Gambogic Acid Efficiently Kills Stem-Like Colorectal Cancer Cells by Upregulating ZFP36 Expression.

BACKGROUND/AIMS: Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported to be a potential novel antitumor drug. Whether GA inhibits putative cancer stem cells (CSCs), which are considered to be the major cause of cancer treatment failure, remains largely unknown. This study investigated whether GA inhibits the CSCs of colorectal cancer (CRC) and its possible mechanisms. METHODS: We performed CCK8 and tumor sphere formation assays, percentage analysis of both side population and CD133+CD44+ cells, and the detection of stem cells markers, in order to assess the role of GA in inhibiting the stem celllike features of CRC. An mRNA microarray was performed to identify the downstream gene affected by GA and rescue assays were performed to further clarify whether the downstream gene is involved in the GA induced decrease of the stem cell-like CRC population. CRC cells were engineered with a CSC detector vector encoding GFP and luciferase (Luc) under the control of the Nanog promoter, which were utilized to investigate the effect of GA on putative CSC in human tumor xenograft-bearing mice using in vivo bioluminescence imaging. RESULTS: Our results showed that GA significantly reduced tumor sphere formation and the percentages of side population and CD133+CD44+ cells, while also decreasing the expression of stemness and EMT-associated markers in CRC cells in vitro. GA killed stem-like CRC cells by upregulating the expression of ZFP36, which is dependent on the inactivation of the EGFR/ ERK signaling pathway. GFP+ cells harboring the PNanog-GFP-T2A-Luc transgene exhibited CSC characteristics. The in vivo results showed that GA significantly inhibited tumor growth in nude mice, accompanied by a remarkable reduction in the putative CSC number, based on whole-body bioluminescence imaging. CONCLUSION: These findings suggest that GA significantly inhibits putative CSCs of CRC both in vitro and in vivo by inhibiting the activation of the EGFR/ ERK/ZFP36 signaling pathway and may be an effective drug candidate for anticancer therapies.

Simple transanal total mesorectal resection versus laparoscopic transabdominal total mesorectal resection for the treatment of low rectal cancer: a single-center retrospective case-control study.

Aim: To evaluate the efficacy and safety of simple TaTNE in the treatment of low rectal cancer compared with laparoscopic transabdominal TME. Methods: We collected patients with low rectal cancer admitted to our hospital between January 2019 and November 2021 who received simple TaTME or laparoscopic transabdominal TME. The main outcome was the integrity of the TME specimen. Secondary outcomes were the number of lymph nodes dissected, intraoperative blood loss, operative time, surgical conversion rate, Specimen resection length, circumferential margin (CRM), and distal resection margin (DRM), complication rate. In addition, the Wexner score and LARS score of fecal incontinence were performed in postoperative follow-up. Results: Pathological tissues were successfully resected in all patients. all circumferential margins of the specimen were negative. Specimen resection length was not statistically significant (9.94 ± 2.85 vs. 8.90 ± 2.49, P > 0.05). The incidence of postoperative complications in group A (n = 0) was significantly lower than that in group B (n = 3) (P > 0.05). There was no significant difference in operation time between group A and group B (296 ± 60.36 vs. 305 ± 58.28, P > 0.05). Among the patients with follow-up time less than 1 year, there was no significant difference in Wexner score and LARS score between group A and group B (P > 0.05). However, in patients who were followed up for more than 1 year, the Wexner score in group A (9.25 ± 2.73) was significantly lower than that in group B (17.36 ± 10.95) and was statistically significant (P < 0.05). Conclusion: For radical resection of low rectal cancer, Simple TaTME resection may be as safe and effective as laparoscopic transabdominal TME, and the long-term prognosis may be better.

Colon cancer patient with long-term colon stent placement: Case report and literature review.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer mortality globally. Large bowel obstruction (occurring in 15-30% of patients with CRCs) accounts for approximately 80% of medical emergencies related to CRC. Currently, there is no standard treatment of this condition. The European Society of Gastrointestinal Endoscopy (ESGE) recommends self-expandable metal stent (SEMS) as a bridge (two weeks) to surgery for left-sided obstructing colon cancer. In the present report, we describe an 81-year-old male with colon cancer who underwent colon stent placement for 32 months, but later underwent radical resection. A follow-up of more than four-months revealed that his condition was normal. The history as well as application and advantages of SEMS are discussed in this report.

