Effects of antioxidant co-supplementation therapy on spermatogenesis dysfunction in relation to the basal oxidation-reduction potential levels in spermatozoa: A pilot study.

Purpose: In this pilot study, the authors compared the effects of antioxidant co-supplementation therapy and methylcobalamin therapy in patients with impaired semen quality. Methods: Eighty-four subjects who visited male infertility clinics and showed abnormal semen test results were randomly subjected to one of the two therapies: antioxidant co-supplementation therapy with vitamin C, vitamin E, coenzyme Q10, and flaxseed oil or methylcobalamin therapy. The oxidation-reduction potential (ORP) and 8-hydroxy-2'-deoxyguanosine levels were used as indicators of oxidative stress levels in semen. Semen analysis was also performed. Results: The authors obtained results from 67 patients who had completed 3 months of treatment. Neither antioxidant co-supplementation therapy nor methylcobalamin therapy changed the semen parameters significantly (except for the sperm concentration, which was increased by the latter therapy). When the pre-treatment ORP value in semen was higher than the cutoff value, both therapies significantly increased the sperm concentration. The 8-hydroxy-2'-deoxyguanosine level did not yield any meaningful predictive value with regard to increased sperm concentrations. Conclusions: Both antioxidant co-supplementation therapy and methylcobalamin therapy increased the sperm concentration in patients with impaired semen quality when the basal ORP levels in their semen were elevated.

Androgen receptor mRNA expression is a predictor for recurrence-free survival in non-muscle invasive bladder cancer.

BACKGROUND: Non-muscular invasive bladder cancer (NMIBC) has a high risk of recurrence. As androgen receptor (AR) reportedly affects bladder cancer, we assessed the correlation between NMIBC recurrence and tumor AR expression in Japanese patients. METHODS: We retrospectively reviewed 53 specimens of non-metastatic NMIBC, with recurrence-free survival (RFS) as the primary endpoint. We used real-time quantitative polymerase chain reaction to quantify AR mRNA expression. Kaplan-Meier product-limit estimators were used to assess RFS distribution, log-rank tests to analyze differences in RFS between high- and low-risk groups; and multivariate analyses of AR mRNA expression and other clinicopathological factors to predict independent factors for RFS. RESULTS: The high AR mRNA-expressing group (n = 43) tended to have a longer median RFS (not reached) than did the low-AR group (n = 10; 9.04 months; P = 0.112). Multivariate analysis showed female sex (hazard ratio [HR]: 7.360, 95% CI: 1.649-32.856, P = 0.009), tumor size ≥3 cm (HR: 23.697, 95% CI: 4.383-128.117, P < 0.001) and low AR mRNA expression (HR: 0.202, 95% CI: 0.048-0.841, P = 0.028) to be independent predictors of shorter RFS. CONCLUSION: Our study showed that low AR mRNA expression level is an independent risk factor for RFS in Japanese patients with NMIBC. Further studies are necessary but AR expression might be a new indicator of recurrence of NMIBC.

PD-1 and PD-L1 are more highly expressed in high-grade bladder cancer than in low-grade cases: PD-L1 might function as a mediator of stage progression in bladder cancer.

BACKGROUND: Bladder cancers have been characterized as a tumor group in which the immunological response is relatively well preserved. Programmed death ligand 1 (PD-L1, B7-H1, CD274) has been shown to be expressed in several malignancies, including bladder cancer. However, the clinicopathological impact of this biomarker has not yet been established. In the present study, a quantitative real-time polymerase chain reaction (qPCR) was performed using paired normal and cancerous bladder cancer tissue to investigate PD-1/PD-L1 gene expression. METHODS: We examined the mRNA expression of PD-1/PD-L1 by a qPCR using 58 pairs of normal and cancerous human bladder tissue specimens. We also examined the correlation with the expressions of the STAT1 and NFAT genes, which are thought to be upstream and downstream of the PD-L1 pathway, respectively. RESULTS: There were no significant differences between normal and cancerous tissue in the expression of the PD-1 and PD-L1 genes (p = 0.724 and p = 0.102, respectively). However, PD-1 and PD-L1 were both more highly expressed in high-grade bladder cancer than in low-grade bladder cancer (p < 0.050 and p < 0.010). PD-L1 was positively correlated with the expressions of both the STAT1 (r = 0.681, p < 0.001) and the NFATc1 genes (r = 0.444. p < 0.001). CONCLUSIONS: PD-1 and PD-L1 might be a new biomarker that correlates with the pathological grade of bladder cancer. PD-L1 might function as a mediator of stage progression in bladder cancer and STAT1-NFAT pathway might associate this function.

