Glucose-regulated protein 78 (GRP78) regulates colon cancer metastasis through EMT biomarkers and the NRF-2/HO-1 pathway.

Glucose-regulated protein 78 (GRP78) is a key chaperone and stress response protein. Previous studies have demonstrated that high GRP78 expression may be correlated with cancer progression and therapeutic response. However, the role of GRP78 in the metastasis of colon cancer is unclear. In this study, we used small interfering RNA (siRNA) to knock down GRP78 expression in colon cancer cells (HT-29 and DLD-1 cells). In wound-healing migration assays, we found that GRP78-knockdown (GRP78KD) cells showed better wound-healing ability than control cells. We also found that GRP78KD cells displayed a better migratory ability than control cells in migration and invasion assays. As we further dissected the underlying molecular mechanism, we found that silencing GRP78 may cause an increase in vimentin expression and a decrease in the E-cadherin level, which was correlated with the increase in migratory ability. In addition, we found that GRP78KD may activate the NRF-2/HO-1 pathway, and this activation was also correlated with the increase in cell invasiveness. Furthermore, we examined GRP78 expression in a tissue array and found that the GRP78 expression in metastatic adenocarcinoma in lymph nodes tended to be weaker than that in primary colonic adenocarcinoma. In conclusion, a low level of GRP78 may cause an increase in metastasis ability in colon cancer cells by altering E-cadherin and vimentin expression and activating the NRF-2/HO-1 signaling pathway. Our study demonstrates that low expression of GRP78 may correlate with a high risk of metastasis in colon cancer.

Glucose-regulated protein 78 mediates the therapeutic efficacy of 17-DMAG in colon cancer cells.

Glucose-regulated protein 78 (GRP78) is expressed as part of the molecular response to endoplasmic reticulum (ER) stress and mediates protein folding within the cell. GRP78 is also an important biomarker of cancer progression and the therapeutic response of patients with different cancer types. However, the role of GRP78 in the cytotoxic effect of 17-DMAG in colon cancer cells remains unclear. GRP78 expression was knocked down by small interfering RNA (siRNA). The anticancer effects of 17-DMAG were assessed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometric cell-cycle analysis, and an Annexin V-propidium iodide (PI) apoptotic assay. We found that HT-29 cells expressed a lower level of GRP78 compared with DLD-1 cells. The MTT assay revealed that HT-29 cells were more sensitive to 17-DMAG treatment than DLD-1 cells. GRP78 knock down (GRP78KD) cells demonstrated an increased sensitivity to 17-DMAG treatment compared with the scrambled control cells. Based on the cell-cycle analysis and Annexin V-PI apoptotic assay, apoptosis dramatically increased in GRP78KD cells compared with scrambled control DLD-1 cells after these cells were treated with 17-DMAG. Finally, we observed a decrease in the level of Bcl-2 and an increase in the levels of Bad and Bax in GRP78KD cells treated with 17-DMAG. These results are consistent with an increased sensitivity to 17-DMAG after knock down of GRP78. The level of GRP78 expression may determine the therapeutic efficacy of 17-DMAG against colon cancer cells.

Thrombomodulin mediates the migratory ability of hormone-independent prostate cancer cells through the regulation of epithelial-to-mesenchymal transition biomarkers.

Thrombomodulin (TM) is highly expressed in endothelial cells and plays the key role in maintaining physical homeostasis. In addition, many pieces of evidence also show that TM contains the diagnostic value for malignant diseases. TM has been found to correlate with metastatic status in multiple cancers, but its role in prostate cancer progression remains unclear. TM expression was determined in prostate cancer cells (DU-145 and PC-3 cells) using real-time PCR and Western blotting. TM expression was manipulated in prostate cancer cells using TM-specific shRNA and an overexpression system. The proliferation, adhesion, and migratory ability of prostate cancer cells expressing various TM levels were determined using the x'Celligence biosensor system and a transwell migration assay. Higher levels of TM transcription and translation were found in DU-145 cells and were negatively correlated with the low migratory ability of DU-145 cells. After silencing TM expression in DU-145 cells, cell growth decreased, but cell adhesion and migration dramatically increased. TM overexpression in PC-3 cells reduced their metastatic ability. We investigated the possible mechanisms of this phenomenon and determined that the enhanced cell migration was mediated through the expression of E-cadherin and vimentin. TM may be a modulator of hormone-independent prostate cancer (HIPC) metastasis. The downregulation of TM expression enhanced the migratory ability of these cells via an increase in vimentin expression and a decrease in E-cadherin expression.

