Effects of TRPV2 on the Expression of PD-L1 and Its Binding Ability to PD-1 in Gastric Cancer.

BACKGROUND: Transient receptor potential vanilloid 2 (TRPV2) is a member of the TRP superfamily of non-specific cation channels with functionally diverse roles. We herein investigated the effects of TRPV2 on the expression of programmed cell death-ligand 1 (PD-L1) and its binding ability to programmed cell death-1 (PD-1) in gastric cancer (GC). METHODS: Knockdown (KD) experiments were performed on human GC cell lines using TRPV2 small-interfering RNA. The surface expression of PD-L1 and its binding ability to PD-1 were analyzed by flow cytometry. Eighty primary tissue samples were assessed by immunohistochemistry (IHC), and the relationships between IHC results, clinicopathological factors, and patient prognosis were analyzed. The molecular mechanisms underlying the effects of TRPV2 on the intracellular ion environment were also investigated. RESULTS: TRPV2-KD decreased the expression level of PD-L1 in NUGC4 and MKN7 cells, thereby inhibiting its binding to PD-1. A survival analysis revealed that 5-year overall survival rates were significantly lower in the TRPV2 high expression and PD-L1-positive groups. In IHC multivariate analysis of GC patients, high TRPV2 expression was identified as an independent prognostic factor. Furthermore, a positive correlation was observed between the expression of TRPV2 and PD-L1. An immunofluorescence analysis showed that TRPV2-KD decreased the intracellular concentration of calcium ([Ca2+]i). Treatment with ionomycin/PMA (phorbol 12-myristate 13-acetate), which increased [Ca2+]i, upregulated the protein expression of PD-L1 and promoted its binding to PD-1. CONCLUSIONS: The surface expression of PD-L1 and its binding ability to PD-1 in GC were regulated by TRPV2 through [Ca2+]i, indicating the potential of TRPV2 as a biomarker and target of immune checkpoint blockage for GC.

NADPH Oxidase 2 Has a Crucial Role in Cell Cycle Progression of Esophageal Squamous Cell Carcinoma.

BACKGROUND: NADPH oxidases (NOXs) are transmembrane proteins that generate reactive oxygen species. Recent studies have reported that NOXs are involved in tumor progression in various cancers. However, the expression and role of NOX2 in esophageal squamous cell carcinoma (ESCC) remain unclear. This study aimed to clarify the pathophysiologic role of NOX2 in patients with ESCC and cell lines. METHODS: Two human ESCC cell lines (TE5 and KYSE170) were used for NOX2 transfection experiments, and the effects on cell proliferation, cell cycle, cell motility, and cell survival were analyzed. An mRNA microarray analysis was also performed to assess gene expression profiles. Additionally, NOX2 immunohistochemistry was performed on 130 primary ESCC tumor samples to assess the prognostic value of NOX2 in patients with ESCC. RESULTS: NOX2 depletion significantly inhibited cell proliferation with the G0/G1 arrest and resulted in apoptosis in two cell lines. Microarray analysis revealed a strong relationship between NOX2 gene expression and the signaling pathway of cell cycle regulation by the B-cell translocation gene 2 (BTG2) family, including BTG2, CCNE2, E2F1, and CDK2 genes. Immunohistochemical staining revealed that high NOX2 protein expression was significantly associated with deeper tumor invasion and selected as one of the independent prognostic factors associated with the 5-year OS rate in patients with ESCC. CONCLUSIONS: NOX2 expression in ESCC cells affects tumorigenesis, especially cell cycle progression via the BTG2-related signaling pathway, as well as the prognosis of patients with ESCC. NOX2 may be a novel biomarker and therapeutic target for ESCC.

The Effect of Preoperative Oral Antibiotics in the Prevention of Surgical Site Infection after Laparoscopic Colorectal Cancer Surgery: A Propensity Score Matching Study.