Restoration of BRAK / CXCL14 gene expression by gefitinib is associated with antitumor efficacy of the drug in head and neck squamous cell carcinoma.

Clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase, has been shown in non-small-cell lung carcinoma patients with EGF receptor mutations, so these mutations are useful marker(s) to find a responder for the drug. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. We previously reported that the expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells was down-regulated by EGF treatment, and that forced expression of BRAK in tumor cells decreased the tumorigenicity of the cells in xenografts. Thus, we investigated the relationship between restoration of BRAK expression by gefitinib and the efficacy of the drug for tumor suppression. We found that EGF down-regulated BRAK expression through the MEK-extracellular signal regulated kinase pathway and that this down-regulated expression was restored by gefitinib in vitro. Oral administration of gefitinib significantly (P < 0.001) reduced tumor growth of xenografts of three HNSCC cell lines (HSC-2, HSC-3, and HSC-4), in female athymic nude mice, accompanied by an increase in BRAK expression specifically in tumor tissue. This tumor-suppressing effect of the drug was not observed in the case of BRAK non-expressing cells. Furthermore introduction of BRAK shRNA vector reduced both the expression levels of BRAK in HSC-3 cells and the antitumor efficacy of gefitinib in vivo. Our data showing an inverse relationship between BRAK expression levels in tumor cells and the tumor growth rate indicate that the gefitinib-induced increase in BRAK expression is beneficial for tumor suppression in vivo.

Angiotensin II induces oxidative stress in prostate cancer.

Angiotensin II has been shown to be a cytokine especially acting as a growth factor. A local renin-angiotensin system has been identified in the prostate gland, and the physiologic function of angiotensin II seems to be similar in prostate cancer, as we previously reported. In the present study, we explored the biological role of angiotensin II in oxidative stress of prostate cancer cells. Activated Akt was determined, and the expression of oxidative stress-related proteins (p47phox, manganese superoxide dismutase 2, glutathione peroxidase) was examined by Western blotting in LNCaP cells, which were stimulated with angiotensin II and/or an angiotensin II receptor type 1 blocker, candesartan. To examine DNA damage induced by angiotensin II, 8-hydroxy-2'-deoxyguanosine was determined, and Western blots were analyzed to detect checkpoint proteins including p53, Chk2, and cdc2. Immunocytochemical studies of inducible nitric oxide synthase and superoxide anion radical (O(2)(-)) were done in LNCaP cells stimulated with angiotensin II. The phosphorylation of Akt was induced by angiotensin II treatment and inhibited by candesartan, as well as by LY294002, an inhibitor of phosphoinositide 3-kinase. Oxidative stress-related proteins were up-regulated by angiotensin II and inhibited by pretreatment with candesartan or catalase. The level of 8-hydroxy-2'-deoxyguanosine was increased by angiotensin II and conversely decreased by candesartan. Immunocytochemical studies showed that angiotensin II enhanced an inflammatory marker, inducible nitric oxide synthase, and the production of O(2)(-) radical. The hypothesis that angiotensin II has the potential to induce oxidative stress, which may be implicated in carcinogenesis of the prostate gland through long-term exposure to chronic inflammation is proposed.

Expression of PD-L1 and CTLA-4 in female urethral carcinoma.

