Exploratory study on the impact of intraesophageal pressure on quality of life in patients following total gastrectomy: a retrospective cohort study.

BACKGROUND: The usefulness of high-resolution impedance manometry (HRIM) in patients who underwent total gastrectomy with Roux-en-Y (R-Y) anastomosis has never been well validated. This study aimed to investigate whether intraesophageal pressure affects quality of life in patients who underwent total gastrectomy with R-Y anastomosis. METHODS: The participants comprised 12 patients who underwent total gastrectomy for gastric cancer between October 2014 and July 2022 and underwent a postsurgical HRIM examination. The association between the HRIM data and Postgastrectomy Syndrome Assessment Scale-37 (PGSAS-37) questionnaires was analyzed. RESULTS: Esophageal body motility was normal in almost all patients. The anastomosis shape (circular stapler and overlap method with linear stapler) did not influence intraesophageal pressure. The integrated relaxation pressure and lower esophageal sphincter (LES) residual pressure during swallowing-induced relaxation were involved in "diarrhea subscale" scores (p = 0.0244 and p = 0.0244, respectively). The average maximum intrabolus pressure was not involved in postgastrectomy symptom. The contractile front velocity correlated with the "indigestion subscale," "diarrhea subscale," and "constipation subscale" (p = 0.0408, p = 0.0143, and p = 0.0060, respectively). The distal latency, i.e., the time from upper esophageal sphincter relaxation to contractile deceleration, was also associated with the "abdominal pain subscale" (p = 0.0399). LES pressure and esophageal body motility affected patients' quality of life after total gastrectomy. CONCLUSIONS: HRIM for the evaluation of intraesophageal pressure is useful for the functional assessment of esophagojejunostomy with the R-Y reconstruction after total gastrectomy.

The c-MYC-ABCB5 axis plays a pivotal role in 5-fluorouracil resistance in human colon cancer cells.

c-MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c-MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c-MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5-fluorouracil (5-FU)-based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c-MYC increased the survival rate following 5-FU treatment in human colon cancer cells, and knockdown of endogenous c-MYC decreased it. Furthermore, c-MYC knockdown decreased the expression level of ABCB5, which is involved in 5-FU resistance. Using a chromatin immunoprecipitation assay, we found that c-MYC bound to the ABCB5 promoter region. c-MYC inhibitor (10058-F4) treatment inhibited c-MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5-FU treatment as expected, and the ABCB5 expression level was increased in 5-FU-resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5-FU and 10058-F4 treatment significantly decreased tumorigenicity in nude mice compared with 5-FU or 10058-F4 treatment alone. 10058-F4 treatment decreased the ABCB5 expression level in the presence or absence of 5-FU. In contrast, 5-FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c-MYC confers resistance to 5-FU through regulating ABCB5 expression in human colon cancer cells.

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model.

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.

JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cells.

Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are the critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target genes. Furthermore, the expression level of nuclear JAB1, but not nuclear STAT3, correlated with unphosphorylated STAT3 DNA-binding activity between COLO205 and LoVo cells. Taken together, these results suggest that nuclear JAB1 positively regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cell line COLO205.

Determination of thymidine phosphorylase expression level facilitates recurrence risk stratification in stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines.

The present study aimed to prospectively clarify the prognostic effect of the expression of several genes that are known to modulate 5-fluorouracil effects in 63 patients who underwent curative resection for stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines between 2008 and 2012. Thymidine phosphorylase (TP) expression in primary tumours was significantly lower in the recurrence group compared with the no-recurrence group (P=0.03), whereas, expression levels of genes that encoded thymidylate synthase, dihydropyrimidine dehydrogenase, folylpolyglutamate synthase, γ-glutamyl hydrolase and dihydrofolate reductase were not statistically different in tumours from the recurrence and no-recurrence groups. In the multivariate analysis using stepwise Cox proportional hazards regression, the following factors were significantly associated with shorter relapse-free survival following adjuvant chemotherapy with oral fluoropyrimidines: Venous invasion [present; hazard ratio (HR)=6.51; 95% confidence interval (CI): 1.55-27.4; P=0.01), Tumour-Node-Metastasis stage (3b; HR=6.18; 95% CI: 1.36-28.2; P=0.02) and TP expression (low; HR=9.61; 95% CI: 1.81-51.0; P=0.04). Patients with two or more risk characteristics had significantly shorter 5-year relapse-free survival compared with patients with one or no risk characteristics (55.8 vs. 91.8%; log-rank P=0.0006). We concluded that low TP expression is an independent predictive factor for poor prognosis in colorectal cancer. Therefore, determining TP expression may help to improve recurrence risk stratification in patients with stage II/III colorectal cancer following adjuvant chemotherapy with oral fluoropyrimidines.

Laparoscopic low anterior resection for rectal cancer after Whitehead's hemorrhoidectomy: A case report.