Glucose-regulated protein 78 mediates hormone-independent prostate cancer progression and metastasis through maspin and COX-2 expression.

Glucose-regulated protein 78 (GRP78) plays an essential role in embryonic development and in the progression and therapeutic resistance of many cancers. However, little is known about the function of GRP78 in hormone-independent prostate cancer. Here, we found that the expression levels of GRP78 were higher in PC-3 cells than in DU-145 cells. When the expression of GRP78 was silenced using a GRP78-specific small interfering RNA in PC-3 cells, the growth rate and adhesive ability were reduced. Cell migration was dramatically decreased in GRP78-depleted cells. Dissection of the involved signal pathways revealed that maspin expression was upregulated after silencing GRP78 expression. The upregulation of maspin and downregulation of COX-2 may cause the decrease in cell proliferation and migration observed after silencing GRP78 expression. Silencing GRP78 expression may suppress the proliferation, adhesion, and migration of prostate cancer cells via maspin and COX-2 regulation.

Single-incision versus conventional laparoscopic appendectomy in 688 patients: a retrospective comparative analysis.

BACKGROUND: Laparoscopic surgery has become the standard for treating appendicitis. The cosmetic benefits of using single-incision laparoscopy are well known, but its duration, complications and time to recovery have not been well documented. We compared 2 laparoscopic approaches for treating appendicitis and evaluated postoperative pain, complications and time to full recovery. METHODS: We retrospectively reviewed the cases of consecutive patients with appendicitis and compared those who underwent conventional laparoscopic appendectomy (CLA) performed using 3 incisions and those who underwent single-incision laparoscopic appendectomy (SILA). During SILA, the single port was prepared to increase visibility of the operative site. RESULTS: Our analysis included 688 consecutive patients: 618 who underwent CLA and 70 who underwent SILA. Postsurgical complications occurred more frequently in the CLA than the SILA group (18.1% v. 7.1%, p = 0.018). Patients who underwent SILA returned to oral feeding sooner than those who underwent CLA (median 12 h v. 22 h, p < 0.001). These between-group differences remained significant after controlling for other factors. Direct comparison of only nonperforated cases, which was determined by pathological examination, revealed that SILA was significantly longer than CLA (60 min v. 50 min, p < 0.001). Patients who underwent SILA had longer in-hospital stays than those who underwent CLA (72 v. 55 h, p < 0.001); however, they had significantly fewer complications (3.0% v. 14.4%, p = 0.006). CONCLUSION: In addition to its cosmetic advantages, SILA led to rapid recovery and no increase in postsurgical pain or complications.