INTRODUCTION: Although the tumors are often easily detected, a considerable number of patients with female urethral carcinoma are diagnosed in an advance stage. Thus, no evidence-based therapeutic approach has been established. We herein report our experience in the treatment of three female patients with urethral carcinoma. We also examined the expression of PD-L1 and CTLA-4. CASE PRESENTATION: Three female patients pathologically diagnosed with urethral carcinoma, including urothelial carcinoma, squamous cell carcinoma, and adenocarcinoma, between 2013 and 2017 were analyzed in this study. Two patients underwent urethrectomy with cystostomy. Immunohistochemistry was performed to assess the levels of PD-L1 and CTLA-4 expression in patients with urethral carcinoma. Eleven control cases of urethral carcinoma tissue were also stained. CONCLUSION: This study revealed the expression of PD-L1 and CTLA-4 in female urethral carcinomas.

Anti-tumor effects of telomelysin for head and neck squamous cell carcinoma.

Telomelysin is a telomerase-specific replication-competent adenovirus with telomerase reverse transcriptase (hTERT) promoter, which has shown strong anti-tumor effects on a variety of human cancer cells. Human head and neck squamous cell carcinoma (HNSCC) cell lines and a murine HNSCC (NR-S1) model were used to investigate whether telomelysin (OBP-301) had a therapeutic efficacy for HNSCC. We examined the cell killing effects of telomelysin and the induction of tumor cell apoptosis by telomelysin in vitro. Based on these data, we examined whether telomelysin therapy produced therapeutic benefits in vivo. The results demonstrated that the treatment of telomelysin led to significant tumor regression on the side with subcutaneous NR-S1 tumor. We first confirmed the direct anti-tumor effect of intratumoral telomelysin injections in a murine HNSCC model. Further analyses of the augmented anti-tumor effects revealed that telomelysin increased the source of tumor antigens for immune cells, resulting in the induction of CD4+ and CD8+ T cells responsible for the in vivo tumor regression of treated and untreated tumors. Subsequently, an elevated IFN-gamma production of spleen cells was observed in mice treated with telomelysin. These results raise the possibility that telomelysin enhances the immune response in addition to its direct tumor cell killing activity. These findings suggest that telomelysin is a potent agent for the treatment of HNSCC patients with multiple metastases.

Involvement of EGFR in the response of squamous cell carcinoma of the head and neck cell lines to gefitinib.

Epidermal growth factor receptor (EGFR) gene mutations are associated with the sensitivity of non-small cell lung carcinomas (NSCLCs) to gefitinib, but such findings have not been reported in squamous cell carcinomas of the head and heck (SCCHNs). Accordingly, we determined whether EGFR gene expression and mutations correlate with the in vitro efficacy of gefitinib in SCCHN cell lines. EGFR status was analyzed in 16 different SCCHN cell lines by polymerase chain reaction (PCR) and direct sequencing for activating mutations, by real-time quantitative RT-PCR, and by Western blot analysis for RNA and protein expression. Using direct sequencing of PCR products from exons 18-23 of 9 SCCHN cell lines, we found a heterozygous EGFR mutation (EGFRmut) with a 2607Gright curved arrow A transition in exon 20 (G/A genotype). The 9 different cell lines that showed this mutation also showed higher sensitivity (lower IC50 values) to gefitinib than cell lines with wild-type EGFR (EGFRwt: G/G genotype) (p=0.016). EGFR protein levels correlated robustly (r=0.76) and significantly (p=0.0007) with EGFR mRNA levels and with IC50 values for gefitinib (r=0.65, p=0.0067). EGFR mRNA correlated with IC50 values (r=0.67, p=0.0046). Our conclusion was that the heterozygous and synonymous transition of the EGFR gene and low EGFR expression levels of mRNA and protein in SCCHN may be reliable predictors of high sensitivity in SCCHN patients to gefitinib.

Antitumor effect of gefitinib on head and neck squamous cell carcinoma enhanced by trastuzumab.

