Expression and potential roles of sodium-potassium ATPase and E-cadherin in human gastric adenocarcinoma.

BACKGROUND: Gastric adenocarcinoma originates from an abnormal epithelium. The aim of this study was to investigate the expression of sodium-potassium ATPase (NKA), a transmembrane protein located in the epithelium for Na+ and K+ transportation, and E-cadherin, which are both crucial for the epithelium and adherens junction, as potential gastric cancer biomarkers. METHODS: 45 patients diagnosed with gastric adenocarcinoma were recruited. Immunohistochemistry and immunofluorescence were conducted to for localization of NKA α1-, ß1-isoform, and E-cadherin. NKA enzyme activity was determined by NADH-linked methods and immunoblotting of NKA α1-, ß1-isoform, and E-cadherin were performed to evaluate protein expression. RESULTS: Immunostaining revealed that NKA was co-localized with E-cadherin in the glands of the gastric epithelium. Both NKA activity and α1-isoform protein expression were reduced in the study group (P < 0.05), indicating impaired NKA functions. In the adherens junctions, the NKA ß1-isoform and E-cadherin were significantly reduced in the study groups (P < 0.05), indicating the adhesion force between cells may have been weakened. CONCLUSIONS: A significant decrease in NKA function (protein and activity) and E-cadherin in tumor lesions appear promising biomarker for gastric adenocarcinoma. Therefore, developing screening methods for detecting NKA function may be beneficial for the early diagnosis of gastric cancer. In our knowledge, this study was the first to investigate the NKA and E-cadherin expression in the relation of gastric adenocarcinoma in human patients.

Increased expression of the sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins.

Objectives Varicocele is characterized by dilatation and tortuosity of the internal spermatic vein. Sonic hedgehog plays an important role in angiogenesis and vascular remodeling under hypoxic stress. We studied the relationship and distribution of SHH and vascular endothelial growth factor in internal spermatic vein in patients diagnosed with varicocele. Methods Specimens of 1 cm were taken from the internal spermatic vein during left varicocele repair (N = 20). The control samples of ISV were obtained from eight male patients who underwent left inguinal herniorrhaphy. We analyzed the sonic hedgehog and vascular endothelial growth factor expression and distribution by immunoblotting, immunohistochemistry, immunofluorescent staining, and confocal laser scanning microscopy. The data were analyzed using the Student's t test. Results Immunoblotting showed higher expression of sonic hedgehog and vascular endothelial growth factor proteins in varicocele veins than in the control group ( P < 0.05) which located over muscle layer and endothelium was demonstrated by immunohistochemical staining. Both proteins with co-localization in the muscle layer and especially distributed in endothelium of varicocele veins were revealed under confocal microscopy. Conclusions These findings showed the upexpression of sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins which imply that the reducing hypoxia or using sonic hedgehog antagonists may be helpful for this vascular disease.

Differential Expression of Renal Outer Medullary K+ Channel and Voltage-gated K+ Channel 7.1 in Bladder Urothelium of Patients With Interstitial Cystitis/Painful Bladder Syndrome.

OBJECTIVE: To investigate the changes including expression and localization of 2 potassium channels, renal outer medullary K+ channel (ROMK) and voltage-gated K+ channel 7.1 (KCNQ1), after increased urinary potassium leakage in patients with interstitial cystitis/painful bladder syndrome (IC/PBS). MATERIALS AND METHODS: The study group included 24 patients with IC/PBS and a control group consisting of 12 volunteers without any IC/PBS symptoms. Bladder biopsies were taken from both groups. We determined the protein expression and distribution of potassium channels using immunoblotting, immunohistochemistry, and immunofluorescent staining under confocal laser microscopy. RESULTS: The results revealed that ROMK was predominantly expressed in apical cells of the bladder urothelium at significantly higher levels (3.3-fold) in the study group than in the control group. In contrast, KCNQ1 was expressed in the basolateral membrane according to confocal microscopy results and did not significantly differ between groups. CONCLUSION: Our data showed that the abundance of ROMK protein in apical cells was increased in the IC/PBS group, whereas KCNQ1, which was distributed in the basolateral membrane of the bladder urothelium, showed similar abundance between groups. These results suggest that upregulation of the ROMK channel in apical cells might permit avid potassium flux into the bladder lumen to maintain intracellular K+ homeostasis in the dysfunctional urothelium.