CONTEXTE: La chirurgie laparoscopique est devenue la norme pour le traitement de l'appendicite. Les avantages de la laparoscopie à simple incision au plan esthétique sont bien connus, mais la durée de l'intervention, ses complications et le temps de récupération n'ont pas été adéquatement documentés. Nous avons comparé 2 approches laparoscopiques pour le traitement de l'appendicite et évalué la douleur et les complications postopératoires, de même que le temps de récupération complète. MÉTHODES: Nous avons passé en revue de manière rétrospective les dossiers de patients consécutifs atteints d'appendicite et comparé ceux qui ont subi une appendicectomie laparoscopique classique (ALC) à 3 incisions à ceux qui ont subi une appendicectomie laparoscopique à simple incision (ALSI). Durant l'ALSI, l'incision était préparée de manière à améliorer la visibilité du champ opératoire. RÉSULTATS: Notre analyse a inclus 688 patients consécutifs : 618 qui ont subi une ALC et 70, une ALSI. Les complications postopératoires ont été plus nombreuses dans le groupe soumis à l'ALC qu'à l'ALSI (18,1 % c. 7,1 %, p = 0,018). Les patients soumis à l'ALSI ont repris l'alimentation orale plus rapidement que ceux qui avaient subi une ALC (temps médian 12 h c. 22 h, p < 0,001). Ces différences entre les groupes sont demeurées significatives après incorporation d'autres facteurs. La comparaison directe des cas non perforés seulement, révélés par l'examen anatomopathologique, a révélé que l'ALSI a demandé significativement plus de temps que l'ALC (60 min c. 50 min, p < 0,001). Les patients soumis à l'ALSI ont séjourné plus longtemps à l'hôpital que les patients soumis à l'ALC (72 h c. 55 h, p < 0,001); toutefois, ils ont présenté significativement moins de complications (3,0 % c. 14,4 %, p = 0,006). CONCLUSION: En plus de ses avantages au plan esthétique, l'ALSI a permis une récupération rapide, sans accroissement de la douleur ou des complications postopératoires.

MicroRNA-200a and -200b mediated hepatocellular carcinoma cell migration through the epithelial to mesenchymal transition markers.

BACKGROUND: MicroRNAs (miRNAs) play an essential role in mediating gene expression in both normal and malignant cells. However, little is known about specific miRNAs during the development of hepatocellular carcinoma (HCC) from well-differentiated to poorly differentiated cells. METHODS: We performed miRNA array analysis of three different HCC cell lines: HepG2, HepJ5, and skHep-1. The expression patterns of miR-200 family members were confirmed by real-time polymerase chain reaction (PCR). We overexpressed miR-200 family members by using a lentivirus system and selected for stably transduced cells using antibiotics. The migration ability of the cells was tested using the Transwell migration assay system. RESULTS: Our miRNA array and real-time PCR results indicated a decrease in the expression of miR-200 family members in poorly differentiated skHep-1 cells compared with well-differentiated HepG2 cells. We overexpressed miR-200a and miR-200b in both HepJ5 and skHep-1 cells and found that the overexpression of the miR-200 family members did not influence proliferation, although migration was decreased in these cells. We found that overexpression of miR-200 family members led to an upregulation of E-cadherin expression in both HepJ5 and skHep-1 cells. Furthermore, we silenced E-cadherin expression by shRNA in miR200a-HepJ5 cells and found that the migratory ability of these cells was enhanced upon the decrease in E-cadherin expression. CONCLUSIONS: Members of the miR-200 family (miR-200a and miR-200b) play important roles in HCC migration by regulating E-cadherin expression.

Glycated hemoglobin A1c is superior to fasting plasma glucose as an independent risk factor for colorectal neoplasia.

OBJECTIVE: To investigate which glycemic index is more strongly associated with colorectal neoplasia. METHOD: This cross-sectional study enrolled 2,776 participants in a comprehensive health management program which included measurement of fasting plasma glucose and HbA1c, along with screening colonoscopy. Primary outcome was colorectal adenoma with or without dysplasia. Risk factors for colorectal neoplasia were determined by the multivariate regression analysis, which evaluated the interrelationship among different glycemic indices in a hierarchical way. RESULTS: Colorectal neoplasms were found in 605 (21.79%) examinees, 68 (2.45%) of whom had high-risk tumors. Glycemic indices including diagnosis of diabetes mellitus, fasting plasma glucose, and HbA1c were all associated with colorectal tumors in the univariate analysis. However, HbA1c outperformed the other two markers as an independent risk factor (adjusted odds ratio, 1.22; 95% confidence interval, 1.10-1.36%) for colorectal neoplasia. Moreover, only HbA1c remained independently associated with colorectal tumor after patients with established diagnosis of diabetes (n = 132) were excluded. We also identified age, male gender, and smoking were independent risk factors for colorectal neoplasia. CONCLUSION: HbA1c as compared with fasting plasma glucose is more strongly and independently associated with colorectal neoplasia. Further research is warranted to elucidate the value of HbA1c in stratifying risk of colorectal cancer.

