Introduction

Strogen (E2) may impair the contraction of colonic smooth muscle leading to constipation. Large conductance Ca²⁺-activated K⁺ channels (BK_Ca) are widely expressed in the smooth muscle cells (SMCs) contributing to hyperpolarization and relaxation of SMCs. Sphingosine kinase 1 (SphK1) is known to influence the expression of BK_Ca.

Aims & Methods

We aimed to elucidate the potential underlying molecular mechanism of BK_Ca and SphK1 that may influence E2-induced colonic dysmotility. In ovariectomized rats, smooth muscle contraction and expression of BK_Ca, SphK1, sphingosine-1-phosphate receptor (S1PR) were analyzed after the treatment with vehicle, BSA-E2, E2, and estrogen receptor antagonist. The role of BK_Ca, SphK1, and S1PR in E2-induced smooth muscle dysmotility was investigated in rat colonic SMCs. The effect of SphK1 on smooth muscle contraction as well as on the expression of BK_Ca and S1PR was analyzed in SphK1 knock-out mutant mice and wild-type mice treated with or without E2.

Results

The E2-treated group exhibited a weak contraction of colonic smooth muscle and a delayed colonic transit. The treatment with E2 significantly upregulated the expression of BK_Ca, SphK1, S1PR1, and S1PR2, but not S1PR3, in colon smooth muscle and SMCs. Inhibition of BK_Ca, SphK1, S1PR1, and S1PR2 expression attenuated the effect of E2 on Ca²⁺ mobilization in rat colon SMCs. Wild-type mice treated with E2 showed impaired gastrointestinal motility and enhanced expression of BK_Ca, S1PR1, and S1PR2 compared with those without E2 treatment. Conversely, in SphK1 knock-out mice treated with E2, these effects were partially reversed. E2 increased the release of S1P which in turn could have activated S1PR1 and S1PR2. Loss of SphK1 attenuated the effect of E2 on the upregulation of S1PR1 and S1PR2 expression.

Conclusion

These findings indicated that E2 impaired the contraction of colon smooth muscle through activation of BK_Ca via the upregulation of SphK1 and the release of S1P. In the E2-induced BK_Ca upregulation, S1PR1 and S1PR2 might also be involved. These results may provide further insights into a therapeutic target and optional treatment approaches for patients with constipation.

Introduction

Visceral hypersensitivity is a hallmark of irritable bowel syndrome (IBS). Transient receptor potential (TRP) channels are known as molecules involved in the pathogenesis of visceral hypersensitivity. A putative role for the Transient Receptor Potential Ankyrin 1 (TRPA1) channel in colonic nociceptor activation and mechanosensitation has been shown in animal studies. We hypothesize there is a correlation in TRPA1 mRNA expression and the symptom-severity in IBS patients.

Aims & Methods

The main objective of this study was to determine TRPA1 mRNA expression in the colonic mucosa and ascertain its correlation with symptom-severity in IBS patients. Second, difference in TRPA1 mRNA expression between IBS patients and healthy controls along with regional differences in TRPA1 mRNA expression was analyzed.
IBS patients (Rome III) and healthy controls were included. Sigmoid biopsies were obtained in all subjects. Additional biopsies of the proximal colon were obtained in 24/30 IBS patients. TRPA1 mRNA levels were measured in duplicate by quantitative reverse-transcriptase–polymerase-chain-reaction (Biorad), using primers from Biolegio and normalized to GAPDH. Levels of mRNA were expressed as relative mRNA values using the -2^ΔCt method. A multivariate regression was utilized to compare mRNA-expression between IBS patients vs healthy controls. Symptoms in IBS patients were assessed using the Gastrointestinal Symptom Rating Scale (GSRS).

Results

30 IBS patients (median age 39.0 yrs, 80% female) and 23 healthy controls (median age 22.7 yrs, 43.5% female) were included. Relative TRPA1 mRNA expression in the sigmoid was significantly higher in IBS patients compared to healthy controls (P<0.001), independent of age and gender. There was no significant difference in relative TRPA1 mRNA expression between IBS subtypes (P>0.05). Within IBS patients expression of TRPA1 mRNA of sigmoid-biopsies was significantly higher compared to biopsies of the proximal colon (p<0.001). No significant correlation between TRPA1 expression in sigmoid or proximal colon in GSRS scales (n=27) was found.

