Combined Inhibition of Rad51 and Wee1 Enhances Cell Killing in HNSCC Through Induction of Apoptosis Associated With Excessive DNA Damage and Replication Stress.

Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.

COTI-2, A Novel Thiosemicarbazone Derivative, Exhibits Antitumor Activity in HNSCC through p53-dependent and -independent Mechanisms.

PURPOSE: TP53 mutations are highly prevalent in head and neck squamous cell carcinoma (HNSCC) and associated with increased resistance to conventional treatment primarily consisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents an attractive therapeutic approach and has been explored in recent years. In this study, the efficacy of a putative p53 reactivator called COTI-2 was evaluated in HNSCC cell lines with different TP53 status.Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of HNSCC cell lines with either wild-type, null, or mutant TP53 to COTI-2 alone, and in combination with cisplatin and/or radiation. Western blotting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and apoptosis analyses were performed to dissect molecular mechanisms. RESULTS: COTI-2 decreased clonogenic survival of HNSCC cells and potentiated response to cisplatin and/or radiation in vitro and in vivo irrespective of TP53 status. Mechanistically, COTI-2 normalized wild-type p53 target gene expression and restored DNA-binding properties to the p53-mutant protein in HNSCC. In addition, COTI-2 induced DNA damage and replication stress responses leading to apoptosis and/or senescence. Furthermore, COTI-2 lead to activation of AMPK and inhibition of the mTOR pathways in vitro in HNSCC cells. CONCLUSIONS: COTI-2 inhibits tumor growth in vitro and in vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cisplatin or radiation may be highly relevant in treating patients with HNSCC harboring TP53 mutations.

Head and neck cancer organoids established by modification of the CTOS method can be used to predict in vivo drug sensitivity.

OBJECTIVES: Currently there are no standard biomarkers of head and neck squamous cell carcinoma (HNSCC) response to therapy. This is, due to a lack of adequate predictive tumor models. To this end, we established cancer organoid lines from individual patient's tumors, and characterized their growth characteristics and response to different drug treatments with the objective of using these models for prediction of treatment response. MATERIALS AND METHODS: Forty-three patients' samples were processed to establish organoids. To analyze the character of these organoids, immunohistochemistry, Western blotting, drug sensitivity assays, clonogenic survival assays, and animal experiments were performed. The HPV status and TP53 mutational status were also confirmed in these lines. RESULTS: HNSCC organoids were successfully established with success rate of 30.2%. Corresponding two-dimensional cell lines were established from HNSCC organoids at higher success rate (53.8%). These organoids showed similar histological features and stem cell, epithelial and mesenchymal marker expression to the original tumors, thus recapitulating many of the characteristics of the original tumor cells. The cisplatin and docetaxel IC50 were determined for HNSCC organoids and the corresponding 2D cell lines using drug sensitivity and clonogenic survival assays. Responses to drug treatment in vivo were found to be similar to the IC50 calculated from organoids by drug sensitivity assays in vitro. CONCLUSION: We established novel in vitro HNSCC cancer organoid lines retaining many properties of the original tumors from they were derived. These organoids can predict in vivo drug sensitivity and may represent useful tools to develop precision treatments for HNSCC.

Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cells by targeting the transcription factors FOXO3a and FOXM1.

Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that promote cancer cell invasive growth and metastasis, yet the mechanisms mediating these functions still largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances the invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in in vitro three-dimensional culture and in an in vivo orthotopic nude mouse model of HNSCC through a novel transcription-independent mechanism. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the AMP-activated protein kinase (AMPK)-mediated phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. This downregulation of FOXO3a's activity, in turn, leads to de-repression of FOXM1 expression. Importantly, we show that either overexpression of FOXO3a or downregulation of FOXM1 impairs both GOF mutant p53-mediated cell invasion in vitro and pulmonary metastases of UM-SCC-1 cells in vivo. Finally, not only do oral cancer patients with p53 mutations exhibit higher levels of FOXM1 expression than patients with wild-type p53, but also HNSCC patients with TP53 mutations and high levels of FOXM1 expression have the poorest survival outcomes. Given our prior demonstration that GOF mutant p53s inhibit AMPK, our current study, establishes and demonstrates a novel transcription-independent GOF mutant p53-AMPK-FOXO3a-FOXM1 signaling cascade that plays an important role in mediating mutant p53s' gain-of-function activities in HNSCCs.