OBJECTIVES: Recent findings suggest that the combination of mechanical bowel preparation (MBP) and preoperative oral antibiotics (OA) decreases the risk of surgical site infection (SSI) in colorectal surgery; however, this remains controversial. The present study examined the efficacy of OA plus MBP in laparoscopic colorectal cancer (CRC) surgery using propensity score matching (PSM). METHODS: A total of 1080 patients with CRC underwent MBP followed by laparoscopic surgery between 2007 and 2019. OA was administered to all patients with CRC who underwent colectomy from 2018. PSM was performed to compare the effects of OA plus MBP (OA) versus MBP only (non-OA) on the rate of superficial SSI. RESULTS: Overall, 128 patients received OA. Significant differences were observed in age, the American Society of Anesthesiologists performance status (ASA-PS), liver disease, and preoperative serum albumin (Alb) between the OA and non-OA groups. The enrolled patients were matched using PSM into two groups based on the following factors: sex, age, body mass index, ASA-PS, diabetes mellitus, liver disease, Alb, and tumor location, which resulted in the disappearance of significant differences. A univariate analysis showed that blood loss of 100 g or more, non-OA, and preoperative chemotherapy or radiation correlated with SSI (p = 0.021, 0.010, 0.038). A multivariate analysis of these three variables identified blood loss of 100 g or more and non-OA as independent risk factors for SSI (hazard ratio (HR): 3.238, p = 0.031; HR: 2.547, p = 0.033). CONCLUSIONS: The present study revealed that OA plus MBP markedly reduced SSI rate. OA with MBP needs to be adopted in laparoscopic CRC surgery.

NADPH oxidase 5 has a crucial role in cellular motility of colon cancer cells.

NADPH oxidases (NOXs) are a family of transmembrane proteins that generate reactive oxygen species. It was previously reported that patients with colon cancer who had high NOX5 expression had poor prognosis. However, no studies have investigated the cellular functions of NOX5 in colon cancer. The present study aimed to clarify the relationship between NOX5 and cancer development using an in vitro model. Reverse transcription­quantitative PCR was performed to determine the NOX5 expression levels of colon cancer cell lines. NOX5­knockdown experiments were conducted, and the effect on cell proliferation, migration, and invasion were analyzed. In addition, mRNA microarray was conducted to assess changes in gene profile. NOX5 mRNA expression was high in HCT116 cells and moderate in SW48 cells. NOX5 knockdown significantly inhibited cell migration and invasion in both HCT116 and SW48 cells; however, NOX5 knockdown reduced cell proliferation in only HCT116 cells. mRNA microarrays revealed a strong relationship between NOX5 expression levels and integrin­linked kinase signaling pathways. The NOX5 expression in colon cancer cells affected cancer progression, especially cell motility. NOX5 may be a novel therapeutic target for the future development of treatments for colon cancer.

Amlodipine and Verapamil, Voltage-Gated Ca2+ Channel Inhibitors, Suppressed the Growth of Gastric Cancer Stem Cells.

BACKGROUND: The membrane transporters activated in cancer stem cells (CSCs) are the target of novel cancer therapies for gastric cancer. The present study investigated ion channel expression profiles in gastric CSCs (GCSCs). METHODS: Cells strongly expressing CD44 were separated from MKN74 cells, a human gastric cancer cell line, by fluorescence-activated cell sorting (FACS), and GCSCs were identified based on tumorsphere formation. Gene expression profiles in GCSCs were examined by a microarray analysis. RESULTS: Among MKN74 cells, CD44 messenger RNA levels were higher in CSCs than in non-CSCs. These CSCs also exhibited resistance to cisplatin. The microarray analysis revealed that the expression of several genes related to voltage-gated Ca2+ channels (VGCCs), including CACNA2D1 and CACNB4, was upregulated. The cytotoxicities of the CACNA2D1 inhibitor amlodipine and the CACNB4 inhibitor verapamil were greater at lower concentrations in CSCs than in non-CSCs, and markedly reduced tumorsphere numbers. Tumor volumes were significantly smaller in a xenograft nude mouse model treated with amlodipine or verapamil in combination with cisplatin than in that treated with cisplatin alone. CONCLUSIONS: The present results indicate that VGCCs play a role in maintaining CSCs, and demonstrated the potential of their specific inhibitors, amlodipine and verapamil, as targeted therapeutic agents against gastric cancer.