Conclusion

In IBS patients relative TRPA1 mRNA expression in colonic biopsies is significantly elevated compared to healthy controls. Furthermore, relative TRPA1 mRNA expression is higher in sigmoid colonic biopsies than in proximal colonic biopsies of IBS patients. These findings suggest a potential for the TRPA1 pathway as a target for IBS treatment in the future. However, no correlation between TRPA1 mRNA expression and symptom severity was found. Therefore, further research towards the clinical relevance of the increased TRPA1 mRNA-expression in IBS-patients along with its location-specific expression is warranted.

Introduction

Radiation therapy is widely used in the treatment of carcinoma in the abdomen and pelvic cavity, but as a side effect, acute and chronic damage to the gastrointestinal tract may occur. It is well known that the rectum is more prone to radiation damage since it is fixed in the pelvis. Acute symptoms of radiation enteritis are mainly associated with inflammation of the intestinal mucosa and loss of normal mucosa, but changes in gastrointestinal motility due to exposure to high-concentration radiation may also be a cause. Previous studies are demonstrating that patients who received radiation therapy had increased bowel movements, symptoms of urgency, and decreased rectal compliance. But there are only limited studies investigated the pattern of changes in the motility of human rectal smooth muscle following irradiation.

Aims & Methods

This study was conducted to investigate changes in motility and their mechanisms after irradiation in human rectum.
Human rectal tissues were obtained immediately in the operating room from patients undergoing colorectal resection. After removing the mucosal layer of rectum, muscle layer was cut in strips of 5-6 mm in length and 2-3 mm in width. The muscle strips were mounted to an isometric force transducer (Biopac Systems, Inc., Goleta, CA, USA) and suspended in 10 ml organ bath containing aerated (97% O₂ and 3% CO₂) and warmed (36.5 ± 0.5°C) KRB solution. The area under the curve (AUC) for 5 min and tone of spontaneous contractions were analyzed to compared the responses to KCl, ACh (acethylcholine), SNP (sodium nitroprusside), a nitric oxide donor, and ATP (adenosine triphosphate), a purinergic receptor agonist. The mechanical responses were recorded and digitized using Acknowledge software (Biopac Systems, Inc., Goleta, CA, USA). Data were analyzed offline using Clampfit (version 10.2. Molecular devices, San Jose, CA, USA). qPCR was performed for expression of VChAT, NOS1 and PDGFRα to quantify cells mediating cholinergic, nitrergic and purinergic neurotransmission.

Results

KCl and ACh increased the tonic contraction and area under the curve (AUC) in a concentration-dependent manner in both normal and irradiated rectum. The difference in contractile pattern between the two groups was not statistically significant. SNP and ATP decreased AUC and reduced tonic contractions in a concentration-dependent manner in both normal and irradiated rectum. The response to SNP between the two groups was not statistically significant. However, the response to ATP reduced significantly in irradiated rectum compared to normal rectum. In addition, expression of VAChT and NOS1 which are related with cholinergic and nitrergic neurotransmission was not different in both normal and irradiated rectum, however, PDGFRα expression which is related with purinergic neurotransmission was significantly decreased in irradiated rectum compared to normal rectum.

Conclusion

In this study, we analyzed contractile changes in irradiated rectum using neurotransmitters. There were no differences in contractile responses to KCl and ACh and in relaxation response to SNP, however, the relaxation response to ATP was significantly reduced in irradiated rectum. Cholinergic and nitrergic neurotransmission were not changed, however, purinergic neurotransmission was decreased in in irradiated rectum.
These results may explain one of the causes by which increased bowel movements, symptoms of urgency, and decreased rectal compliance in irradiated patients.

Introduction

The intestinal epithelium serves as a site for digestion and absorption of the nutrients and dietary factors, while forming the selective barrier to separate the body’s internal environment from the external luminal environment. The intestinal barrier is established by a combination of several mechanisms, including the mucous layer, antimicrobial peptides, and the intercellular tight junction (TJ)⁽¹⁾. The TJ structure is a multiprotein complex composed of both transmembrane proteins such as claudins and occludin, and cytosolic plaque proteins such as zonula occludens and cingulin, and regulates the paracellular passage of macromolecules, ions, and water. Our and other research groups have shown that the regulation of the intestinal TJ barrier is closely related to intestinal microbial metabolism^(2-4), however, the precise underlying mechanisms are still unclear.