Risk Factors for Recurrence of Radically Resected Mucinous Colorectal Adenocarcinoma.

BACKGROUND: Mucinous adenocarcinoma (MA) is a subtype of colorectal cancer (CRC) associated with a higher incidence of local extension and worse survival compared to non-mucinous adenocarcinoma, but few studies have investigated surgery-related predictors for recurrence of MA. Therefore, we aimed to elucidate the predictors for local recurrence and remote metastasis of MA after surgery. PATIENTS AND METHODS: This study retrospectively analyzed 162 patients with mucinous colorectal adenocarcinoma (MAC) after radical resection. Analysis variables included demographics, clinical indicators, pathologic stage, surgical procedure, adjuvant therapy, and recurrence. Univariate and multivariate analyses were performed to investigate the risk factors for local and distant tumor relapse. RESULTS: A total of 162 patients (86 male) with a mean age of 58.26 years were included; 70.37% of patients had colonic tumors, and 29.63% had rectal tumors. The 5-year disease-free survival (DFS) rates for these patients were as follows: 100% for TNM stage I, 71.2% for stage II, and 47.8% for stage III. Five-year DFS rates of MAC, colonic and rectal MA were 62.0%, 65.8%, and 51.7%, respectively. Local recurrence occurred in 38 patients and distant metastasis in 33 patients. In univariate analysis, predictors for local recurrence of MAC were intraoperative blood loss, intraoperative transfusion, and N2 stage; and predictors for distant metastasis were male sex, CA199, CEA, intraoperative blood loss, T4 stage, and N2 stage. In multivariate analysis, predictors for local recurrence of MAC were intraoperative transfusion (P=0.04, OR=4.175) and N2 stage (P=0.000, OR=5.291), and predictors for distant metastasis were male sex (P=0.049, OR=2.410), CA199 (P=0.02, OR=1.003), and T4 stage (P=0.007, OR=4.006). CONCLUSION: Intraoperative transfusion and N2 stage were significant predictors for local recurrence. Male sex, CA199, and T4 stage were significant predictors for distant metastasis. Knowledge of the risk factors for postoperative recurrence provides a basis for logical approaches to treatment and follow-up of MAC.

Outcomes of Laparoscopic Surgery for Mucinous Colorectal Adenocarcinoma.

Background: Mucinous colorectal adenocarcinoma (MAC) has a higher incidence of local extension, leading to lower overall resection rates. Few studies have investigated the outcomes of laparoscopic surgery for MACs to date. Therefore, we aimed to elucidate the validity of laparoscopic surgery for mucinous adenocarcinoma (MAC). Methods: This study analyzed short-term and long-term outcomes between laparoscopic and open surgery for MACs from 2008 to 2018. Multivariate analyses were used to define prognostic factors of overall survival (OS) and disease-free survival (DFS). Results: Patients in the laparoscopy (LAP) group had significantly less blood loss, fewer days to first flatus and to diet, and shorter length of hospital stay. The 3-year and 5-year DFS rates for all stages combined were 65.7% and 62.5% in the LAP group compared with 60.5% and 57.6% in the open (OPEN) surgery group (P = .521). The 3-year and 5-year OS rates for all stages combined were 72.3% and 67.3% in the LAP group compared with 72.6% and 67.8% in the OPEN group (P = .934). OS and DFS in stage II, stage III, and pathological T4 (pT4) stage patients who underwent laparoscopic surgery did not differ from patients who underwent open surgery. Multivariate analysis showed that stage pT4, pN2, and carcinoembryonic antigen (CEA) were significant predictors of OS. Independent factors, including intraoperative blood transfusion, stage pT4, pN2, CEA, and CA19-9, carbohydrate antigen 19-9, have a great effect on DFS. Conclusions: Laparoscopic surgery is a safe and feasible option for mucinous colorectal AC, which provides faster postoperative recovery and less intraoperative blood loss.

The Clinical Significance of Mesenteric Lymphocytes in Human Colorectal Cancer.