ANO9 regulates PD-L2 expression and binding ability to PD-1 in gastric cancer.

The function of ANO9 in gastrointestinal cancer remains unclear. We investigated the biological behaviors and clinical prognostic values of ANO9 in gastric cancer (GC). Knockdown experiments were performed on human GC cell lines using ANO9 siRNA. Eighty-four primary tissue samples from patients with advanced GC were examined immunohistochemically (IHC). Knockdown of ANO9 reduced the progression of cancer cells in MKN7 and MKN74 cells. A microarray analysis revealed that ANO9 regulated PD-L2 via interferon (IFN)-related genes. We confirmed using flow cytometry that the depletion of ANO9 reduced the binding ability to PD-1 by downregulating the expression of PD-L2 in MKN7 and MKN74 cells. IHC revealed a correlation between the expression of ANO9 and PD-L2 and also that the strong expression of ANO9 was an independent poor prognostic factor in patients with advanced GC. The present results indicate that ANO9 regulates PD-L2 and binding ability to PD-1 via IFN-related genes in GC. Therefore, ANO9 has potential as a biomarker and target of immune checkpoint blockage (ICB) for GC.

Clinical significance of the distance between the cricoid cartilage and upper edge of the tumor using PET-CT in cervical esophageal cancer.

Cervical esophageal squamous cell carcinoma (CESCC) is less common compared with thoracic esophageal cancer, and few studies have investigated the clinicopathological features of CESCC. The present study analyzed 69 patients with CESCC who underwent various therapies at the University Hospital of Kyoto Prefectural University of Medicine between January 2000 and December 2016. The distance between the inferior border of the cricoid cartilage and upper edge of the tumor was evaluated using positron emission tomography and computed tomography. Positive and negative values indicated oral and anal directions, respectively. Using receiver operating characteristic curves, the cut-off value for laryngeal preservation was calculated as -5 mm. According to this value, the patients were divided into two groups: The short group (distance from the cricoid cartilage ≥-5 mm) and long group (distance from the cricoid cartilage <-5 mm). There were no significant differences in clinicopathological factors between the two groups except for body mass index. In univariate analysis, the 3-year overall survival rate was significantly lower in short group (45.4 vs. 79.6%; P=0.009). In multivariate analysis, short group was an independent prognostic risk factor (hazard ratio=2.65; P=0.039). This may be due to lymphatic flow around the cervical esophagus.

ANO9 Regulated Cell Cycle in Human Esophageal Squamous Cell Carcinoma.

BACKGROUND: Few studies have reported the function and activation mechanism of ANO9 in esophageal squamous cell carcinoma (ESCC). The current study aimed to investigate the role of ANO9 in the regulation of tumor progression. METHODS: Knockdown experiments with human ESCC cell lines were performed using ANO9 siRNA, and the effects on cell proliferation, the cell cycle, apoptosis, and cellular movement were analyzed. Immunohistochemistry (IHC) analysis was performed on 57 primary tumor samples obtained from ESCC patients. RESULTS: In an in vitro study, depletion of ANO9 reduced cell proliferation, invasion, and migration in KYSE150 and KYSE 790 cells. In the cell cycle analysis, depletion of ANO9 increased the number of cells in G0/G1 arrest. In addition, the knockdown of ANO9 increased apoptosis. The results of the microarray analysis indicated that various centrosome-related genes such as CEP120, CNTRL, and SPAST were up- or downregulated in ANO9-depleted KYSE150 cells. The IHC results showed that high expression of ANO9 was associated with poor prognosis. CONCLUSIONS: The results of the current study suggest that ANO9 regulates the cell cycle via centrosome-related genes in ESCC.