Impaired Na(+)/K(+)-ATPase Function in Patients with Interstitial Cystitis/Painful Bladder Syndrome.

Na(+)/K(+)-ATPase (NKA) is abundantly expressed in the basolateral membrane of epithelial cells, which is necessary for tight junction formation. The tight junction is an urothelial barrier between urine and the underlying bladder. Impairment of tight junctions allows migration of urinary solutes in patients with interstitial cystitis/painful bladder syndrome (IC/PBS). We evaluated NKA expression and activity in bladder samples from patients with IC/PBS. The study group consisted of 85 patients with IC/PBS, and the control group consisted of 20 volunteers. Bladder biopsies were taken from both groups. We determined the expression and distribution of NKA using NKA activity assays, immunoblotting, immunohistochemical staining, and immunofluorescent staining. The protein levels and activity of NKA in the study group were significantly lower than the control group (1.08 ± 0.06 vs. 2.39 ± 0.29 and 0.60 ± 0.04 vs. 1.81 ± 0.18 µmol ADP/mg protein/hour, respectively; P < 0.05). Additionally, immunofluorescent staining for detection of CK7, a marker of the bladder urothelium, predominantly colocalized with NKA in patients in the study group. Our results demonstrated the expression and activity of NKA were decreased in bladder biopsies of patients with IC/PBS. These findings suggest that NKA function is impaired in the bladders from patients with IC/PBS.

Metallothionein overexpression of bladder biopsies associated with tissue hypoxia in patients with interstitial cystitis/painful bladder syndrome.

OBJECTIVE: To study the relationship between hypoxia and metallothionein expression in bladder biopsies of interstitial cystitis/painful bladder syndrome patients. METHODS: The study group consisted of 41 patients with interstitial cystitis/painful bladder syndrome, and the control group consisted of 12 volunteers without any interstitial cystitis/painful bladder syndrome symptoms. All biopsy specimens were analyzed for both proteins of hypoxia-inducible factor-1α and metallothionein expression by immunoblotting, immunostaining and confocal laser scanning microscopy. Data were analyzed using the Mann-Whitney U-test. RESULTS: An increased expression of hypoxia-inducible factor-1α and metallothionein was noted in the study group compared with the control group (P < 0.05). Both proteins of hypoxia-inducible factor-1α and metallothionein mainly distributed over bladder urothelium by immunohistochemical staining, and showed co-localization under confocal microscopy. CONCLUSIONS: High expression and co-localization of metallothionein and hypoxia-inducible factor-1 alpha in the bladder mucosa of patients with interstitial cystitis suggest that overexpression of metallothionein is associated with the bladder hypoxia related to this disease.

Decreased expression of zonula occludens-1 and occludin in the bladder urothelium of patients with interstitial cystitis/painful bladder syndrome.

BACKGROUND/PURPOSE: Unique barrier properties of the urothelial surface membrane permit urine storage without contents leak into the bloodstream. Previous reports suggested that the bladder urothelial barrier might be compromised in interstitial cystitis/painful bladder syndrome (IC/PBS). We examined the changes of tight junction proteins (zonula occludens-1 (ZO-1) and occludin) in IC/PBS patients. METHODS: Bladder samples were derived from of 32 patients with IC/PBS and eight controls. We detected the tight junction proteins of ZO-1 and occludin expression by immunoblotting, immunohistochemical (IHC) staining and double immunofluorescent (IF) staining with confocal microscopy. Data were analyzed using the Mann-Whitney U-test. RESULTS: Expression of ZO-1 and occludin in the IC/PBS group was reduced compared to the control group by immunoblotting and IHC staining. Also, the thinning and denudation of urothelium were demonstrated in the IC/PBS group by histological study. IF staining showed the interruption of bladder urothelium in IC/PBS patients under confocal microscopy. CONCLUSION: Our data showed that decreased expression of tight junction proteins (ZO-1 and occludin) and interruption of bladder urothelium in IC/PBS patients. Treatment to repair the discontinuous urothelium may be useful to relieve some clinical symptoms of patients with IC/PBS.

Activation of extrinsic apoptotic pathway from bladder biopsy in patients with interstitial cystitis/painful bladder syndrome.