OBJECTIVE: The mesentery is a potential site of residual tumor in patients with colorectal cancer (CRC). However, the mesenteric immune microenvironment remains unclear. In this study, we investigated the immune landscape of the mesentery, particularly the role of lymphocytes and its association with the clinicopathological characteristics of CRC. METHODS: Flow cytometry was used to detect lymphocytes in the paired mesenteric tissue specimens adjacent to the colorectal tumors and normal mesenteric tissue specimens 10 cm away from the colorectal tumor edge and preoperative peripheral blood samples obtained from patients with CRC who underwent surgery. T-distributed stochastic neighbor embedding was utilized to analyze multiparameter flow cytometry data. Multiplex immunohistochemistry was performed to evaluate T cells subsets in the paired mesentery adjacent to the colorectal tumors and normal mesentery. The Fisher's exact test and non-parametric Wilcoxon's matched-pairs tests were used for statistical analysis. The non-parametric Mann-Whitney U test was used to determine associations between percentage data and clinical parameters of patients with CRC. RESULTS: We found that immune cells in the normal mesentery were mainly of lymphoid lineage. Compared with peripheral blood, the normal mesentery showed decreased NK cells and the CD4/CD8 ratio and increased CD3+ CD56+, memory CD4+ T, memory CD8+ T, CD4+ tissue-resident memory T (TRM), and CD8+ TRM cells. Compared with the normal mesentery, the mesentery adjacent to the colorectal tumor showed increased B and regulatory T cells and decreased NK, CD3+ CD56+, CD4+ TRM, and CD8+ TRM cells. Moreover, memory CD8+ T cells and plasmablasts are negatively correlated with the depth of invasion of CRC. Increased memory CD4+ T cells are associated with distant metastasis of CRC and high preoperative serum carcinoembryonic antigen levels. CONCLUSION: The mesentery shows a specific immune microenvironment, which differs from that observed in peripheral blood. CRC can alter the mesenteric immune response to promote tumor progression.

PD-L1 promotes colorectal cancer stem cell expansion by activating HMGA1-dependent signaling pathways.

PD-L1 is critical for tumor cell escape from immune surveillance by inhibiting T cell function via the PD-1 receptor. Accumulating evidence demonstrates that anti-PD-L1 monoclonal antibodies might potently enhance antitumor effects in various tumors, but the effect of PD-L1 on colorectal cancer stem cells (CSCs) remains unclear. We observed high PD-L1 expression in CD133+CD44+ colorectal CSCs and CSC-enriched tumorspheres. Altering PD-L1 expression promoted colorectal CSC self-renewal by increasing the expression of stemness genes, the CD133+CD44+ cell population sizes and the ability to form tumorspheres. Additionally, PD-L1 expression was markedly increased in chemoresistant colorectal cancer (CRC) cells in vitro and in vivo. More importantly, PD-L1 enhanced CRC cell tumorigenicity in nude mice; the inoculation of 1 × 104 cells resulted in high tumor formation efficiency. Mechanistically, PD-L1 directly interacted with HMGA1, and HMGA1 upregulation by PD-L1 activated HMGA1-dependent pathways, including the PI3K/Akt and MEK/ERK pathways, and promoted CSC expansion. HMGA1 downregulation rescued the PD-L1-induced phenotypes, highlighting the role of HMGA1 in PD-L1-mediated colorectal CSC self-renewal. Moreover, PD-L1 expression was correlated with the expression of CSC markers and HMGA1 in clinical CRC specimens. Thus, PD-L1 could crucially contribute to the maintenance of CSC self-renewal by activating HMGA1-dependent signaling pathways.

Long non­coding RNA 00152 functions as a competing endogenous RNA to regulate NRP1 expression by sponging with miRNA­206 in colorectal cancer.

Colorectal cancer (CRC) is the third most common type of cancer; however, the molecular mechanisms underlying colorectal tumor metastasis and growth remain elusive. Recently, accumulating evidence has indicated that long non­coding RNAs (lncRNAs) play a critical role in CRC progression and metastasis; however, the biological role and clinical significance of lncRNA 00152 (lnc00152) in CRC remains largely unknown. Thus, in this study, lnc00152 expression was measured in 80 human CRC tissue samples, 40 non­cancerous tissue samples, and 3 CRC cell lines (SW480, SW620 and LoVo) using RT­qPCR. We examined the effects of lnc00152 on CRC cells following transfection with lnc00152 overexpression plasmid or respective siRNA in vitro and in vivo. Luciferase assays revealed the mechanism driving competitive endogenous RNA (ceRNA). We identified that lnc00152 was aberrantly overexpressed in colorectal tumors and cancer cells and that lnc00152 was modulated by miRNA­206. lnc00152 overexpression enhanced the proliferative and invasive ability of CRC cells in vitro, promoted tumor growth in vivo, and was associated with the shorter overall survival of patients with CRC. In addition, lnc00152 overexpression promoted epithelial-mesenchymal transition (EMT) and increased neuropilin­1 (NRP1) expression in the CRC cells. By contrast, lnc00152 silencing exerted a counteractive effect. Collectively, these findings demonstrate the critical role of lnc00152 in tumor growth and progression in CRC, and identify a novel therapeutic target associated with CRC development and progression.

