment group and solvent control group respectively since 12th week,and mice were sacrificed at week 14th and 16th,and no treatment to fibrosis model group and negative control group.The injury and fibrosis in mice colon tissue were detected with HE staining and Masson collagen staining.The expressions of collagen Ⅰ,glucose-regulated protein78(GRP78),C/EBP homologous protein(CHOP)and a-smooth muscle actin(α-SMA)and E-cadherin(E-cad)at mRNA level were determined by realtime PCR.The expression of α-SMA and E-cad in mice colon tissue was examined by immunohistochemical staining.Results At 16th week,the colonic tissue injury scores(7.00±0.90,7.17±1.17)and collagen area ratio(17.78％±4.15％,18.56％±3.81％)of fibrosis model group and solvent control group significantly increased compared with negative control group(1.50±1.38 and 9.11％±2.99％)and IL-10 treatment qroup(4.33±0.82 and 12.56％±1.39％)(F=36.150,F=11.280； P=0.000).At week 12th,14th and 16th,the expressions of GRP78,α-SMA,collagen Ⅰ in fibrosis model group and solvent control group significantly increased compared with negative control group(all P＜0.05),however the expression of E-cad significantly decreased(P＜0.05).The expression of CHOP mRNA in fibrosis model group(0.95％ ±0.12％)significantly increased compared with negative control group(0.21％ ± 0.12％)at week 12th(t=5.188,P=0.000),however there was no statistical significant difference in groups at week 14th and 16th(P＞0.05).At week 14th and 16th,the expressions of GRP78,α-SMA and collagen Ⅰ(at week 14th:0.73％±0.31％,1.18％±0.11％ and 1.10％±0.49％； at week 16th:0.57％±0.16％,0.81％ ±0.50 ％ and 0.76 ％ ± 0.25 ％)in IL-10 treatment group were significantly lower than that of fibrosis model group(P＜0.05).The expression of E-cad(at week 14th:0.73％ ±0.29％ ； at week 16th:0.97％ ±0.25％)significantly increased compared with fibrosis model group(at week 14th:0.37％±0.17％； at week 16th:0.35％±0.20％)(F=6.524,P=0.003； F=17.493,P=0.000).However at week 16th,the expression of α-SMA in IL-10 treatment group was lower than that of solvent control group(1.82±0.22)％(F=9.842,P=0.000),and the expression of E-cad significantly increased than in solvent control group(0.47 ± 0.25)％(F=17.493,P =0.000).Conclusion IL-10 may have a role in inhibiting EMT and reducing intestinal fibrosis in mice,which may be related to the regulation of ERS by IL-10.

The effect of Lactobacillus plantarum on the treatment of colitis in interleukin-10 knockout mice / 中华消化杂志

Objective To evaluate the role of Lactobacillus plantarum (LP) in the treatment of colitis in interleukin (IL)-10 knockout (IL-10-/-) mice and to explore its possible mechanisms.Methods Eight weeks old female wildtype (WT) mice and IL-10-/- mice, twenty mice of each type,were randomly assigned to four groups, WT group, WT+ LP group, IL-10-/- group and IL-I0-/- +LP group. The WT and IL-10-/- mice were gavaged with 0.5 ml saline, WT+Lp and IL-10-/- +Lp groups were gavaged with Lp 0.02 g (0.5 ml) ,took Lp 1 × 109 cfu everyday,continued for 4 weeks and then the experiment finished. The fresh mice faeces was collected once every week before (week 0) and during the experiment. The mice were executed at the end of experiments, the change of mice weight Was recorded, the length and the wet weight of colon were measured, fresh colon tissue specimens were taken for biological slices and proinflammatory cytokines TNF-a and IFN-γ were measured in colon mucosa. The fresh faeces were selectively cultured. The colonization of Lp in normal and colitis mice and its regulation role in intestinal flora were observed. Results Compared with WT mice, IL-10-/- mice demonstrated severe diarrhea, significantly decreased in body weight (P ＜0.05)and serious malnutrition. After Lp treatment, diarrhea relieved in IL-10-/- mice and the body weight increased significantly (P＜0.05). Pathological examination suggested that 100％ of IL-10-/-mice had intestinal inflammation, however after Lp treatment intestinal inflammation improved significantly. Mucosal ulcer, epithelial hyperplasia, the infiltration of neutrophils and lymphocytes in the lamina propria were also significantly reduced.The histopathological score was significantly lowered (P＜0.01). After Lp treatment, colon wet weight and the ratio of wet weight to length of IL-10-/- mice changed significantly (P＜0.01). Colon edema and thickening improved remarkably. The TNF-a and IFN-γ concentration of colon in IL-10-/- mice were 377.4±84.4 μg/g and 602.6±108.1 μg/g,which increased obviously than WT group (139.2 ± 32. 7 μg/g and 173.0± 52.4 μg/g, P＜0.05). After treated with Lp for four weeks, the TNF-α and IFN-γ concentration of colon in IL-10-/-+Lp group mice were 207.2±65.7 μg/g and 442.1 ± 138.4 μg/g, both were lower than that of IL-10-/- group mice (P＜0.05). The intestinal flora was disrupted in IL-10-/- mice. Conclusion Lp can effectively reduce intestinal inflammation in IL-10-/- mice, which take certain part in treatment in colitis. This treatment effect is associated with intestinal flora regulation and the inhibition of proinflammation cytokines expression.