Replication Stress Leading to Apoptosis within the S-phase Contributes to Synergism between Vorinostat and AZD1775 in HNSCC Harboring High-Risk TP53 Mutation.

Purpose: The cure rate for patients with advanced head and neck squamous cell carcinoma (HNSCC) remains poor due to resistance to standard therapy primarily consisting of chemoradiation. As mutation of TP53 in HNSCC occurs in 60% to 80% of non-HPV-associated cases and is in turn associated with resistance to these treatments, more effective therapies are needed. In this study, we evaluated the efficacy of a regimen combining vorinostat and AZD1775 in HNSCC cells with a variety of p53 mutations.Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of high-risk mutant p53 HNSCC cell lines to vorinostat in combination with AZD1775. Cell cycle, replication stress, homologous recombination (HR), live cell imaging, RNA sequencing, and apoptosis analyses were performed to dissect molecular mechanisms.Results: We found that vorinostat synergizes with AZD1775 in vitro to inhibit growth of HNSCC cells harboring high-risk mutp53. These drugs interact synergistically to induce DNA damage, replication stress associated with impaired Rad51-mediated HR through activation of CDK1, and inhibition of Chk1 phosphorylation, culminating in an early apoptotic cell death during the S-phase of the cell cycle. The combination of vorinostat and AZD1775 inhibits tumor growth and angiogenesis in vivo in an orthotopic mouse model of oral cancer and prolongs animal survival.Conclusions: Vorinostat synergizes with AZD1775 in HNSCC cells with mutant p53 in vitro and in vivo A strategy combining HDAC and WEE1 inhibition deserves further clinical investigation in patients with advanced HNSCC. Clin Cancer Res; 23(21); 6541-54. ©2017 AACR.

Use of NRP1, a novel biomarker, along with VEGF-C, VEGFR-3, CCR7 and SEMA3E, to predict lymph node metastasis in squamous cell carcinoma of the tongue.

Lymph node (LN) metastasis has been suggested as a major prognostic factor for oral cancer. Knockdown of the growth factors and receptors involved in these metastatic mechanisms could significantly reduce LN metastasis and improve the survival of oral cancer patients after treatment. The present study, therefore, aimed to evaluate the expression levels of the following growth factors and receptors in squamous cell carcinoma (SCC) of the tongue: the vascular endothelial growth factor (VEGF)­C and VEGF­D, which bind to the cell surface tyrosine kinase receptor VEGF receptor­3 (VEGFR­3); C­C motif chemokine receptor 7 (CCR7); neuropilin (NRP)1 and NRP2; and semaphorin 3E (SEMA3E). Furthermore, we assessed microvessel density (MVD) and lymphatic vessel density (LVD) to demonstrate the correlation between these factors and regional LN metastasis, with respect to the clinicopathological features. Finally, we analyzed the correlation between these proteins and overall or disease­free survival, in order to demonstrate their prognostic value. Univariate analysis revealed a significant association between LN metastasis and the expression levels of VEGF­C, VEGFR­3, CCR7, NRP1, and SEMA3E, as well as LVD, in SCC cells. In contrast, multivariate analysis identified associations between LN metastasis and NRP1 expression, as well as between LN metastasis and LVD; however, no correlation was found between LN metastasis and the expression levels of the other proteins. The expression levels of VEGF­C, VEGFR­3, NRP1, and SEMA3E, as well as LVD, were correlated with disease­free survival time. These results indicate that LN metastasis is associated with poor survival in SCC. This study suggests that NRP1 expression and LVD are independent factors that are likely to predict the risk of LN metastasis in SCC of the tongue, whereas the expression of VEGF­C, VEGFR­3, CCR7, and SEMA3E are non­independent predictive factors.

Comparative study of ¹8F-FDG-PET/CT imaging and serum hTERT mRNA quantification in cancer diagnosis.

