Shiro Kohi

Prognostic Impact of Hyaluronan and Its Regulators in Pancreatic Ductal Adenocarcinoma

Background Although pancreatic ductal adenocarcinoma is characterized by an abundant stroma enriched with hyaluronan (HA), the prognostic impact of HA and its regulators remains unknown. Methods Using immunohistochemistry, expression patterns of HA and its regulators, including a synthesizing enzyme (HAS2), and a degrading enzyme (HYAL1) were investigated in patients who received surgical resection. The prognostic significance of these markers and other clinicopathological variables was determined using univariate and multivariate analyses. The HA levels were determined quantitatively by enzyme-linked immunosorbent assay (ELISA). Results We found that strong expressions of HA (P=0.008) and HAS2 (P=0.022) were significantly associated with shorter survival time after surgery. By contrast, weak expression of HYAL1 was significantly associated with poor survival (P=0.001). In multivariate analysis, tumor stage (hazard ratio (HR)=2.76, 95% confidence interval (CI): 1.14-6.66 P=0.024), strong HA expression (HR=6.04, 95%CI: 1.42-25.69 P=0.015), and weak HYAL1 expression (HR=3.16, 95%CI: 1.19-8.40 P=0.021) were independent factors predicting poor survival. ELISA revealed higher concentration of HA in pancreatic cancer tissues than in normal pancreatic tissues (P=0.001). Conclusion These findings suggest, for the first time, that HA and its regulators may have prognostic impact in patients with pancreatic cancer.

Role of hyaluronan in pancreatic cancer biology and therapy: Once again in the spotlight

Pancreatic ductal adenocarcinoma (PDAC) remains the most deadly disease worldwide, with the lowest survival rate among all cancer types. Recent evidence suggests that hyaluronan (HA), a major component of ECM, provides a favorable microenvironment for cancer progression. Pancreatic ductal adenocarcinoma is typically characterized by a dense desmoplastic stroma containing a large amount of HA. Accumulation of HA promotes tumor growth in mice and correlates with poor prognosis in patients with PDAC. Because HA is involved in various malignant behaviors of cancer (such as increased cell proliferation, migration, invasion, angiogenesis, and chemoresistance), inhibiting HA synthesis/signaling or depleting HA in tumor stroma could represent a promising therapeutic strategy against PDAC. In this review article, we summarize our current understanding of the role of HA in the progression of PDAC and discuss possible therapeutic approaches targeting HA.

Hyaluronan stimulates pancreatic cancer cell motility

Hyaluronan (HA) accumulates in pancreatic ductal adenocarcinoma (PDAC), but functional significance of HA in the aggressive phenotype remains unknown. We used different models to investigate the effect of HA on PDAC cell motility by wound healing and transwell migration assay. Changes in cell motility were examined in 8 PDAC cell lines in response to inhibition of HA production by treatment with 4-methylumbelliferone (4-MU) and to promotion by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by co-culture with tumor-derived stromal fibroblasts. We also investigated changes in cell motility by adding exogenous HA. Additionally, mRNA expressions of hyaluronan synthases and hyaluronidases were examined using real time RT-PCR. Inhibition of HA by 4-MU significantly decreased the migration, whereas promotion of HA by TPA or co-culture with tumor-derived fibroblasts significantly increased the migration of PDAC cells. The changes in HA production by these treatments tended to be associated with changes in HAS3 mRNA expression. Furthermore, addition of exogenous HA, especially low-molecular-weight HA, significantly increased the migration of PDAC cells. These findings suggest that HA stimulates PDAC cell migration and thus represents an ideal therapeutic target to prevent invasion and metastasis.

Targeting hyaluronan for the treatment of pancreatic ductal adenocarcinoma

Progression of cancer is often associated with interactions between cancer cells and extracellular matrix (ECM) surrounding them. Increasing evidence has suggested that accumulation of hyaluronan (HA), a major component of ECM, provides a favorable microenvironment for cancer progression. Pancreatic ductal adenocarcinoma (PDAC) is characterized typically by a dense desmoplastic stroma with a large amount of HA, making this molecule as an attractive target for therapy. Several studies have shown efficacy of inhibitors of HA synthesis or signaling for the treatment of PDAC. Recent studies have also demonstrated substantial improvements in the effects of chemotherapy by a targeted depletion of stromal HA in PDAC using an enzymatic agent. Thus, targeting HA has been recognized as a promising therapeutic strategy to treat this highly aggressive neoplasm. In this review article, we summarize our current understanding of the role of HA in the progression of PDAC and discuss possible therapeutic approaches targeting HA.