The overexpression of EGFR and/or HER-2 is associated with tumor cell resistance to chemotherapy, radiotherapy, disease progression and poor prognosis in patients with a variety of malignant tumors. Treatment combining the EGFR-targeting drug, gefitinib (ZD1839, Iressa) with the HER-2-targeting drug, trastuzumab (Herceptin) has been reported to improve therapeutic efficacy in patients with breast cancer. The purpose of this study was to examine the antitumor effect of this combination on head and neck squamous cell carcinoma (HNSCC) in vitro. Cell proliferation was inhibited significantly in two cell lines. Although IC50 of gefitinib alone against some cell lines was not reached, it was achieved after being combined with trastuzumab. Furthermore, IC50 was lower for the combination than for gefitinib alone in several cell lines. These results suggest that the combination may improve efficacy against HNSCC.

Correlation between FDG-PET findings and GLUT1 expression in salivary gland pleomorphic adenomas.

OBJECTIVE: One reason for the difficulty in accurate preoperative pathological diagnosis of major salivary gland tumors with fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) is the tendency of pleomorphic adenomas to have a high, standardized uptake value (SUV). The expression of glucose transporter 1 (GLUT1) and the quantity of GLUT1 messenger RNA (mRNA) were analyzed in specimens of pleomorphic adenoma to identify whether GLUT1 is responsible for the increased glucose uptake in FDG-PET examinations of these tumors. METHODS: Eighty salivary gland tumors resected at Yokohama City University Hospital were retrospectively investigated. FDG-PET was performed prior to surgery. PET images were evaluated by two experienced radiologists. GLUT1 was immunohistochemically stained, and GLUT1 mRNA density was quantified using real-time polymerase chain reaction in 10 of 40 pleomorphic adenomas. RESULTS: The pleomorphic adenomas stained positively for GLUT1, and there was significant correlation between the GLUT1 index and the SUV in FDG-PET. CONCLUSIONS: GLUT1 is expressed in salivary gland pleomorphic adenomas. Furthermore, the GLUT1 index shows significant correlation with the SUV of FDGPET. This result suggests that GLUT1 plays an important role in increasing FDG uptake in salivary gland pleomorphic adenomas.

Oxidative stress marker 8-hydroxyguanosine is more highly expressed in prostate cancer than in benign prostatic hyperplasia.

Oxidative stress is a primary cause of vascular endothelial damage. In the prostate, ischemia increases the levels of reactive oxygen species, growth factors and cytokines, and induces the development of angiogenesis, which results in cancer progression. The expression levels of an oxidative stress marker, 8-hydroxyguanosine (8-OHdG), were compared between prostate cancer and non-neoplastic prostate tissues. A prostate tissue microarray composed of 10 cases of prostatic adenocarcinoma and 70 cases of benign prostatic hyperplasia was immunohistochemically stained for 8-OHdG. All cases expressed 8-OHdG. The levels of 8-OHdG expression in prostatic cancer (30.0% moderate and 70.0% strong) were significantly higher than those in benign prostatic hyperplasia (71.4% moderate and 28.6% strong; (p<0.01). Notably, 8-OHdG is expressed more highly in prostate cancer tissues in comparison to benign prostate tissues.

Chemopreventive effects of angiotensin II receptor type 2 agonist on prostate carcinogenesis by the down-regulation of the androgen receptor.

We recently reported that angiotensin II receptor blockers (ARBs) have chemopreventive and chemotherapeutic potential against prostate cancer via the reduction of androgen receptor (AR) expression. In this study, we investigated the effects of the angiotensin II receptor type 2 (AT2R) agonist Compound 21 (C21), which is expected to play similar roles to an ARB, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model previously established in our laboratory. In vitro analyses of the cell growth, Western blotting and reporter gene assays were performed using LNCaP cells. TRAP rats at 6 weeks of age were randomly divided into 3 groups of 12 animals each and treated with C21 at 1 or 2 mg/kg/day in drinking water for 12 weeks. C21 reduced the proliferation activity of prostate cancer cells and down-regulated the PSA promoter activity and the AR protein expression. We discovered that C21 inhibited the progression of prostate carcinogenesis in TRAP rats and decreased the incidence of adenocarcinoma in the lateral prostate. A significant increase in the apoptotic index with activation of caspase 3 and 7 were observed by immunohistochemistry and Western blotting analyses. C21 also down-regulated the expression of AR significantly in TRAP rat prostate. C21 decreased the expression of AR and reduced the proliferation activity effectively in prostate cancer cells and TRAP rat prostate. These findings suggest that AT2R agonist may be a candidate novel chemopreventive agent against human prostate cancer.