The expression and role of TRPV2 in esophageal squamous cell carcinoma.

BACKGROUND: Transient receptor potential vanilloid 2 (TRPV2) was recently shown to be involved in migrant potentials. The present study aimed to investigate its role in esophageal squamous cell carcinoma (ESCC). METHODS: Knockdown experiments were conducted using TRPV2 siRNA in human ESCC cell lines, and anti-tumor effects were analyzed. The gene expression profiles of cells were analyzed using a microarray method. An immunohistochemical staining was performed on 62 primary tumor samples. RESULTS: TRPV2 overexpression was observed in TE15 and KYSE170 cells. TRPV2 depletion suppressed proliferation, cell cycle progression, and invasion/migration ability, and induced apoptosis. A pathway analysis of microarray data showed that TRPV2 depletion down-regulated WNT/ß-catenin signaling-related genes and basal cell carcinoma signaling-related genes. The suppression of tumor functions, such as proliferation, invasion, and angiogenesis, was predicted in the ontology analysis. Immunohistochemical analysis revealed a correlation between strong TRPV2 expression and a poor prognosis in ESCC patients. CONCLUSION: The present results suggest that TRPV2 regulates cancer progression by affecting WNT/ß-catenin or basal cell carcinoma signaling, and that TRPV2 strong expression is associated with a worse prognosis in ESCC patients. These results provide an insight into the role of TRPV2 as a novel therapeutic target or biomarker for ESCC.

Functional Outcomes of Billroth I Gastroduodenostomy Using Linear Staplers in Totally Laparoscopic Distal Gastrectomy.

BACKGROUND/AIM: This study examined whether functional outcomes of linear-stapled Billroth I (LS-BI) in totally laparoscopic distal gastrectomy (TLDG) are comparable to those of circular-stapled Billroth I (CS-BI) in laparoscopy-assisted distal gastrectomy (LADG). PATIENTS AND METHODS: This was a retrospective study of patients with gastric cancer undergoing TLDG with LS-BI (n=50) or LADG with CS-BI (n=50). Postoperative endoscopic findings of the remnant stomach and nutritional status were evaluated. RESULTS: The occurrence of grade 2 or more severe remnant gastritis in the LS-BI group (46.0%) was significantly higher than that in the CS-BI group (18.0%) (p=0.005), whereas there was no significant difference in the incidence of residual food and bile reflux between the two groups. Postoperative changes in body weight, and serum albumin and total protein levels were similar between the two groups. CONCLUSION: TLDG with LS-BI may be a good alternative to LADG with CS-BI because of its comparable nutritional outcomes, but with a higher occurrence of remnant gastritis.

Relationship Between Postoperative CRP and Prognosis in Thoracic Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIM: Few studies have examined postoperative CRP in esophageal cancer. We investigated the relationship between postoperative CRP values according to the postoperative period and prognosis in esophageal cancer. PATIENTS AND METHODS: We performed a retrospective cohort study including 187 patients who underwent esophagectomy for esophageal squamous cell carcinoma (ESCC) between January 2008 and October 2016. RESULTS: CRP within 1 month of surgery was not related to overall survival (OS) or recurrence-free survival (RFS). In a univariate analysis, postoperative 2 months (2M)-CRP ≥0.15 ml/dl was associated with poorer OS (41.4 vs. 71.4%, p=0.0002) and RFS (28.9 vs. 51.3%, p=0.007). In a multivariate analysis, 2M-CRP ≥0.15 ml/dl was an independent factor for poorer OS (HR=2.27, 95%CI=1.03-3.34, p=0.005) and RFS (HR=1.65, 95%CI=1.08-2.52, p=0.020). The incidence of postoperative pneumonia was significantly higher in the 2M-CRP ≥0.15 ml/dl group (p=0.026). CONCLUSION: 2M-CRP ≥0.15 ml/dl is an independent prognostic factor for ESCC. Furthermore, postoperative pneumonia may be associated with patient prognosis after esophagectomy.