Receptor for Hyaluronic Acid-Mediated Motility is Associated with Poor Survival in Pancreatic Ductal Adenocarcinoma.

Receptor for hyaluronic acid (HA)-mediated motility (RHAMM) is a nonintegral cell surface receptor involved in the aggressive phenotype in a wide spectrum of human malignancies, but the significance of RHAMM in pancreatic ductal adenocarcinoma (PDAC) remains unknown. In this study, we investigated the expression of RHAMM and its clinical relevance in PDAC. RHAMM mRNA expression was examined in 8 PDAC cell lines and in primary pancreatic cancer and adjacent non-tumor tissues from 14 patients using real-time RT-PCR. Western blotting was carried out to analyze the expression of RHAMM protein in PDAC cell lines. We also investigated the expression patterns of RHAMM protein in tissue samples from 70 PDAC patients using immunohistochemistry. The RHAMM mRNA expression was increased in some PDAC cell lines as compared to a non-tumorous pancreatic epithelial cell line HPDE. The RHAMM mRNA expression was significantly higher in PDAC tissues as compared to corresponding non-tumorous pancreatic tissues (P < 0.0001). The RHAMM protein expression was higher in the vast majority of PDAC cell lines relative to the expression in HPDE. The immunohistochemical analysis revealed strong expression of RHAMM in 52 (74%) PDAC tissues. Strong expression of RHAMM was significantly associated with a shorter survival time (P = 0.038). In multivariate analysis, tumor stage (P = 0.039), residual tumor (P = 0.015), and strong RHAMM expression (P = 0.034) were independent factors predicting poor survival. Strong expression of RHAMM may predict poor survival in PDAC patients and may provide prognostic and, possibly, therapeutic value.

A novel epigenetic mechanism regulating hyaluronan production in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma enriched with hyaluronan (HA), a major component of extracellular matrix known to play a critical role in tumor progression. The mechanisms that regulate HA synthesis in PDAC are poorly understood. To investigate whether DNA methylation and HA production from PDAC cells are associated, we studied the effect of 5-aza-2′-deoxycitidine (5-aza-dC), an inhibitor of DNA methylation, or DNA methyltransferase 1 (DNMT1) knockdown by small interfering RNA, on the HA production from PDAC cells. HA production into the conditioned medium was evaluated in PDAC cells treated with 5-aza-dC or DNMT1 knockdown. mRNA expression of HA synthase (HAS) genes was investigated by real-time RT-PCR. Treatment of PDAC cells with 5-aza-dC led to a significant increase in the HA production (up to 2.5-fold increase) in all 4 cell lines tested. This enhanced HA production by 5-aza-dC treatment was accompanied by increased mRNA expression of HAS2 and HAS3. Furthermore, increased HA production and HAS2/HAS3 mRNA expression was also observed in PDAC cells by knockdown of DNMT1. These findings provide evidence, for the first time, that epigenetic mechanism is involved in the regulation of HA synthesis in PDAC cells.

Stapled gastro/duodenojejunostomy shortens reconstruction time during pylorus-preserving pancreaticoduodenectomy

AIM To investigate whether a stapled technique is superior to the conventional hand-sewn technique for gastro/duodenojejunostomy during pylorus-preserving pancreaticoduodenectomy (PpPD). METHODS In October 2010, we introduced a mechanical anastomotic technique of gastro- or duodenojejunostomy using staplers during PpPD. We compared clinical outcomes between 19 patients who underwent PpPD with a stapled gastro/duodenojejunostomy (stapled anastomosis group) and 19 patients who underwent PpPD with a conventional hand-sewn duodenojejunostomy (hand-sewn anastomosis group). RESULTS The time required for reconstruction was significantly shorter in the stapled anastomosis group than in the hand-sewn anastomosis group (186.0 ± 29.4 min vs 219.7 ± 50.0 min, P = 0.02). In addition, intraoperative blood loss was significantly less (391.0 ± 212.0 mL vs 647.1 ± 482.1 mL, P = 0.03) and the time to oral intake was significantly shorter (5.4 ± 1.7 d vs 11.3 ± 7.9 d, P = 0.002) in the stapled anastomosis group than in the hand-sewn anastomosis group. There were no differences in the incidences of delayed gastric emptying and other postoperative complications between the groups. CONCLUSION These results suggest that stapled gastro/duodenojejunostomy shortens reconstruction time during PpPD without affecting the incidence of delayed gastric emptying.