OBJECTIVE: To investigate the intrinsic or extrinsic pathway of apoptosis from bladder biopsy that was performed in interstitial cystitis/painful bladder syndrome (IC/PBS). Although previous studies have reported observations of the dysfunction, denudation, and thinning of the bladder urothelium in patients associated with an increase of cell apoptosis, the molecular mechanism is unclear. METHODS: The study group consisted of 32 patients with IC/PBS, and the control group consisted of 12 volunteers without any symptoms of IC. Bladder biopsies were obtained from both the groups. The expression of apoptosis-associated proteins was observed by detecting the Bcl-2/Bax ratio and the levels of cleaved caspase-9, Fas, cleaved caspase-8, and cleaved caspase-3 to differentiate intrinsic or extrinsic pathway. The data were analyzed using Mann-Whitney U test. RESULTS: Increased levels of cleaved caspase-3 were found in the IC/PBS group relative to the control group (P <.05). The levels of the extrinsic apoptotic pathway proteins, Fas and cleaved caspase-8, were also increased in the study group compared with the control group (P <.05). There was no significant difference in the levels of the intrinsic apoptotic pathway proteins, including cleaved caspase-9 and the Bcl-2/Bax ratio, between the control and study groups. CONCLUSION: Our findings demonstrate the activation of extrinsic apoptotic pathway from bladder biopsy in patients with IC/PBS. This study might help us to clarify the molecular changes and lead to a better understanding of this bladder disease.

Increased expression of hypoxia-inducible factor-1alpha and Bcl-2 in varicocele and varicose veins.

BACKGROUND: Primary vein wall abnormalities leading to secondary blood stasis and increased venous pressure that cause tissue hypoxia are observed in varicocele and varicose veins. Both types of diseased vessels are characterized by dilated thickened vein walls. Hypoxia upregulates Bcl-2 (antiapoptosis protein) expression in different human cell types. We studied the expression of hypoxia-inducible factor-1alpha (HIF-1α) and Bcl-2 in both venous diseases. METHODS: All vascular specimens, including the saphenous and internal spermatic veins, from patients with either varicocele or left inguinal herniorrhaphy (control group) were studied using immunoblotting, immunohistochemical staining, and double immunofluorescence staining. The data were analyzed using 1-way analysis of variance with Tukey comparison test. RESULTS: Protein analysis revealed that both venous diseases had a higher expression of HIF-1α and Bcl-2 compared with the control group (P < 0.05). Immunohistochemical staining and double immunofluorescence staining revealed that the greatest degree of HIF-1α and Bcl-2 colocalization occurred in the muscle layer of both diseased vessels. Moreover, under confocal microscopy, elevated Bcl-2 expression was found in the endothelium of both study groups compared with the control group. CONCLUSIONS: Our findings revealed increased expression of HIF-1α and Bcl-2 in varicocele and varicose veins and increased Bcl-2 expression especially in the endothelium under hypoxia. Thus, Bcl-2 overexpression may protect cells against apoptosis and contribute to the dilated thickened walls seen in both types of diseased vessels.

Increased extrinsic apoptotic pathway activity in patients with hepatocellular carcinoma following transarterial embolization.

AIM: To determine the apoptosis pathway in residual viable hepatocellular carcinoma (HCC) tissues following transarterial embolization (TAE). METHODS: Ten patients with HCC who received surgical resection after TAE were enrolled in the study group, and 24 patients with HCC who received surgical resection only served as the control group. In the study group, we measured the changes in tumor size and α fetoprotein (AFP) levels after TAE. All tissue samples were taken from the residual tumors. The expression of various apoptotic proteins was evaluated via immunoblotting procedures. The results were analyzed using a Student's t test. RESULTS: Tumor size and the AFP level decreased by 46.2% and 55.3% after TAE, respectively. There was no significant difference detected for the Bcl-2/Bax ratio or the cleaved caspase-9 expression levels in either group. However, extrinsic apoptopic pathway-related expression of Fas and cleaved caspase-8 expression were significantly higher in the study group than in the control group (P < 0.05). In addition, cleaved caspase-3 expression in the study group was higher (1.62-fold) than in control group (P < 0.05). CONCLUSION: TAE is an effective palliative therapy that decreases tumor size and AFP levels via an increase in extrinsic apoptosis pathway in patients with unresectable HCC.