We have reported on the clinical usefulness of human telomerase reverse transcriptase (hTERT) mRNA quantification in sera in patients with several cancers. Positron emission tomography-computed tomography (PET/CT) using ¹8F-fluorodeoxyglucose (FDG) has recently become an excellent modality for detecting cancer. We performed a diagnostic comparative study of FDG-PET/CT and hTERT mRNA quantification in patients with cancer. Four hundred seventy subjects, including 125 healthy individuals and 345 outpatients with cancer who had received medical treatments for cancer in their own or other hospitals, were enrolled. The subjects were diagnosed by FDG-PET/CT, and we measured their serum hTERT mRNA levels using real-time RT-PCR, correlating the quantified values with the clinical course. In this prospective study, we statistically assessed the sensitivity and specificity, and their clinical significance. hTERT mRNA and FDG-PET/CT were demonstrated to be correlated with the clinical parameters of metastasis and recurrence (P < 0.001), and of recurrence and tumor number in cancer compared with noncancer patients, respectively. A multivariate analysis showed a significant difference in the detection by FDG-PET/CT, ¹8F-FDG uptake, the detection by hTERT mRNA, and age. The use of both FDG-PET/CT and hTERT mRNA resulted in a positivity of 94.4% (221/234) for the detection of viable tumor cells. FDG-PET/CT is superior to hTERT mRNA quantification in the early detection of cancer and combinative use of FDG-PET/CT and hTERT mRNA may improve the diagnostic accuracy of cancer.

Wee-1 Kinase Inhibition Sensitizes High-Risk HPV+ HNSCC to Apoptosis Accompanied by Downregulation of MCl-1 and XIAP Antiapoptotic Proteins.

PURPOSE: Although the majority of patients with HPV(+) oropharyngeal cancers have a favorable prognosis, there are some patients with tumors that are resistant to aggressive chemoradiotherapy with unusual patterns of locoregional and systemic recurrences. Therefore, more effective therapies are needed. In this study, we investigated the chemosensitizing efficacy of the selective Wee-1 kinase inhibitor, AZD-1775, in HPV(+) head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: Clonogenic survival assays and an orthotopic mouse model of HPV(+) oral cancer were used to examine the in vitro and in vivo sensitivity of HPV(+) HNSCC cell lines to AZD-1775 in combination with cisplatin, respectively. Cell-cycle analysis, DNA damage (γH2AX), homologous recombination (HR), and apoptosis were examined to dissect molecular mechanisms. RESULTS: We found that AZD-1775 displays single-agent activity and enhances the response of HPV(+) HNSCC cells to cisplatin both in vitro and in vivo. The sensitivity of the HPV(+) HNSCC cells to AZD-1775 alone or in combination with cisplatin was associated with G2 checkpoint abrogation, persistent DNA damage, and apoptosis induction. This finding of AZD-1775 increasing the sensitivity of HPV(+) HNSCC cells to cisplatin through apoptosis was not seen previously in the HPV(-) HNSCC cancer cells and is accompanied by a decreased expression of the antiapoptotic proteins, MCl-1and XIAP, which appear to be cleaved following AZD-1775 treatment. CONCLUSIONS: AZD-1775 selectively sensitizes HPV(+) HNSCC cells and orthotopic oral xenografts to cisplatin through apoptosis and support the clinical investigation of AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV(+) HNSCC tumors.

Gain-of-function mutant p53 promotes cell growth and cancer cell metabolism via inhibition of AMPK activation.

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined. We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells. Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth. Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation. Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function.

A metabolomics-based approach for predicting stages of chronic kidney disease.

Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.3% accuracy (range, 73.9-87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.

Exploration of novel predictive markers in rat plasma of the early stages of chronic renal failure.

To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.

Impact of CYP3A5*3 on plasma exposure and urinary excretion of fentanyl and norfentanyl in the early postsurgical period.