Antitumor effects of ZD6474 on head and neck squamous cell carcinoma.

Angiogenesis is required for tumor growth and metastasis and, therefore, represents a target for cancer treatment. While many factors have been implicated in promoting angiogenesis, vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. ZD6474 is a potent VEGF receptor-2 (VEGFR-2) tyrosine kinase inhibitor which also has activity against the epidermal growth factor receptor (EGFR) tyrosine kinase. The purpose of this study was to investigate the sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to ZD6474, and to evaluate its antitumor efficacy on HNSCC xenografts. This is the first demonstration of antitumor effects of ZD6474 on HNSCC. In vitro ZD6474 displayed antiproliferative effects on HNSCC cells and inhibition of VEGFR-2 and EGFR pathways. In vivo ZD6474 displayed antitumor activity, induced apoptosis and antiangiogenic activity on nude mice bearing an established xenograft of YCU-H891 cells. These results suggest that ZD6474 has the potential to inhibit two key pathways in tumor growth via inhibition of VEGF-dependent tumor angiogenesis and via inhibition of EGFR-dependent tumor cell proliferation.

Anti-tumor effects of bevacizumab in combination with paclitaxel on head and neck squamous cell carcinoma.

Human tumors are dependent on angiogenesis for growth, and the vascular endothelial growth factor (VEGF) is a major regulator of this process. Bevacizumab (Avastin), a monoclonal antibody directed against VEGF, has shown promise in treating a variety of cancers. In this study, we first examined the anti-tumor effects of bevacizumab on head and neck squamous cell carcinoma (HNSCC). Then we examined the effects of bevacizumab combined with paclitaxel, a chemotherapeutic agent, in HNSCC. This is the first demonstration of the anti-tumor effects of bevacizumab on HNSCC. In vitro, bevacizumab did not show any antiproliferative effects against the HNSCC cell lines. However, in vivo, bevacizumab showed dramatic anti-tumor effects against HNSCC tumor xenografts in mice. In addition, treatment with a bevacizumab-paclitaxel combination resulted in a remarkable inhibition of the HNSCC tumor xenografts, compared to the effects of each agent separately. A decreased blood vessel density and an increased apoptotic index were seen in the shrunken tumors. These results suggest that bevacizumab in combination with paclitaxel could have useful clinical application in HNSCC.

Antitumor effects of Nafamostat mesilate on head and neck squamous cell carcinoma.

OBJECTIVE: Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, has antitumor activities toward adenocarcinoma, e.g., colon cancer. However, its antitumor effects on other types of cancer have been less extensively studied. We investigated the biological activities of Nafamostat mesilate on cell proliferation, cell-invasive potential and growth factor production in head and neck squamous cell carcinoma (HNSCC). METHODS: Two human HNSCC cell lines established in our department, YCU-L891 and -H891, and a human vulvar squamous cell carcinoma cell line, A431, were examined for the effect of Nafamostat mesilate. The effects on cell growth were evaluated using the MTT assay. The effects on the relative expression levels of mRNA were measured by quantitative RT-PCR. Cytokine secretion was analyzed by enzyme-linked immunosorbent assay. RESULTS: Nafamostat mesilate inhibited the proliferation of two HNSCC cell lines, YCU-L891 and YCU-H891, and A431. In these cell lines, Nafamostat mesilate down-regulated both matrix metalloproteinase (MMP)-2 and -9. In addition, it reduced the productions of vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1) by the tumor cells. CONCLUSION: Our results suggest that Nafamostat mesilate has potential for use as a treatment against local growth, invasion and metastasis of squamous cell carcinoma.

Antitumor effects of IDN5109 on head and neck squamous cell carcinoma.