Increased expression of hypoxia-inducible factor-1α and vascular endothelial growth factor associated with glomerulation formation in patients with interstitial cystitis.

OBJECTIVE: To examine whether hypoxia occurs in the bladders of patients with interstitial cystitis (IC) by monitoring the expression of hypoxia-inducible factor-1 (HIF-1) and VEGF. Previous studies have reported that bladder perfusion is decreased in patients with IC. Hypoxia induces overexpression of vascular endothelial growth factor (VEGF), which has been reported to be associated with the formation of glomerulations in patients with IC. METHODS: The study group consisted of 32 patients with IC, and the control group consisted of 8 volunteers. We obtained bladder biopsies from both groups and studied the expression of HIF-1α and VEGF proteins by immunoblotting and immunohistochemical and double immunofluorescent staining. Data were analyzed using the Mann-Whitney U test. RESULTS: Immunoblotting and immunostaining revealed that the expression of HIF-1α and VEGF proteins was increased in the study group compared with the control group. The relative intensities of HIF-1α and VEGF proteins were 60.60 ± 7.81 and 43.60 ± 5.37 in the study group and 26.20 ± 4.72 and 20.25 ± 1.45 in the control group, respectively. The overexpression of VEGF in study group biopsies was particularly evident in umbrella cells examined by confocal microscopy. CONCLUSION: Our findings identified increased expression of HIF-1α in bladder tissue and overexpression of VEGF in umbrella cells from patients with IC. These events may be associated with glomerulation formation during hydrodistention in IC bladders. Thus, these molecular findings could offer the therapeutic mechanism for hyperbaric oxygenation application to patients with IC.

Involved intrinsic apoptotic pathway in the varicocele and varicose veins.

BACKGROUND: Disordered programmed cell death may play a role in the development of venous diseases. Tissue hypoxia caused by blood stagnation and venous hypertension is the similar etiology of varicocele and varicose veins. We studied the vascular histopathology and determined whether there is the same apoptotic pathway in both venous diseases. METHODS: The study groups consisted of 1-cm venous segments obtained from 10 patients during vascular stripping surgery for varicose saphenous vein and 1 cm of internal spermatic veins obtained from 12 patients during left varicocele repair. The control samples of 1 cm internal spermatic vein were obtained from 10 male patients who underwent left inguinal herniorrhaphy. The three layers of vascular histology were measured and compared by Masson trichrome stain, and the apoptotic proteins including Bcl-2, Fas, cleaved caspase-9, cleaved caspase-8, and cleaved caspase-3 were detected. Data were analyzed using the one-way analysis of variance with Tukey's comparison test. RESULTS: The relative thickness of intima and adventitia layer was smaller in both study groups than in the control group. But a significant hypertrophy of media layer was observed in the varicocele and varicose veins than in the control group (p < 0.05). Overexpression of Bcl-2 and decreased expressions of cleaved caspase-9 and cleaved caspase-3 was observed in both study groups. There is no statistical difference in Fas and cleaved caspase-8 expressions in the control and study groups. CONCLUSION: Our data showed vascular smooth muscle hypertrophy in the diseased vessels. The same dysregulation of apoptosis through intrinsic pathway was demonstrated in varicocele and varicose veins under tissues hypoxia. This mechanism of reduced apoptosis might contribute to the dilated and thickened walls of both venous diseases.

Dysregulated apoptosis through the intrinsic pathway in the internal spermatic vein of patients with varicocele.