Decreased fragile histidine triad expression in colorectal cancer and its association with apoptosis inhibition.

AIM: To detect the expression of fragile histidine triad (FHIT) in normal colorectal tissue, colorectal adenoma and colorectal cancer (CRC) tissue, and to analyze its relationship with the clinicopathological features of CRC, and apoptosis-associated proteins (Bcl-2, Bax, survivin) and apoptosis in colorectal cancer. METHODS: FHIT mRNA analysis was performed by nested reverse transcription-polymerase chain reaction (RT-PCR) assay. Tissue microarray (TMA) was established to detect the expression of FHIT, Bcl-2, Bax and survivin genes in 80 CRC tissue specimens, 16 colorectal adenoma tissue specimens and 16 hemorrhoid (PPH) tissue specimens during the same period of time as the control. Citrate-microwave-SP was used as immunohistochemical method. The relationship between clinicopathological factors, such as differentiation grades and 5-year survival rate was observed. TUNEL assay was used to detect the apoptosis index in 80 CRC tissue specimens. RESULTS: Ten out of 26 (38.5%) CRC tissue specimens expressed aberrant FHIT transcripts, none of the aberrant FHIT transcripts was observed in the matched normal tissue and colorectal adenoma tissue by nested RT-PCR assay. The positive rate of FHIT gene expression in normal colorectal tissue, colorectal adenoma and carcinoma tissue was 93.75%, 68.75% and 46.25%, respectively. Clinicopathological analysis of patients showed that the decreased FHIT gene expression was not associated with age, sex, serum CEA levels, tumor site and size, histological classification. However, the expression of FHIT was correlated with differentiation grades, pathological stages, lymph node metastases and 5-year survival rate after operation. The positive rate of apoptosis-associated proteins (Bax, Bcl-2 and survivin) in CRC tissue was 72.50%, 51.25% and 77.50%, respectively. The expression of these apoptosis-associated proteins in CRC tissue was correlated with the expression of FHIT. The mean apoptosis index in FHIT negative tumors was significantly lower than that in FHIT positive tumors (5.41 +/- 0.23 vs 0.56 +/- 0.10, P < 0.01). CONCLUSION: The FHIT gene plays an important role in the regulation of apoptosis and decreased FHIT expression plays a key role in the initiation and progression of colorectal carcinoma.

miR-892a regulated PPP2R2A expression and promoted cell proliferation of human colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common malignancy cancers in the world. Aberrant microRNA expression is involved in human diseases including cancer. In the present study, we investigated the miR-892a's role in CRC cell proliferation. We found that miR-892a was frequently upregulated in human colorectal cancer tissues and cell lines compared with the matched tumor adjacent tissues and normal colonic cell line FHC. Overexpression of miR-892a promoted cell proliferation and colony formation of CRC. Bioinformatics analysis further revealed PPP2R2A was identified as a potential miR-892a. Overexpression of miR-892a-in SW480 cells reduced PPP2R2A protein expression. Subsequently, data from luciferase reporter assays showed that PPP2R2A 3'-untranslated region (3'-UTR) carried the directly binding site of miR-892a. Furthermore, siRNA-mediated silencing of PPP2R2A blocked the inhibitory effect of miR-892a-in on CRC cell growth. In sum, our data provided compelling evidence that overexpression of miR-892a may provide a selective growth promotion for CRC cells by direct suppression of PPP2R2A expression.

[Effect of Trichinella spiralis infection on expression and distribution of colonic epithelial E-cadherin in mice and its mechanism].

