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Ken Ando - Top 30 Publications

Association Between Laryngopharyngeal Reflux and Radiation-induced Mucositis in Head and Neck Cancer.

We investigated whether laryngopharyngeal reflux (LPR) is a risk factor for radiation-induced mucositis.

Intravenous dendritic cell administration enhances suppression of lung metastasis induced by carbon-ion irradiation.

Carbon-ion radiotherapy (CIRT) is an advanced radiotherapy and has achieved good local control, even in tumors that are resistant to conventional photon beam radiotherapy (PBRT). However, distant metastasis control is an important issue. Recently, the combination of radiotherapy and immunotherapy has attracted the attention. In immunotherapy, dendritic cells (DCs) play a pivotal role in the anti-tumor immune system. However, the mechanisms underlying the combination therapy of DCs and radiotherapy have been unclear. In the present study, we evaluated anti-metastatic effects of this combination therapy, focused on the irradiation type and the route of DC administration, using a mouse model. C3H/He mice bearing NR-S1 cells were treated with CIRT or PBRT, using biologically equivalent doses. Subsequently, DCs were administered intratumorally (IT) or intravenously (IV). IV and IT DC administrations combined with CIRT to the local tumor, but not alone, significantly suppressed pulmonary metastasis, whereas the combination of DCs with PBRT suppressed metastasis at a relatively higher dose. Additionally, the anti-metastatic effect was greater in IV DC administration compared with in IT DC administration in both CIRT and PBRT. The expression levels of CD40 and IL-12 in DCs were significantly increased after co-culturing with CIRT-treated NR-S1 cells. In addition, IV administration of those co-cultured DCs significantly suppressed pulmonary metastasis. Furthermore, ecto-calreticulin levels from CIRT-treated NR-S1 cells significantly increased compared with those of a PBRT-treated tumor. Taken together, these results suggest that local CIRT combined with IV DCs augments an immunogenicity of the tumor cells by ecto-calreticulin expression and the maturation of DCs to stimulate anti-tumor immunity to decrease lung metastases.

In-room computed tomography-based brachytherapy for uterine cervical cancer: results of a 5-year retrospective study.

Herein, we investigate the long-term clinical outcomes for cervical cancer patients treated with in-room computed tomography-based brachytherapy. Eighty patients with Stage IB1-IVA cervical cancer, who had undergone treatment with combined 3D high-dose rate brachytherapy and conformal radiotherapy between October 2008 and May 2011, were retrospectively analyzed. External beam radiotherapy (50 Gy) with central shielding after 20-40 Gy was performed for each patient. Cisplatin-based chemotherapy was administered concurrently to advanced-stage patients aged ≤75 years. Brachytherapy was delivered in four fractions of 6 Gy per week. In-room computed tomography imaging with applicator insertion was performed for treatment planning. Information from physical examinations at diagnosis, and brachytherapy and magnetic resonance imaging at diagnosis and just before the first brachytherapy session, were referred to for contouring of the high-risk clinical target volume. The median follow-up duration was 60 months. The 5-year local control, pelvic progression-free survival and overall survival rates were 94%, 90% and 86%, respectively. No significant differences in 5-year local control rates were observed between Stage I, Stage II and Stage III-IVA patients. Conversely, a significant difference in the 5-year overall survival rate was observed between Stage II and III-IVA patients (97% vs 72%; P = 0.006). One patient developed Grade 3 late bladder toxicity. No other Grade 3 or higher late toxicities were reported in the rectum or bladder. In conclusion, excellent local control rates were achieved with minimal late toxicities in the rectum or bladder, irrespective of clinical stage.

Successful treatment of radiation-induced mucositis with proton pump inhibitor administration: A report of two laryngeal cancer cases.