BACKGROUND/PURPOSE: Apoptosis plays a critical role in various physiological processes. Varicocele is the most common cause of male infertility in adults. The dilated and thickened wall of the internal spermatic vein (ISV) in varicocele is considered similar to that in varicose veins. We investigated apoptotic protein expression in the ISV of varicocele, including Bcl-2, Fas, caspase-8 and caspase-9, to determine the intrinsic or extrinsic pathway. METHODS: The study group consisted of 10 patients with grade 3 left varicocele. The control group consisted of 10 patients with left indirect inguinal hernia. A 1-cm section of ISV was resected, using left inguinal incision, from each patient in both groups. The ISV sections were used to detect the mediators that regulate the intrinsic (Bcl-2 and caspase-9) and extrinsic (Fas and caspase-8) apoptotic pathways, by immunoblotting and immunohistochemical staining. Results were analyzed using Student's t tests. RESULTS: Bcl-2, Fas, caspase-8 and caspase-9 immunoblots from both groups revealed a single band. The relative intensities of the Bcl-2 and caspase-9 protein bands differed significantly between the varicocele and control groups. Thickening of the smooth muscle layer of the ISV was found in patients with varicocele compared with the control group. Bcl-2 overexpression and downregulation of caspase-9 expression were noted in the varicocele group. There was no significant difference in Fas or caspase-8 expression in either group. CONCLUSION: We showed overexpression of Bcl-2 and downregulation of caspase-9 expression in the ISV under hypoxic stress. This indicated dysregulated apoptosis through the intrinsic pathway in the ISV of patients with varicocele. To the best of our knowledge, this is the first study of the apoptotic pathway in the human ISV. Additional studies are needed to establish whether adjunctive hyperbaric oxygen therapy reduces the recurrence rate after varicocelectomy.

Cadmium concentration and metallothionein expression in prostate cancer and benign prostatic hyperplasia of humans.

BACKGROUND/PURPOSE: Cadmium (Cd) causes various genitourinary disorders and is a carcinogen for prostate cancer. Metallothionein (MT) is a protein that detoxifies heavy metals. We evaluated changes in Cd concentration and MT expression in human prostate cancer (CaP) and benign prostatic hyperplasia (BPH). Our goal was to clarify the relationship between Cd concentration and MT expression in prostatic diseases. METHODS: The experimental group consisted of 18 patients who underwent radical prostatectomy for CaP. The control group consisted of 35 patients who underwent transurethral resection of the prostate for BPH. Tissue samples were acquired from the gross tumor site and from resected chips. We determined Cd concentration by atomic absorption, MT expression by immunoblotting, and immunohistochemical staining. The significance of between-group differences for these outcomes was analyzed using Student's t tests. RESULTS: There was no statistically significant difference in Cd concentration between the CaP and BPH groups. Immunoblots from both groups revealed a single band. The relative intensity of the MT band was 0.58 +/- 0.09 in the BPH group and 0.17 +/- 0.03 in the CaP group. MT expression in patients with BPH was 3.4-fold higher than in those with CaP. CONCLUSION: MT may bind heavy metals and protect patients from CaP. Additional studies are needed to reveal the factors that influence the expression of MT in prostate epithelial cells, and to analyze the free and compound forms of Cd at the same time.

Cadmium accumulation and metallothionein overexpression in internal spermatic vein of patients with varicocele.

OBJECTIVES: To determine the possible molecular mechanism for the thickened wall in the internal spermatic vein (ISV) of patients with varicocele, we examined the cadmium (Cd) content and metallothionein (MT) expression in these diseased vessels. Previous studies have shown that Cd might play a role in the etiology of varicocele-associated infertility. MT, a metal-binding protein, protects against cell apoptosis during hypoxia. METHODS: The study group consisted of 20 patients with grade 3 left varicocele. The control group consisted of 15 volunteers with left-sided indirect inguinal hernia. Through a left inguinal incision, a 1-cm section of the ISV was resected from each patient to measure the Cd and MT levels. The results were analyzed using Student's t test. RESULTS: The Cd content in the ISV was 59.84 +/- 5.7 ng/g in the control group and 192.1 +/- 24.2 ng/g in the varicocele group. The relative intensity of the MT band was 40.52 +/- 3.74 in the control group and 78.26 +/- 5.61 in the varicocele group. MT expression was greater in the varicocele group than in the control group, and its deposition in the vascular endothelial layer was predominant using immunohistochemistry staining and confocal laser scanning. CONCLUSIONS: The results of the present study have demonstrated a greater accumulation of Cd in the ISV of the varicocele group than in the control group. The high Cd content and hypoxic conditions would induce overexpression of MT in the diseased vessels to protect the vascular cells from apoptosis. This might be a mechanism for the thickened wall of the ISV in patients with varicocele.

Involved intrinsic apoptotic pathway of testicular tissues in varicocele-induced rats.