Aims & Methods

The aim of this study was to investigate the role of intestinal microflora in the regulation of the colonic TJ barrier using mice and Caco-2 and MDCKII epithelial cells. In particular, we focused on the molecular mechanisms of n-butyrate-mediated expression of claudin-23. To investigate the effect of intestinal microflora on the TJ expression, RNA sequencing analysis was performed in the colonic tissues from germ-free (GF) and specific pathogen-free (SPF) mice. Claudin-23 expression was also examined in the colon of experimental colitis mice. Human intestinal Caco-2 cells were treated with various microbial metabolites and components including n-butyrate. The TJ barrier function was assessed by measuring the transepithelial resistance (TER) and FITC-dextran (FD) permeability. Claudin-23 expression and transcriptional activity were examined by qRT-PCR, immunoblot, immunofluorescence, and luciferase reporter analyses. The siRNA targeting SP1 and the selective inhibitor of AMPK were used in combination in some experiments. To investigate the role of claudin-23 in the epithelial barrier function, TER and FD permeability were measured in MDCKII epithelial cells stably expressing human claudin-23.

Results

Among several TJ molecules, the claudin-23 mRNA was lower in the colon of GF mice than that of the SPF mice. Claudin-23 mRNA expression was higher in the lower intestinal segments, such as ileum, cecum, and colon, compared with those in the duodenum and jejunum. Claudin-23 expression in the colitis mice was downregulated compared to the normal mice. n-butyrate, but not other microbial metabolites or components, upregulated the claudin-23 expression at both mRNA and protein levels in intestinal Caco-2 cells. N-butyrate enhanced the intestinal TJ barrier integrity, indicated by increased TER and decreased FD permeability. Luciferase reporter assay showed that n-butyrate increased the claudin-23 transcriptional activity. The SP1 siRNA and AMPK inhibitor reduced the n-butyrate-mediated claudin-23 expression and promoter activity. Exogenously expressed claudin-23 localized to the junctional region of MDCKII cells, but did not affect the TER and FD permeability.

Conclusion

Claudin-23 expression in the mouse colon is regulated by a microbial metabolite, n-butyrate. Our results suggest that the transcription factor, SP1, and the AMPK pathway are involved in its transcriptional regulation. Although the precise role of claudin-23 in the intestine is still unclear, the n-butyrate-mediated claudin-23 expression may contribute to the intestinal homeostasis.

References

(1) Suzuki T. Anim Sci J. 2020;91(1):e13357.
(2) Isayama K et al. Exp Cell Res. 2023;425(2):113528.
(3) Yokoo K et al. FASEB J. 2021;35(11):e21854.
(4) Tinh NTT et al. Foods. 2021;10(2):240.

Introduction

Colorectal cancer ranks second in cancer-related deaths worldwide. O-GlcNAc glycosylation, as a crucial post-translational modification, is overexpressed in various cancer. Hippo pathway effector YAP can be O-GlcNAc glycosylated to promote development of some types of cancer. However, the effects of YAP's O-GlcNAc glycosylation on colorectal cancer growth are poorly understood, and the development of targeted drugs is still blank.

Aims & Methods

We aim to develop a novel anti-colorectal cancer drug targeting OGT and investigate its anti-tumor mechanism. The expression level of OGT, YAP and YAP's phosphorylation were examined through WB, IHC and bioinformatics analysis. YAP's O-GlcNAc glycosylation and OGT/YAP interaction were assessed by molecular dynamics simulations and Co-IP. OSMI-1, an OGT inhibitor, was used to construct an OSMI-1's folate-targeted diselenide-responsive liposome. Basic characteristics of liposome were identified. Next, the effect of OSMI-1's liposome on colon cancer development and invasiveness was examined, and the change of Hippo-YAP pathway as well as downstream gene expression were assessed. Finally, the metabolism, drug distribution, toxicity, and therapeutic effect of OSMI-1's liposome were investigated in subcutaneous tumor mice model.