NNK enhances cell migration through α7-nicotinic acetylcholine receptor accompanied by increased of fibronectin expression in gastric cancer.

BACKGROUND: In this study, we intended to dissect the mechanism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-enhanced migration of gastric cancer. Smoking has been defined as a risk factor for gastric cancer. Tobacco-specific carcinogen, NNK, was reported to enhance cancer progression in gastric cancer. Currently, metastasis is the major issue for clinical cancer therapy, but the influence of NNK on the migration of gastric cancer remains to be determined. METHODS: The expression of nicotinic receptor in gastric cancer cells was identified by real-time polymerase chain reaction and Western blotting. The influence of NNK on migration of gastric cancer cells was evaluated by the transwell migration assay system. Receptor-mediated migration was studied by both inhibitor and small interfering RNA. RESULTS: Alpha7 nicotinic acetylcholine receptor, alpha7-nicotinic acetylcholine receptor (nAChR), was identified higher than alpha9-nAChR in gastric cancer cell lines, AGS cells. NNK enhanced significantly gastric cancer cell migration in transwell assay. We used inhibitor and siRNA to demonstrate that alpha7-nAChR mediated NNK-enhanced gastric cancer cell migration and upregulation of fibronectin were involved in NNK-enhanced migration of gastric cancer cells. Finally, we found that silenced fibronectin expression level inhibited the migratory ability in AGS cells. CONCLUSIONS: NNK enhanced gastric cancer metastasis through alpha7-nAChR and fibronectin-one of the hallmarks of epithelial mesenchymal transition.

Survivin-mediated therapeutic efficacy of gemcitabine through glucose-regulated protein 78 in hepatocellular carcinoma.

BACKGROUND: Survivin is an antiapoptotic molecule that is widely expressed in cancers, including hepatocellular carcinoma (HCC). Survivin has become a general therapeutic target for cancers because of its selective overexpression in a majority of tumors. However, little is known regarding the effect of survivin expression in combination with gemcitabine on HCC. METHODS: We generated survivin knockdown cells (survivin-KD) via a short interfering RNA (siRNA) technique. The antiproliferation effects of gemcitabine were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay, and cell cycle evaluation. RESULTS: According to the MTT assay, we found that survivin-KD cells were more sensitive than parental cells and scrambled control cells to gemcitabine treatment. The apoptotic cell population increased in survivin-KD cells that were treated with gemcitabine in comparison to scrambled control cells, as observed by the cell cycle distribution and TUNEL assays. We found that survivin knockdown resulted in a reduction of glucose-regulated protein 78 (GRP78), which may be responsible for the observed increased survivin-KD cell sensitivity to gemcitabine. CONCLUSIONS: We conclude that survivin knockdown may contribute to a therapeutic effect of gemcitabine through GRP78 on HCC cells.

Knockdown survivin expression reduces the efficacy of curcumin treatment in hepatocellular carcinoma cells.

BACKGROUND: Survivin is a potential therapeutic target for cancer. Increased survivin expression promotes cell survival and therapeutic resistance. However, there is little information regarding whether the expression level of survivin affects curcumin treatment in hepatocellular carcinoma (HCC). METHODS: Survivin expression was suppressed in HCC cells using a short interfering RNA (siRNA) technique. The anticancer effects of curcumin were examined using a biosensor system, MTT assay, TUNEL assay, and cell cycle analysis. RESULTS: Curcumin resistance developed in cells with suppressed survivin, in contrast to the parental cells, as determined by survival assays. Cell cycle analysis and TUNEL assays revealed that the apoptotic cell population was increased in the scrambled-siRNA cells treated with curcumin compared with the survivin-siRNA cells. Suppression of survivin expression resulted in curcumin resistance via the modulation of Bcl-2 and Bax expression. CONCLUSIONS: We conclude that the expression levels of survivin may mediate the therapeutic efficacy of curcumin in HCC cells.