OBJECTIVE: To study the effect of Trichinella spiralis (T.spiralis) infection on the expression and distribution of colonic epithelial E-cadherin in mice and its mechanism. METHODS: BALB/c mice and STAT6-/- mice were infected with T.spiralis, and mice without infection were used as control. Seven days later, the horseradish peroxidase (HRP) was infused by rectal enema. Serum HRP was detected in the subsequent 0, 60 and 120 minutes. Then the mice were sacrificed and colon was taken out. The distribution of E-cadherin in colon was detected by immunofluorescence staining, and the expression of E-cadherin was detected by Western blot. The expression of interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA. RESULTS: Serum HRP level in infected BALB/c mice was significantly higher than that in control mice (P<0.05), while it was not significantly different between infected STAT6-/- mice and controls (P>0.05). In infected BALB/c mice, E-cadherin located in cytoplasm of colonic epithelial cells, while in controls, it located in cellular membrane. E-cadherin expression down-regulated significantly in infected BALB/c mice as compared to controls. E-cadherin expression and distribution did not change obviously in infected STAT6-/- and control mice. IL-4 level in mesenteric lymph nodes of infected BALB/c mice [(193.0±12.5) µg/L] was significantly higher as compared to control BALB/c and infected STAT6-/- mice [(21.0±2.3) µg/L and (15.0±3.1) µg/L, all P<0.05]. CONCLUSION: T.spiralis infection can increase colonic epithelial permeability of mice, which may be associated with induction of Th2 cytokine secretion.

[Impact of fragile histidine triad gene transfection on the proliferation and apoptosis of human colorectal cancer cell].

OBJECTIVE: To investigate the effect of fragile histidine triad (FHIT) gene transfection on human colorectal cancer cell line SW480 through up-regulation of caspase-8 expression. METHODS: The eukaryotic expression plasmid containing FHIT, pRc/CMV2-FHIT was prepared and purified, and then identified by restrictive enzyme digestion. pRc/CMV2-FHIT was transfected into SW480 cells, and positive cell clones (SW480-FHIT, study group) were selected and amplified. Empty plasmid-transfected SW480 cells(SW480-pRc/CMV2, negative control) and normal SW480 cells (bland control) were used as control. Methyl thiazolyl tetrazolium (MTT) assay was used to test the changes in the proliferation of SW480 cells. Cell-cycle kinetics and apoptosis were analyzed by flow cytometry (FCM). The changes of pro-caspase-8, caspase-8 mRNA and caspase-8 relative activity were analyzed by Western blot, semi-quantitative RT-PCR and colorimetric assay with pan labeled substrate, respectively. RESULTS: At 96 hours after transfection, cell inhibition rates of the study group and the negative control group were 71.7% and 16.9%. G0/G1 ratio was (63.2±3.5)% and (50.6±2.1)%, optical density of caspase-8 mRNA band 107 and 41, and relative activity of caspase-8 0.43 and 0.25, respectively. All the differences above were statistically significant (P<0.05). When FHIT inhibitor was added, the relative activity of caspase-8 decreased to 0.22, comparable to that in the control group. CONCLUSIONS: FHIT gene transfection can significantly inhibit the proliferation and induce G0/G1 arrest in human colon cancer cell line SW480. The mechanism is related to the up-regulation of caspase-8 expression.

[Fragile histidine triad protein expression and correlation with apoptosis in rectal carcinoma].

OBJECTIVE: To investigate the relationship between the expression of fragile histidine triad (FHIT) protein and the clinicopathological characteristics of rectal carcinoma. The relationship between FHIT protein expression and Bcl-2, Bax and survivin expression, as well as cell apoptosis in rectal carcinoma were explored. METHODS: Tissue microarray and immunohistochemistry SP were used to detect the expression of FHIT, Bcl-2, Bax and Survivin in 16 cases of normal rectal tissue, 16 cases of rectal adenoma and 80 cases of rectal carcinoma. TUNEL was used to detect apoptosis index (AI) in 80 cases of rectal carcinoma. RESULTS: The positive rates of FHIT expression in normal rectal tissue, rectal adenoma and adenocarcinoma were 93.8%, 75.0% and 46.3% respectively. There were no significant differences between FHIT expression and histological types, gender and age (P>0.05). FHIT expression was significantly correlated with lymph node metastasis, Duke's stage and 5-year survival rate. The expression of FHIT was positively correlated with that of Bcl-2, Bax and survivin in rectal cancer. The mean AI in FHIT-negative tumors was significantly lower than that in FHIT-positive tumors (P<0.01). CONCLUSIONS: The reduction of FHIT protein expression may play an important role in the development of rectal carcinoma, and FHIT protein may be associated with the regulation of cell apoptosis.