BACKGROUND: The pharmacokinetic characteristics of intravenous fentanyl have not been fully clarified in the early postsurgical period. The aim of this study was to evaluate the plasma exposure and urinary excretion of fentanyl and norfentanyl according to cytochrome P450 (CYP) 3A5 genetic polymorphism. METHODS: Fifty-two adult Japanese postoperative patients receiving a continuous intravenous fentanyl infusion were enrolled. Plasma concentrations of fentanyl and norfentanyl were determined at 24 hours after the operation, and their urinary excretion from 12 to 36 hours was evaluated. RESULTS: Plasma concentrations of fentanyl normalized for infusion rate were significantly higher in the *3/*3 group than in the *1 carrier group. The plasma concentration ratio of norfentanyl to fentanyl was significantly lower in the *3/*3 group than in the *1 carrier group. Urinary excretion rates of fentanyl and norfentanyl were 4.4% and 71%, respectively, and no significant differences were observed between the CYP3A5 genotypes. Renal clearance ratios of fentanyl and norfentanyl to creatinine were 0.34 and 3.4, respectively. There were no significant differences in the renal clearance ratios between the genotypes. Free fractions of fentanyl and norfentanyl in human plasma were 4.9% and 95%, respectively. Total and nonrenal clearance of fentanyl were significantly lower in the *3/*3 group than in the *1 carrier group. CONCLUSIONS: CYP3A5*3 affected the plasma exposure of fentanyl but not urinary excretion in our postoperative patients. The renal clearance ratios of fentanyl and norfentanyl to creatinine were much higher than their free fractions in plasma. These findings suggest a slight contribution of renal tubular secretion of fentanyl and norfentanyl to their plasma exposures.

Effect of a nitric oxide synthase inhibitor and a CXC chemokine receptor-4 antagonist on tumor growth and metastasis in a xenotransplanted mouse model of adenoid cystic carcinoma of the oral floor.

Nitric oxide (NO) is related to angiogenesis and tumor progression and chemokine receptor-4 (CXCR4) plays a central role in cell migration in metastasis and dissemination of cancer. The present study evaluated the effectiveness of a NOS inhibitor and a CXCR4 antagonist, given as single agents or in combination, in a xenotransplanted mouse model of adenoid cystic carcinoma (ACC) of the oral floor. A metastatic tumor (ACCIM) derived from a cervical metastatic lesion of human ACC that was transplantable in nude mice was used. ACCIM showed a high frequency of spontaneous metastasis to the lung when transplanted subcutaneously in nude mice. Mice with subcutaneous transplants of ACCIM were subdivided into six groups and intraperitoneally received one of the following treatments daily for 5 weeks: a) PBS (control), b) AMD3100 (CXCR4 antagonist), c) L-NAME (NOS inhibitor), d) 1400W (iNOS inhibitor), e) both AMD3100 and L-NAME (AMD3100+L-NAME) and f) both AMD3100 and 1400W (AMD3100+1400W). Tumor growth was evaluated during treatment and metastasis was assessed at 28 weeks. Single-agent treatment with AMD3100, L-NAME or 1400W inhibited tumor growth by 20.8, 26.5 and 54.5%, respectively. Combined treatment with AMD3100+L-NAME and AMD3100+1400W inhibited tumor growth remarkably by 48.0 and 50.2%, respectively. Immunohistochemical analysis revealed lower expression of CXCR4, iNOS and eNOS in tumor cells treated with AMD3100+L-NAME or AMD3100+1400W compared to control tumor cells and increased numbers of apoptotic tumor cells were demonstrated using the TUNEL method. CXCR4 expression decreased in 1400W-treated tumors using western blot analysis. When the effect of each agent on tumor-induced angiogenesis in tumor stroma was examined histologically, microvessel density was significantly lower in the groups treated with 1400W, AMD3100+L-NAME or AMD3100+1400W compared to the control, AMD3100 and L-NAME groups. Moreover, treatment with AMD3100 or 1400W markedly inhibited lung metastasis. Our results indicated that single-agent treatment with 1400W and combined treatment with AMD3100+L-NAME or AMD3100+1400W induced apoptosis and significantly inhibited tumor-induced angiogenesis and proliferation of ACCIM in vivo. Blockade of CXCR4 and iNOS was suggested to inhibit lung metastases from ACCIM. CXCR4 and iNOS may, thus, be important prognostic factors for long-term survival in ACC.

Inferior alveolar nerve regeneration after bifocal distraction osteogenesis in dogs.