Silencing of glucose-regulated protein 78 (GRP78) enhances cell migration through the upregulation of vimentin in hepatocellular carcinoma cells.

BACKGROUND: Glucose-regulated protein 78 (GRP78) plays an important role in embryonic development and cancer progression. However, there is little information regarding the regulation of GRP78 in hepatocellular carcinoma (HCC) metastasis. METHODS: We used RNA silencing and cDNA expression vectors to manipulate target gene expression in HCC cells. The transwell migration assay and xCelligence biosensor system were applied to determine the proliferatory and migratory ability of the HCC cells. RESULTS: In this study, we found that GRP78 silencing enhanced cell migration in both HepJ5 and Mahlavu cells. Overexpressed GRP78 in skHep1 cells suppressed the migratory ability. In the insight mechanism dissection for GRP78-mediated cancer migration, we found that downregulation of GRP78 caused the increase of vimentin expression on HCC cells. Suppressed vimentin expression also decreased the migratory ability on HCC, indicating that vimentin expression levels modulated the cell migratory ability. CONCLUSION: We found that silencing GRP78 in HCC cells may enhance cell migration through the increase of vimentin expression.

Prevalence of advanced colonic polyps in asymptomatic Chinese.

AIM: To investigate the prevalence of advanced polyps in asymptomatic Chinese and to determine the risk of proximal advanced colonic polyps in subjects with and without polyps in the distal colon. METHODS: Data were collected prospectively during colonoscopic examinations performed in 5 973 subjects as part of health evaluation at our unit from December 1997 to December 2003. Polyps were considered advanced, if they were larger than 10 mm or were tubovillous, villous or malignant. Proximal colon was defined as the splenic flexure and more proximal portions of the colon. RESULTS: Colon polyps were detected in 971 (16.3%) subjects (613 males and 358 females) with their mean age being 56.6+/-10.7 years. Advanced polyps were noted in 199 (3.3%) individuals. Subjects were sub-classified according to the location of polyps into three groups: distal (569, 58.6%), proximal (284, 29.2%), and combined proximal and distal (118, 12.2%) groups. Subjects with advanced polyps in these three groups were 95 (9.8%), 56 (5.8%), and 48 (4.9%) respectively. In the 48 subjects with advanced combined polyps, 13 advanced polyps were distributed at the distal colon, 17 at the proximal colon, and 18 at both. Eighteen colon cancers including 12 at sigmoid and 6 at ascending colon were confirmed by final pathology. The relative risk for advanced proximal polyp according to distal findings was 3.1 (95%CI: 1.3-7.4) for hyperplastic polyp, 2.7 (95%CI: 1.4-5.3) for tubular polyp and 13.5 (95%CI: 5.1-35.4) for advanced polyp as compared to that for no polyp. However, 56 (28.2%) of 199 subjects with advanced polyps had no index polyps at the distal colon and might go undetected under sigmoidoscopic screening. CONCLUSION: Although distal lesions can predict the risk of advanced proximal polyps, a substantial portion of Chinese with advanced proximal polyps is not associated with any distal sentinel lesions. These data have implications for screening policy of colon cancers in Taiwanese Chinese.

Clinicopathologic significance of immunohistochemical fecal occult blood test in subjects receiving bidirectional endoscopy.