Results

Based on TCGA and GTEx database, gene of YAP (p<0.05) and OGT (p<0.01) were remarkably overexpressed in colon cancer. High level OGT gene expression was linked with poor OS (p=0.016) and DFS (p=0.02). OGT (p=0.05) and YAP (P<0.01) expression raised with stage. WB and IHC revealed an over-expression of YAP and OGT in both colon cancer tissue and cell lines. Co-IP verified the YAP/OGT interaction and molecular dynamics simulations predicted glycosylation sites of serine 103, 366, and 216, and threonine 213 and 41, which might affect the neighboring amino acids phosphorylation. OSMI-1's folate-targeted diselenide-responsive liposome appeared as stable particles of average 205.5 nm in size. Drug loading amount was 280ug/ml. Mass spectrum indicated a standard (Cholesterol-Se)₂ in liposome and it stably released over 90% of OMSI-1 within 16 hours in GSH/H2O2 environment. IC50 of the drug for colon cancer was 1240ug/ml, while it presented low toxicity to normal colon cell. OSMI-1's liposome effectively inhibited the proliferation and invasion ability of colon cancer cell CACO2 and HCT116. It suppressed glycosylation of YAP but increased its phosphorylation level relatively and YAP presented more cytoplasmic translocation. The downstream target genes CTGF, Bcl-2, E-cadherin, and Vitemin were all significantly downregulated. In nude mice, the drug was significant accumulated at the subcutaneous tumor site within 12 hours and its half-life time was 20 hours. After treated with OSMI-1's liposome for 15 days, the average subcutaneous tumor volume of mice was significantly smaller than OSMI-1 group and the control group (16.9mm³ vs 27.6mm³ vs 46.6mm³, p<0.05, n=15), and there was no significant change in the body weight of the mice.

Conclusion

In summary, we successfully constructed an OSMI-1's folate-targeted diselenide-responsive liposome, which exhibited well targetability and efficacy against colon cancer tissue with low toxicity to normal tissue. Furthermore, we demonstrating that OGT inhibition can reduce the YAP's O-GlcNAcylation and enhance its phosphorylation thus suppress YAP nuclear translocation, to suppress the development of colon cancer. Our research provides a brand new targeted therapy for colon cancer, with significant application value and broad development prospects.

References

1. Ferlay J, Colombet M, Soerjomataram I, et al. Cancer statistics for the year 2020: An overview [published online ahead of print, 2021 Apr 5]. Int J Cancer. 2021;10.1002/ijc.33588. doi:10.1002/ijc.33588
2. Ohta Y, Fujii M, Takahashi S, et al. Cell-matrix interface regulates dormancy in human colon cancer stem cells. Nature. 2022;608(7924):784-794. doi:10.1038/s41586-022-05043-y
3. Yang Y, Yan Y, Yin J, et al. O-GlcNAcylation of YTHDF2 promotes HBV-related hepatocellular carcinoma progression in an N6-methyladenosine-dependent manner. Signal Transduct Target Ther. 2023;8(1):63. Published 2023 Feb 10. doi:10.1038/s41392-023-01316-8
4. Li X, Wu Z, He J, et al. OGT regulated O-GlcNAcylation promotes papillary thyroid cancer malignancy via activating YAP. Oncogene. 2021;40(30):4859-4871. doi:10.1038/s41388-021-01901-7

Introduction

Colorectal serrated lesions (SLs) include hyperplastic polyp (HP), traditional serrated adenoma (TSA) and sessile serrated lesion (SSL). Aberrant DNA methylation is prevalent in colorectal SLs. We previously reported that SMOC1 (SPARC-related molecular calcium-binding 1) was frequently methylated and silenced in TSAs as well as in high-grade adenomas and colorectal cancers (CRCs) but was rarely methylated and silenced in SSLs. [1] However, the expression of SMOC1 in colorectal lesions, including SLs, has not yet been studied in detail.

Aims & Methods

To evaluate the potential of SMOC1 as a biomarker for diagnosis of SLs and risk prediction in colorectal tumors, we aimed to characterize the expression of SMOC1 in colorectal lesions. Specimens of colorectal lesions (n = 199) and adjacent normal colorectal tissues (n = 112) were collected from 173 patients who underwent endoscopic or surgical resection. Colorectal lesions were randomly divided into two groups: a training set and a validation set. SMOC1 expression was analyzed by immunohistochemistry. A rabbit anti-SMOC1 polyclonal antibody (1:1000 dilution, C-20; Sigma-Aldrich) was used for immunohistochemical staining. The intensity of SMOC1 staining was graded as strong (3), moderate (2), weak (1) or negative (0). The proportions of positively stained tumor cells were assigned a value of 0 to 10. Because neoplasm heterogeneity caused varying degrees of immunoreactivity in the slides, we used the sum of each intensity × proportion as an immunohistochemistry (IHC) score (e.g., intensity × proportion = (3) × 5 + (2) × 3 + (1) × 1 + (0) × 1 = IHC score 22; maximum score = 30) to improve accuracy. When TSAs had both flat and protruding components, SMOC1 expression was evaluated in the protruding component. Thereafter, levels of SMOC1 expression were compared between flat and protruding components in order to evaluate its involvement in the progression of TSAs. All slides were independently evaluated by gastrointestinal pathologists who were blinded to the clinical data.