A 65-year-old man presented with bloody stool. Colonoscopy revealed a raised tumor in the rectum, above the peritoneal reflection. He underwent endoscopic mucosal resection, but the pathological findings suggested the possibility of residual cancer. We performed laparoscopic low anterior resection using a circular stapling instrument for additional curative surgery. However, we could not insert the shaft of the endoscopic circular stapler from the anus because of anal stenosis due to Whitehead's hemorrhoidectomy the patient had undergone 20 years earlier. Therefore, we planned to use a linear stapler to insert an anvil into the rectum. The cartridge-carrying instrument was inserted from the sigmoidal side, and we performed a side-to-end anastomosis. The patient was discharged without anastomotic leakage or defecation disorder. We present this case because laparoscopic low anterior resection for rectal cancer with anal stenosis has not been previously reported.

Loss of skeletal muscle mass after curative gastrectomy is a poor prognostic factor.

Sarcopenia has been reported to relate to poor prognosis in various malignant cancer types. The present study aimed to clarify the prognostic impact of skeletal muscle mass (SMM) loss after curative gastrectomy in patients with gastric cancer. A total of 119 patients who underwent curative gastrectomy for gastric cancer between 2009 and 2016 were analyzed. The SMM loss at 6 months postoperatively compared with the SMM prior to surgery was calculated using the hospital records. The median loss of SMM was 3.8%. Multivariate logistic regression analysis demonstrated that total gastrectomy was a significant and independent risk factor for SMM loss of ≥5% (odds ratio=2.58; P=0.02). Results from multivariate analysis using stepwise Cox proportional hazards regression indicated that the following factors were significantly associated with shorter overall survival after curative gastrectomy: Age [>70 years; hazard ratio (HR)=2.46, P=0.04], TNM stage (≥2; HR=2.65, P=0.04) and loss of SMM (≥5%; HR=2.57, P=0.03). The present findings suggested that loss of SMM after curative gastrectomy for gastric cancer is an independent predictive factor for poor prognosis.

Dclk1 Inhibition Cancels 5-FU-induced Cell-cycle Arrest and Decreases Cell Survival in Colorectal Cancer.

BACKGROUND/AIM: 5-Fluorouracil (5-FU) is frequently used in colorectal cancer treatment, but with limited success. The aim of the present study was to explore the cytotoxic effects of 5-FU, in combination with inhibition of doublecortin-like kinase 1 (Dclk1), a tumor stem cell marker that regulates pro-survival signaling in colorectal cancer cells, in the human colon cancer cell line, COLO-320. MATERIALS AND METHODS: The effects of 5-FU treatment plus Dclk1 inhibition on the phosphorylation of checkpoint kinase 1 (Chk1), cell cycle, DNA damage, apoptosis, and cell survival in COLO-320 cells were evaluated. RESULTS: Combined treatment with 5-FU and a Dclk1 inhibitor, LRRK2-IN-1 (LRRK), decreased 5-FU-induced phosphorylation of Chk1 and canceled 5-FU-induced cell-cycle arrest at the S phase. Combined treatment with 5-FU and LRRK failed to induce poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, but tended to decrease cell survival compared to individual treatment with 5-FU or LRRK. CONCLUSION: These results indicate that a combination of 5-FU and LRRK may be an effective, novel approach for colorectal cancer therapy.

JAB1-STAT3 activation loop is associated with recurrence following 5-fluorouracil-based adjuvant chemotherapy in human colorectal cancer.

Jun activation domain-binding protein 1 (JAB1) has been shown to have multiple roles in tumorigenesis, including the degradation of tumor suppressor proteins such as p53, Smad7, Runx3 and the cyclin-dependent kinase inhibitor p27Kip1, and the activation of oncogenic transcription factors, such as c-Jun and hypoxia-inducible factor-1α. In addition, our previous study revealed that JAB1 positively regulates signal transducer and activator of transcription 3 (STAT3) DNA-binding activity in human colon cancer cells. In turn, the oncogenic transcription factor STAT3 positively regulates JAB1 expression, indicative of a positive feedback loop. Furthermore, high JAB1 expression is associated with a poor prognosis in numerous malignant carcinomas. However, the association between JAB1 expression and prognosis in colorectal cancer remains unclear. The aim of the present study was to elucidate the association between JAB1 and STAT3 expression and recurrence in colorectal cancer. In the present study, it was found that high JAB1 expression in primary colorectal cancer tissues is an independent predictor of recurrence following 5-fluorouracil (5-FU)-based adjuvant chemotherapy in colorectal cancer patients, and that high expression of both JAB1 and STAT3 in primary colorectal cancer tissues is associated with a lower recurrence-free survival rate following 5-FU-based adjuvant chemotherapy compared to high expression of only JAB1 or STAT3. Overall, these results suggest that JAB1 is a novel predictive marker of recurrence following 5-FU-based adjuvant chemotherapy in colorectal cancer patients, and that the JAB1-STAT3 activation loop may be a potential therapeutic target in recurrent colorectal cancer following 5-FU-based adjuvant chemotherapy.

Enhancement of cytotoxic effects of gemcitabine by Dclk1 inhibition through suppression of Chk1 phosphorylation in human pancreatic cancer cells.