PURPOSE: Bifocal distraction osteogenesis has been shown to be a reliable method for reconstructing missing bone segments. However, no reports have been published regarding inferior alveolar nerve regeneration during this procedure. We assumed that the nerve could regenerate with the bone regeneration during bifocal distraction, if the nerve had been saved at a mesial site of the transport disc. In the present study, we investigated that possibility in dogs. MATERIALS AND METHODS: Using a bifocal distraction osteogenesis method, we produced a 10-mm mandibular defect, including the nerve defect, and distracted the transport disc at a rate of 1 mm/day in 12 dogs. The nerve was saved at the mesial site of the transport disc. The regenerated nerve was evaluated by a jaw opening reflex examination performed once daily. Histologic examinations with hematoxylin-eosin and immunohistochemical staining with neurofilament and S-100 antibody were also performed on all dogs after death at 3, 6, and 12 months after the first operation. RESULTS: The jaw opening reflex had recovered in all dogs. The average period of recovery was 109.5 ± 24.7 days. On histologic examination, although consecutive nerves were observed in all areas, cellular nerve fascicles were seen, consistent with wallerian degeneration at 3 and 6 months in the nerve connection area on the distal side of the transport disc. CONCLUSIONS: Our results have indicated that inferior alveolar nerve regeneration after bifocal distraction osteogenesis is successful in dogs. Although our research is still at the stage of animal experiments, future application in humans can be considered to be possible.

Antiproliferative effect of a novel mTOR inhibitor temsirolimus contributes to the prolonged survival of orthotopic esophageal cancer-bearing mice.

Esophageal squamous cell carcinoma (ESCC) remains one of the most aggressive cancers with poor prognosis regardless of a several reports that indicate a better therapeutic efficacy using some new chemotherapeutic agents. Recent drug development has contributed to an improved specificity to suppress mTOR activity by which many types of malignancies can be explosively progressed. Temsirolimus (CCI-779, TricelTM) is one of recently synthesized analogs of rapamycin and has provided better outcomes for patients with renal cell carcinoma. In this study, we experimentally evaluated an efficacy of targeting mTOR by temsirolimus for ESCC treatment, with an assessment of its survival advantage using an advanced ESCC animal model. First, we confirmed that the expression of phosphorylated mTOR was increased in 46 of 58 clinical ESCC tumor tissues (79.3%) and appeared to get strengthened with tumor progression. All of ESCC cell lines used in this study revealed an increase of mTOR phosphorylation, accompanied with the upregulation of hypoxia inducible factor-I α (HIF-1α), one of the critical effectors regulated by mTOR. Temsirolimus treatment apparently suppressed the activation of mTOR and its downstream effectors, resulting in the reduced ability of ESCC cell proliferation. Finally, the weekly administration of temsirolimus significantly diminished the size of subcutaneous tumors (vehicle, 3261.6 ± 722.0; temsirolimus, 599.2 ± 122.9; p = 0.007) in nude mice and effectively prolonged orthotopic esophageal cancer-bearing mice (median survival periods: control, 31 d; temsirolimus, 43 d; p = 0.0024). These data suggests that targeting mTOR by temsirolimus may become a therapeutic alternative for esophageal cancer, with a contribution to a better outcome.

Differential expression of aquaporin 5 and aquaporin 3 in squamous cell carcinoma and adenoid cystic carcinoma.

Aquaporins (AQPs) are a membrane protein family involved in the selective transport of water across cell membranes. Recent studies have reported the expression of AQP5 in several tumor types such as gastric, pulmonary, ovarian, pancreatic and colorectal cancer. We have previously reported the expression on tumor cells and the important role of AQP3 on cell growth in tongue cancer. However, little is known about the expression and precise role of AQP5 on squamous cell carcinoma (SCC) of the tongue. We investigated the expression of AQP5 and AQP3 in human oral SCC and adenoid cystic carcinoma (ACC). Overexpression of both AQP5 and AQP3 were immunohistochemically observed on tumor cells in SCC, whereas ACC cells were faintly stained with those antibodies against AQPs. Treatment with pan-AQP inhibitor or specific AQP5-siRNA showed inhibition of cell growth in SCC cell lines via the inhibition of integrins and the mitogen-activated protein kinase pathway. AQPs play important roles in cell growth in SCC rather than ACC.