Results

Histology of the 199 colorectal lesions was as follows: HP, n = 26; SSL, n = 50; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), n = 14; TSA, n = 51; TSA with high grade dysplasia (HGD)/EIC, n = 3; low grade adenoma, n = 17; high grade adenoma, n = 14; HGD, n = 16; EIC, n = 8. Mean IHC scores in normal colonic tissues and colorectal lesions in the training set were as follows: normal colon, 24.2; HP, 21.6; SSL, 24.8; SSLD/SSL with EIC, 17.5; TSA, 7.3; low grade adenoma, 21.4; high grade adenoma, 11.7; HGD, 12.1; EIC, 10.9. These results suggest that SMOC1 is abundantly expressed in normal colon, HPs, SSLs and low grade adenomas, whereas it is significantly downregulated in TSAs, high grade adenomas, HGDs and EICs (p < 0.05). Mean IHC scores for colorectal lesions in the validation set were as follows: HP, 17.7; SSL, 22.8; SSLD/SSL with EIC, 11.5; TSA, 3.1; TSA with HGD/EIC, 4.7. Again, we observed abundant expression of SMOC1 in HPs and SSLs, whereas it was significantly downregulated in TSAs and TSAs with HGD/EIC (p < 0.05). Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components both the training set (p < 0.001) and the validation set (p < 0.05).

Conclusion

Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 may be a diagnostic marker of SLs as well as a predictive marker of colorectal tumors at high risk of developing into cancer.

References

[1] Aoki H, Yamamoto E, Takasawa A, Niinuma T, Yamano HO et al. Epigenetic silencing of SMOC1 in traditional serrated adenoma and colorectal cancer. Oncotarget. 2018

Introduction

Endoscopic submucosal dissection has become the preferred technique for managing large colonic lesions or those suspected of superficial tumor invasion. Macronodular lesions may present with a muscular retraction sign (MRS), which is associated with reduced curative resection rates due to lower R0 resection rates and increased rates of deep submucosal invasion. A few data are available about how to differentiate fibrosis and cancer and about technical and oncological outcomes of ESD in this situation.

Aims & Methods

We conducted a prospective study to evaluate the prevalence of the muscular retraction sign and its clinical outcomes in patients with colorectal macronodular lesions. This analysis utilized data from a large prospective French colorectal ESD cohort (FECCo).

Results

A total of 1,238 macronodular lesions were treated, of which 254 (20.5%) exhibited a muscular retraction sign. The prevalence of MRS increased with the size of the largest macronodule, reaching up to 50% for nodules larger than 40 mm. Enbloc, R0 and curative resection rates were significantly lower in the MRS+ group (82.68% vs 97.86%, 70.97% vs 89.76% and 56.31% vs 83.52%, p<0.001 respectively). MRS+ lesions were more frequently associated with at least submucosal cancer (30.32% vs 9.65%, p<0.001). Regardless of MRS, the presence of a nodule larger than 4cm is associated with a decrease in enbloc resection rates, particularly in the colon, as well as reductions in R0 resection rates and curative resection rates in comparison with nodules smaller than 4cm. The curative resection rate for lesions with ≥T1 cancer and presenting with MRS was 1.5% (p<0.001).
In multivariate analysis, protruding lesions (OR = 1.91 [1.19–3.06], p = 0.007) and nodules larger than 20mm (OR = 2.48 [1.74–3.52], p<0.001) or 40mm (OR 5.98 [3.55-10.08], p<0.001) were identified as risk factors for MRS. The size of the nodule did not increase the risk of submucosal cancer, unlike sigmoid location (OR 2.66 [1.15-6.17] p=0.022) and nodules classified as JNET 3 (OR 6.53 [2.07-20.57], p=0.001).

Conclusion

The prevalence of MRS is 20.5% in cases of macronodular lesions, a rate that increases significantly with the size of the macronodule. MRS negatively impacts the outcomes of ESD in terms of enbloc, R0 and curative resections. ESD should be reconsidered in cases of large macronodular colonic lesions, especially for sigmoid locations or in the presence of JNET 3 vascular pattern anomalies. For rectal large macronodular lesions, ESD should be proposed in expert centers with the option, if necessary, of intermuscular dissection to optimize the R0 resection rate and thereby improve the chances of proposing an organ-preservation strategy.