Although gemcitabine (GEM) is frequently used in the treatment of pancreatic cancer, the effects are limited. To increase the inhibitory effect of GEM, the identification of a molecular target is needed. Recent studies have revealed that doublecortin-like kinase 1 (Dclk1) positively regulates tumor growth, invasion, metastasis, factors related to epithelial-mesenchymal transition (EMT), pluripotency, angiogenesis, and anti-apoptosis in pancreatic cancer cells. Therefore, Dclk1 is a potential therapeutic target for pancreatic cancer. However, the Dclk1-signaling pathway including its substrate proteins remains to be elucidated. To identify the candidate substrate proteins phosphorylated by Dclk1, we performed a cancer-related phosphorylated protein microarray using Dclk1-inhibited MIA Paca2 cells. Expression levels of phosphorylated cdc25A (p-cdc25A) and phosphorylated Chk1 (p-Chk1), belonging to the ATR pathway, were decreased by treatment with Dclk1 inhibitor LRRK2-IN-1 (LRRK), indicating Dclk1 involvement in the ATR pathway. Consistent with this finding, the GEM-induced p-Chk1 expression was significantly decreased by treatment with LRRK. Notably, combined treatment with GEM and LRRK allowed cell cycle progression without arresting at S phase, while individual treatment with GEM induced cell cycle arrest at S phase. In addition, combined treatment with GEM and LRRK increased the number of γ-H2AX-positive cells compared with that upon individual treatments. Moreover, LRRK alone, and combined treatment with GEM and LRRK, induced caspase-3 activation and PARP1 cleavage, in contrast to treatment with GEM alone. Finally, combined treatment with GEM and LRRK significantly reduced cell survival compared to individual treatment with GEM. These results indicate that Dclk1 inhibition in combination with GEM treatment offers a novel approach to treat pancreatic cancer cells.

Mitogenic and antiapoptotic effects of various growth factors on human corneal fibroblasts.

PURPOSE: To investigate both the effects of various growth factors on the proliferation of human corneal fibroblasts and the abilities of these factors to protect the cells from apoptosis. METHODS: Cultured human corneal fibroblasts were incubated separately with 11 different growth factors whose receptors are expressed by these cells. Cell proliferation was evaluated by measurement of [(3)H]thymidine incorporation. The activation of the protein kinase Akt, which plays an important role in antiapoptotic signaling, was assessed by immunoblot analysis with antibodies specific for a phosphorylated form of the enzyme. Apoptosis was quantitated by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay. RESULTS: Of the 11 growth factors examined, platelet-derived growth factor, insulin, insulin-like growth factors-1 and -2, and epidermal growth factor, each stimulated the proliferation of corneal fibroblasts, induced the activation of Akt in these cells, and protected them from apoptosis induced by sodium nitroprusside (SNP). Basic fibroblast growth factor, keratinocyte growth factor, nerve growth factor, and hepatocyte growth factor stimulated cell proliferation but did not induce Akt activation or protect the cells from SNP-induced apoptosis. Transforming growth factor-beta1 and -beta2 did not stimulate proliferation and had no effect on Akt activity or on SNP-induced apoptosis. CONCLUSIONS: In terms of their modulatory effects on the proliferation and apoptosis of human corneal fibroblasts, the 11 growth factors examined can be classified into three groups. These growth factors may both contribute to maintenance of the cornea and coordinate the proliferative and apoptotic responses of corneal fibroblasts during corneal wound healing.

HIF-1α activation under glucose deprivation plays a central role in the acquisition of anti-apoptosis in human colon cancer cells.

A poor vascular network development in a tumor mass leads to poor oxygen and nutrient supply. To adapt to a hypoxic microenvironment, it is well-known that cancer cells activate the transcription factor hypoxia-inducible factor-1α (HIF-1α). HIF-1α plays a central role in hypoxia-induced metabolic switching, anti-apoptosis, angiogenesis and drug resistance. Glucose deprivation, another major stressful microenvironment, protects cancer cells from drug-induced apoptosis. However, the molecular mechanisms by which cancer cells adapt to poor nutrient conditions remain poorly understood. In this study, we focused on HIF-1α, signal transducer and activator of transcription 3 (STAT3) and trans-cription factor 4 (TCF4), which are involved in cell survival, anti-apoptosis and drug resistance. We examined their activities and the relationships among these transcription factors under glucose deprivation. Our results showed that glucose deprivation increased HIF-1α, STAT3 and TCF4 DNA-binding activity, as well as the expression levels of their target genes OCT4, BCL-2 and VEGF. HIF-1α knockdown significantly increased poly(ADP-ribose) polymerase 1 (PARP-1) cleavage at higher levels than STAT3 knockdown under glucose deprivation. Furthermore, HIF-1α knockdown led to a significant decrease in the expression levels of both STAT3 and TCF4, although STAT3 knockdown decreased only HIF-1α expression level. Our data indicated that activation of the HIF-1α signaling pathway under glucose deprivation leads to the acquisition of anti-apoptotic properties in human colon cancer cells, and targeting the HIF-1α signaling pathway may provide an effective avenue for treating cancers resistant to conventional therapy.