[A case of primary adenocarcinoma of small intestine responding to XELOX chemotherapy and leading to a partial metabolic response].

We report a case of adenocarcinoma of the small intestine responding to XELOX chemotherapy, leading to a partial metabolic response(PMR). The patient was a 58-year-old male with multiple peritoneal dissemination of adenocarcinoma of the small intestine. Chemotherapy with XELOX(L-OHP 130 mg/m² on day 1 , and capecitabine 1,000 mg/m2 on days 1-14)was performed. After 4 courses, a significant tumor reduction was obtained. This case suggests that chemotherapy with XELOX is a potential regimen for small intestinal adenocarcinoma.

Steroid-free living donor liver transplantation for HCV--a multicenter prospective cohort study in Japan.

This prospective, non-randomized, multicenter cohort study analyzed the safety and efficacy of a steroid-free immunosuppressive (IS) protocol for hepatitis C virus (HCV)-positive living donor liver transplant (LDLT) recipients in Japan. Of 68 patients enrolled from 13 transplant centers, 56 fulfilled the inclusion/exclusion criteria; 27 were assigned the steroid-free IS protocol (Fr group) and 29 the traditional steroid-containing IS protocol (St group). Serum HCV RNA levels increased over time and were higher in the St group until postoperative day 90 (POD 14, p=0.013). Preemptive anti-HCV therapy was started in a higher percentage of recipients (59.3%) in the Fr group than in the St group (31.0%, p=0.031), mainly due to early HCV recurrence. The incidence of HCV recurrence at one yr was lower in the Fr group (22.2%) than in the St group (41.4%; p=0.066). The incidence of acute cellular rejection was similar between groups. New onset diabetes after transplant, cytomegalovirus infection, and renal dysfunction were significantly less frequent in the Fr group than in the St group (p=0.022, p<0.0001, p=0.012, respectively). The steroid-free IS protocol safely reduced postoperative morbidity and effectively suppressed both the HCV viral load in the early post-transplant period and HCV recurrence in HCV-positive LDLT recipients.

[A case of resected rectal cancer with hepatic node and multiple liver metastases effectively treated by preoperative modified FOLFOX6 and sLV5FU2 chemotherapy].

A 55-year-old male had complained of melena.Colonoscopy revealed a type 2 tumor at the rectum.CT demonstrated hepatic lymph nodes and multiple liver metastases(stage IV).Low anterior resection was performed(tub2, RsRa, circ, type 2, pSS, pN1, sH3, cHN1, sP0, cM0: fstage IV).The patient was treated with mFOLFOX6 and sLV5FU2 after operation.CT revealed a partial response after 14 courses of systemic chemotherapy.sLV5 FU2 therapy was converted to capecitabine because he experienced bone marrow suppression.CT showed that the liver metastases had enlarged but the hepatic lymph nodes disappeared.Right portal vein embolization was performed.After 4 weeks, right hepatectomy and hepatic lymph node dissection were performed.Preoperative chemotherapy with mFOLFOX6 seems beneficial as a neoadjuvant chemotherapy for hepatic lymph node-positive advanced colorectal cancer.

Role of endothelin receptor signalling in squamous cell carcinoma.

Endothelin plays important roles in various physiological functions including vascular constriction. Recent studies reported that the endothelin receptors ETA and ETB are highly expressed in lung and skin tumor tissues. In contrast, there are few reports on endothelin signalling in the proliferation of head and neck cancer. We found that both ETA and ETB endothelin receptors were overexpressed in tumor cells of tongue cancer samples by immunohistochemistry. ETA and ETB were expressed in cultured lingual and esophageal squamous cell carcinoma (SCCs) cell lines. When both cultured cell lines were treated with an ETA selective antagonist (BQ123) or an ETB selective antagonist (BQ788), inhibition of cell growth was observed. Similar results were observed when SCCs were treated with specific siRNA for the suppression of ETA or ETB. Furthermore, inhibition of the mitogen-activated protein (MAP) kinase pathway by the treatments with ET receptor antagonists and siRNA was also observed. These results indicate that endothelin signalling may, in part, play important roles in cell growth in SCCs through the MAP kinase pathway.