Increased Expression of HYAL1 in Pancreatic Ductal Adenocarcinoma.

Objectives Increased production and processing (degradation) of hyaluronan (HA) is critical for cancer invasion and metastasis. Although HA is known to be overexpressed in pancreatic ductal adenocarcinoma (PDAC), little is known about the expression and biological significance of HA-degrading enzymes, hyaluronidases (HYALs), in PDAC. Methods Expression of HYALs mRNA was examined in PDAC cells by quantitative real-time RT-PCR. HYAL1 protein expression was examined in primary PDAC tumors by enzyme-linked immuno-sorbent assay. The migratory ability of PDAC cells was determined by a transwell cell migration assay. Results Screening of mRNA expression of three major HYAL genes (HYAL1, 2, and 3) identified HYAL1 as a gene overexpressed in PDAC cells. Treatment of PDAC cells with 5-aza-2′-deoxycytidine and/or trichostatin A further increased the HYAL1 expression, suggesting a possible involvement of epigenetic mechanisms in the transcriptional regulation of this gene. HYAL1 protein concentrations were significantly higher in primary PDAC tissues as compared with nontumor pancreatic tissues (P = 0.049). Importantly, inhibition of HYAL activity by dextran sulfate significantly inhibited the migration of PDAC cells showing strong HYAL1 expression (P = 0.002). Conclusions These findings suggest that overexpression of HYAL1 is a common mechanism that may contribute to the aggressive phenotype of PDAC.

Hyaluromycin, a Novel Hyaluronidase Inhibitor, Attenuates Pancreatic Cancer Cell Migration and Proliferation

Pancreatic ductal adenocarcinoma (PDAC) is characterized by accelerated production and degradation of hyaluronan (HA), a major component of extracellular matrix involved in the malignant phenotype of cancer. In particular, increased hyaluronidase (HYAL) activity plays a critical role in cancer progression, at least in part, by producing low-molecular-weight- (LMW-) HA or small fragments of HA, suggesting HYAL as a target for cancer treatment. Hyaluromycin, a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces hyaluromycini as a HYAL inhibitor. We investigated the antitumor effects of hyaluromycin in PDAC cells. We examined the effects of hyaluromycin on the proliferation and migration of PDAC cells. To elucidate the mechanisms underlying the effect of hyaluromycin on PDAC cells, we examined the concentration of LMW-HA in the conditioned media after treating PDAC cells with hyaluromycin. We demonstrate that hyaluromycin inhibits proliferation and migration of PDAC cells. We also found that these antitumor effects of hyaluromycin were associated with a decreased concentration of LMW-HA and a decreased phosphorylation of ribosomal protein S6. Our results suggest that hyaluromycin is a promising new drug against this highly aggressive neoplasm.

Single-incision laparoscopic cholecystectomy for acute cholecystitis: A retrospective cohort study of 52 consecutive patients

BACKGROUND Single-incision laparoscopic cholecystectomy (SILC) has become increasingly popular but its role in acute cholecystitis remains controversial. METHODS We compared the clinical features and outcomes of SILC procedures between 52 patients with acute cholecystitis (the AC group) and 308 patients without acute cholecystitis (the NAC group). We also analyzed clinical variables to identify factors affecting difficulties associated with SILC for acute cholecystitis. RESULTS The patients in the AC group were significantly older than those in the NAC group (72 vs. 61 years, median, P = 0.0005). The preoperative levels of white blood cell counts were significantly higher in the AC group than in the NAC group (6600 vs. 5500/μL, P = 0.0004). The operative time was significantly longer in the AC group than in the NAC group (188 vs. 135 min, P < 0.0001). The volume of intraoperative blood loss was significantly larger in the AC group than in the NAC group (20 vs. 5 mL, P < 0.001). Furthermore, additional trocar insertion was required in 12% in the NAC group, whereas it was required in 60% in the AC group (P < 0.0001). Regarding the difficulties of SILC for acute cholecystitis, delayed operation (after 72 h from the onset) was significantly associated with a prolonged operative time, while a higher grade of acute cholecystitis (grade II or III) was significantly associated with an increased blood loss during surgery. CONCLUSIONS These findings suggest that when compared to SILC for gallbladder diseases without acute inflammation, SILC for acute cholecystitis was associated with a longer operative time, increased blood loss, higher rate of additional trocar requirement, higher rate of postoperative complications, and longer hospital stay. The difficulties associated with SILC for acute cholecystitis were affected by the timing of surgery and the grade of inflammation.