INTRODUCTION: Increased testicular germ cell apoptosis has been reported in varicocele-induced rats. We studied intrinsic or extrinsic pathway of apoptosis by detecting Bcl-2, caspase-9, caspase-8, and activated caspase-3 expressions in the bilateral testes of experimental varicocele-induced rats. MATERIALS AND METHODS: Experimental left varicocele (ELV) was created by partial ligation of left renal vein in a study group of 24 adult male Sprague-Dawley rats. The other 24 rats were as control group. Eight rats from each group were killed at 4, 8, and 12 weeks following varicocele creation. Testicular tissues of both groups were sampled for TUNEL assay and immunoblotting. RESULTS: Increased apoptotic germ cell was found in the ipsilateral testis of varicocele group at 8 and 12 weeks after operation (P < 0.05). Increased activated caspase-3 expression in the contralateral (right) testis was noted at 12 weeks following varicocele creation (P < 0.05). CONCLUSIONS: Our study demonstrates down-regulation of Bcl-2 expression and increased expressions of caspase-9 and activated caspase-3 in the ipsilateral testis of ELV rats at 8 and 12 weeks, indicating gradually increased testicular tissues apoptosis through the intrinsic pathway in varicocele-induced rats. Simultaneously, increased apoptosis in the contralateral testis was observed at 12 weeks (P < 0.05) following varicocele creation also.

Bcl-2 overexpression in the internal spermatic vein of patients with varicocele.

BACKGROUND/PURPOSE: Varicocele is characterized by dilatation and tortuosity of the internal spermatic vein (ISV). Cross sections of ISV showed marked thickening of smooth muscle layer under microscopy. This study examined Bcl-2 (anti-apoptotic protein) expression in the ISV of patients with varicocele. METHODS: The study group consisted of eight patients with left varicocele, grade 3. The control group consisted of six volunteers with left indirect inguinal hernia. Using a left inguinal surgical incision, a 1-cm section of ISV was resected from each patient in both groups as specimens for immunoblotting and immunohistochemical staining of Bcl-2. Results were analyzed using Student's t test. RESULTS: Bcl-2 immunoblots from both groups revealed one single band. alpha-tubulin was used as loading control. The average relative intensity of the Bcl-2 was 25.82 +/- 10.53 in the control group and 113.49 +/- 27.49 in the varicocele group. Hematoxylin staining revealed a thickening ISV in the patients with varicocele, predominantly in the muscle layer, which was not found in the control group. Moreover, the intensity of Bcl-2 immunostaining was markedly higher in the study group than in the control group. CONCLUSION: This study showed Bcl-2 overexpression in the ISV of patients with varicocele. Additional studies are necessary to clarify the molecular mechanisms of varicocele formation and recurrence.

Increased expression of hypoxia-inducible factor-1alpha in the internal spermatic vein of patients with varicocele.

PURPOSE: Varicocele is recognized as a cause of male infertility. Testis hypoxia may be one of the possible mechanisms of varicocele. We examined whether tissue hypoxia occurred in the ISV of patients with varicocele by detecting the expression of HIF-1alpha. MATERIALS AND METHODS: The study group consisted of 8 patients with grade 3 left varicocele. The control group consisted of 6 volunteers with left indirect inguinal hernia. Using a left inguinal surgical incision, a 1 cm section of ISV was resected from each patient in both groups as specimens for immunoblotting and immunohistochemical staining of HIF-1alpha. Results were analyzed using Student's t test. RESULTS: HIF-1alpha immunoblots from both groups revealed a single band. The relative intensity of the HIF-1alpha protein band was 10.92 +/- 2.70 in the control group and 73.15 +/- 8.93 in patients with varicocele (ie 7-fold higher). CONCLUSIONS: HIF-1alpha expression in the ISV of patients with varicocele was significantly higher than in the control group. This directly shows that hypoxia related pathophysiological changes have occurred in the ISV of patients with varicocele and that hypoxia may have also occurred in the testicular tissue. Thus, it would be of interest to investigate whether decreasing HIF-1alpha activation and testis hypoxia could reduce the recurrence of varicocele. To our knowledge, this is the first report on HIF-1alpha expression in human ISV. Additional studies will be necessary to clarify the relationship between testis hypoxia and male infertility in patients with varicocele.