Effect of probiotics on intestinal flora disturbance and bacterial translocation in mice with spontaneous colitis / 上海交通大学学报（医学版）

Objective To investigate the effect of Lactobacillus plantarum(LP)on intestinal flora and bacterial translocation in mice with spontaneous inflammatory bowel disease(IBD). Methods Interleukin 10 knockout mice(IL-10~(-/-))were used as models of IBD.Eight-week old female mice were randomized to control group, IL-10~(-/-)group and IL-10~(-/-)+LP group.IL-10~(-/-)+LP group received 0.5 mL LP(1.0×10~9CFU/mL)per day for 4 weeks,and the other groups received 0.5 mL Ringer buffer.Intestinal flora including Bifidobacteria,Lactobacilli,Enterobacteriaceae and Clostridium perfringens in the feces and bacterial translocation in mesenteric lymph nodes and spleens were detected. Results The contents of Bifidobacteria and Lactobacilli significantly decreased in the intestine of IL-10~(-/-)mice,while those of Enterobacteriaceae and Clostridium perfringens significantly increased,and the bacterial translocation significantly increased.Four weeks after LP treatment, the disturbed intestinal flora was restored, and the bacterial translocation decreased. Conclusion LP administration can modulate the imbalance of intestinal flora and decrease the bacterial translocation,thus enhance intestinal barrier function in mice with IBD.

Functional expression of oligopeptide transporter PepT1 in the gastrointestinal tract and its regulatory factors / 中华临床营养杂志

Many peptide transporters have been identified in mammals, among which PepT1 has been widely studied. PepT1, a member of proton-coupled oligopeptide transporter (POT) superfamily, is a peptide transporter of low affinity and high capacity and is mainly expressed in the brush border membrane of intestinal epithelial cells. PepT1 plays an important role in the absorption of di/tri-peptide (the degradation products of protein in intestinal tract). Meanwhile, it mediates the transport of peptide-like drugs and the bacterial products. Therefore,the changes of the functional expression of PepT1 in the gastrointestinal tract may dramatically affect the internal and external environmental stability and drug absorption. This paper reviews the structural features and function,distribution, transport mechanisms, and regulatory factors of PepT1.

Advances in the effect of probiotics on gut barrier and microflora of experimental inflammatory bowel diseases / 肠外与肠内营养

The aetiology of inflammatory bowel diseases(IBD) is still unknown at the present time.However,along with more and more experimental models of IBD developed in recent ten years,the therapeutic effect of probiotics on IBD and the possible mechanisms were widely explored.A lot of experiments have shown that probiotics administration can significantly ameliorate the IBD in many kinds of animal models and this beneficial effect of probiotics may be associated with inhibiting microbial growth,enhancing gut-barrier function,modulating immune response of intestinal mucosa and decomposing luminal pathogenic antigens.