BACKGROUND/AIMS: Fecal occult blood test has been utilized to screen for lower gastrointestinal pathologies, such as colorectal cancer and polyps that bleed. Recent studies have revealed a relatively high frequency of upper gastrointestinal abnormalities in subjects with positive fecal occult blood by guaiac-based method. Although immunohistochemical tests of fecal occult blood were assumed to have greater diagnostic validity, the distribution of gastrointestinal pathology using such examinations is not well established. This study aims to investigate the efficacy of immunohistochemical analysis of fecal occult blood in detecting upper and lower gastrointestinal lesions in asymptomatic individuals. METHODOLOGY: Subjects who underwent regular health checkups were enrolled if they received both esophagogastroscopic and colonoscopic examinations. Each subject was tested by an immunohistochemical fecal occult blood test. The fecal occult blood results were evaluated and correlated with lesions identified in endoscopic examinations. RESULTS: In total 655 males and 722 females with age 46.2 +/- 12.1 years were enrolled, 287 cases (20.7%) had polypoid lesions of colon, including 6 colon cancers, 37 with polyps > or = 1 cm, 104 with polyp 5-9 mm, and 140 with polyp < 5 mm. FOB was positive in 31 cases, of which 15 (15/31, 48.4%) were polypoid lesions of colon, 1 was colonic ulcer, 9 (29.0%) were active gastroduodenal ulcers but 6 (19.4%) had no significant lesions. The positive and negative predictive value for colon polyps was 48.4% and 80%, respectively. The sensitivity was 50% (3/6) for colon cancer and varied among polyps with different sizes: 16.2% (6/37) for polyps > or = 1 cm; 5.8% (6/104) for polyps 5-9 mm and 0% (0/140) for polyps < 5 mm. CONCLUSIONS: A substantial portion of subjects (29%) with positive fecal occult blood reaction of immunohistochemical analysis but negative colonoscopy still needs esophagogastroscopic examination to disclose upper gastrointestinal lesions. Immunohistochemical determination of fecal occult blood remains imperfect for polypoid lesions of colon in view of its sensitivity and specificity.

Detection and treatment of an asymptomatic case of early esophageal cancer using chromoendoscopy and endoscopic mucosal resection.

Recent trends in the management of superficial esophageal cancer consist of improved detection and curative endoscopic therapy. However, successful endoscopic therapy has not been reported in Taiwanese patients with this disease. We describe the case of a male, 38-year-old habitual drinker admitted for a general health check-up, whose endoscopic examination revealed a slightly depressed discolored lesion in the middle esophagus. Chromoendoscopy with 3% Lugol's iodine solution showed a mesh-like unstained pattern occupying approximately two-thirds of the circumferential esophageal mucosa. Spraying with 2% toluidine blue solution stained a 3 x 6 cm suspect area pale blue. Endoscopic biopsy confirmed squamous cell carcinoma. Histopathologic examination revealed the lesion was a type IIc superficial esophageal cancer. Endoscopic ultrasonography showed the lesion was limited to the epithelial layer with no evidence of lymph node involvement. The lesion was removed en bloc using endoscopic mucosectomy. Microscopic examination of the resected specimen demonstrated that the depth of invasion was confined to the epithelial layer except for some areas with small nests of tumor cells within the lamina propria. Balloon dilatation to prevent post mucosectomy stricture was performed and the patient recovered uneventfully. At 1 year of follow-up, the patient was alive without any endoscopic signs of local recurrence. This case suggests that chromoendoscopy in combination with endoscopic resection is likely to benefit patients with early-stage esophageal cancer.

Pit pattern analysis by magnifying chromoendoscopy for the diagnosis of colorectal polyps.

BACKGROUND AND PURPOSE: The development of magnifying chromoendoscopy has facilitated the observation of mucosal pit patterns. This study investigated the value of this technology in predicting the histologic findings of colorectal lesions. METHODS: A total of 954 colorectal polyps were included. After identifying the lesions at colonoscopy, 0.2% indigocarmine solution was sprayed and then the zoom apparatus was switched to make a magnified view of the stained crypt orifice at a maximum 100 times magnification. The observed pit patterns were classified into 6 categories (I, II, IIIL, IIIS, IV, and V) according to Kudo's classification. Type I and II were designated as non-neoplastic patterns whereas other types were neoplastic. Correlation of the pit pattern with the findings of histologic examinations of resected or biopsied polyps was performed. RESULTS: There were 678 diminutive (