Taxanes, a new class of antitumor drugs, are effective against a large number of human tumors, although there are problems with drug resistance. The novel taxane, IDN5109, is characterized by its high tolerability, antitumor efficacy, ability to overcome multidrug resistance, and oral bioavailabilty. We investigated the cellular response of IDN5109 to head and neck squamous cell carcinoma (HNSCC), and compared the antitumor activity of IDN5109 with that of paclitaxel. This is the first demonstration of antitumor effects of IDN5109 on HNSCC. In in vitro experiments, IDN5109 showed antiproliferative effects against HNSCC cell lines. After treatment with IDN5109, Bcl-2 and Bcl-XL were down-regulated, Bax was up-regulated, and caspase-3 was activated. After treatment with IDN5109, concentrations of both VEGF and IL-8 in the culture supernatant of HNSCC cells decreased. In in vivo experiments, the oral administration of IDN5109 showed antitumor effects against HNSCC tumor xenografts. Immunohistochemistry showed that IDN5109 inhibited tumor angiogenesis and induced apoptosis in HNSCC cells, producing a decreased blood vessel density and increased apoptosis index. On the basis of these results, IDN5109 is useful as a chemotherapeutic agent against HNSCC.

[Corrigendum] Antitumor effects of IDN5109 on head and neck squamous cell carcinoma.

Oncol Rep 15: [Related article:] 329­334, 2006; DOI: 10.3892/or.15.2.329 After the publication of the article, the authors noted that the relevance of the findings reported in this article on IDN5109 inhibition of growth of head and neck squamous cell carcinoma (HNSCC) is now in question. To examine the antitumor effect of IDN5109, this study was conducted using YCU-H891 and KCC-MS871 cell lines that the authors believed to be HNSCC cell lines. Because different human leukocyte antigen (HLA) was detected in 2 cell lines (KCC-TCM901 and KCC-T873) which were derived from the same patient in an experiment after publication, 16 cell lines established in our institution were analyzed by short tandem repeat (STR) analysis. STR analysis revealed that genotype of KCC-TCM901, YCU-H891 and KCC-MS871 was identical to that of HeLa cells, and that genotype of YCU-T891 was identical to that of YCU-L891, which was considered to be cross-contamination.

[Corrigendum] Antitumor effects of ZD6474 on head and neck squamous cell carcinoma.

Oncol Rep 17: [Related article:] 289­295, 2007; DOI: 10.3892/or.17.2.289 After the publication of the article, the authors noted that the relevance of the findings reported in this article on ZD6474 inhibition of growth of head and neck squamous cell carcinoma (HNSCC) is now in question. To examine the antitumor effect of ZD6474 in vitro and in vivo, this study was conducted using YCU-H891 cell line that the authors believed to be HNSCC cell line. Because different human leukocyte antigen (HLA) was detected in 2 cell lines (KCC-TCM901 and KCC-T873) which were derived from the same patient in an experiment after publication, 16 cell lines established in our institution were analyzed by short tandem repeat (STR) analysis. STR analysis revealed that genotype of KCC-TCM901, YCU-H891 and KCC-MS871 was identical to that of HeLa cells, and that genotype of YCU-T891 was identical to that of YCU-L891, which was considered to be cross-contamination.

Expression of SPARC in tongue carcinoma of stage II is associated with poor prognosis: an immunohistochemical study of 86 cases.

SPARC (secretory protein acidic and rich in cysteine), also known as osteonectin or BM-40, associates with progression in various kinds of tumors. We have examined whether SPARC expression can be a prognostic marker for patients with head and neck squamous cell carcinomas (HN-SCC). We examined immunolocalization of SPARC in 86 clinical specimens of tongue carcinoma. Although there was no correlation between SPARC positivity in the tumor cells and tumor stages, the 5-year overall survival rate was significantly lower in the SPARC positive cases (28.6%) than in the SPARC negative cases (91.7%), confined to stage II patients (p < 0.001, Wilcoxon test). Additionally, in stage II cases (n = 3), frequency of the postoperative metastasis was significantly higher in SPARC positive cases (5/8, 62.5%) than in the negative cases (1/15, 6.7%) (p < 0.01, chi2 test). Together with these results, SPARC can be a beneficial prognostic marker for the stage II tongue carcinoma, of which clinical outcomes are sometimes difficult to predict.