Presently, the relationship between laryngopharyngeal reflux (LPR) and radiation-induced mucositis has not been fully explored. In the present study, we report 2 cases of laryngeal cancer in which radiation-induced mucositis ameliorated after proton pump inhibitor (PPI) administration. Case 1 was diagnosed with T1aN0M0 right glottis carcinoma and was treated with radiation therapy. Grade 3 mucositis occurred after administration of 46Gy irradiation. PPI was administered and mucositis ameliorated quickly without cessation of radiation therapy. Case 2 was diagnosed with T2N0M0 supraglottic cancer and was treated with concurrent chemoradiation therapy. Grade 3 mucositis occurred after administration of 44Gy irradiation. PPI was administered and mucositis ameliorated quickly without cessation of chemoradiation therapy. In both cases, a remarkable therapeutic effect of PPI was observed in the perilaryngeal areas including the epiglottic vallecula, arytenoid, and postcricoid area. In both cases, LPR involvement was suspected before the onset of radiation therapy. The two cases presented here, indicated a causal relationship between LPR and radiation-induced mucositis. In cases of severe mucositis in the perilaryngeal area in patients with LPR prior to radiation therapy, PPI administration may be an effective therapeutic option.

The prognostic value of rectal invasion for stage IVA uterine cervical cancer treated with radiation therapy.

The prognostic value of rectal invasion is still unclear in stage IVA cervical cancer. The objective of this study is to evaluate patient outcome and prognostic factors in stage IVA cervical cancer treated with radiation therapy.

Correction: Clinical Trial of Prophylactic Extended-Field Carbon-Ion Radiotherapy for Locally Advanced Uterine Cervical Cancer (Protocol 0508).

Evaluation of therapeutic gain for fractionated carbon-ion radiotherapy using the tumor growth delay and crypt survival assays.

The aim of the study was to evaluate the therapeutic gain of carbon ion (C-ion) radiotherapy using a mouse model.

Farnesoid X receptor knockdown provides significant growth inhibition in hepatocellular carcinoma cells while it does not interfere with the proliferation of primary human hepatocyte-derived cells.

Identification of substances with specific toxicity for carcinoma cells promises to facilitate the development of cancer chemotherapeutics that cause minimal side effects. Here, we show that knockdown of the farnesoid X receptor (FXR) effectively suppresses the proliferation of human hepatocellular carcinoma cell lines HepG2 and HLE accompanied by elevated expression of cyclin-dependent kinase (CDK) inhibitor p16/INK4a and p21/Cip1 proteins. On the other hand, the growth of the primary human hepatocyte-derived cell line Fa2N-4 is not affected by the treatment with FXR siRNA irrespective of marked increases in the mRNAs of p16/INK4a and p21/Cip1. Surprisingly, the expression levels of p16/INK and p21/Cip1 proteins are left unchanged in Fa2N-4 cells that are subjected to the FXR siRNA treatment. Since the expression levels of these CDK inhibitor proteins in FXR-knockdown Fa2N-4 cells were elevated in the presence of proteasomal inhibitor MG132, these CDK inhibitors may be subjected to the proteasomal degradation, thereby counteracting the increased expression of their cognate mRNAs, therefore similar levels of p16 and p21 proteins were observed in control and FXR-knockdown Fa2N-4 cells. These results suggest that FXR-knockdown is effective for inhibiting the proliferation of hepatocellular carcinoma cells, not interfering with the regulatory mechanism of normal hepatocyte growth.

Clinical trial of prophylactic extended-field carbon-ion radiotherapy for locally advanced uterine cervical cancer (protocol 0508).

To evaluate the efficacy and the toxicity of prophylactic extended-field carbon-ion radiotherapy (C-ion RT, Protocol 0508) for locally advanced squamous cell carcinoma of the uterine cervix in phase I / II clinical trial. Between May 2006 and January 2012, 26 patients of Protocol 0508 were treated with C-ion RT. The numbers of patients with stage IIB, IIIB, and IVA disease were 13, 11, and 2, respectively. Twenty patients had pelvic lymph node metastases. Median tumor size was 6.1 cm (range, 4.0–10.0 cm). The treatment consisted of extended-field irradiation of 39.0 gray equivalents (GyE) in 13 fractions, and additional 15.0 GyE in 5 fractions was given to the gross tumor volume (GTV) and surrounding tissues. With regard to local boost, 18.0 GyE in 2 fractions was given to GTV only. Total dose to the cervical tumor was 72.0 GyE over 20 fractions. The median follow-up period was 37 months. Twenty-one patients had grade 1 or 2 acute gastrointestinal toxicity, but all patients completed the treatment on schedule. There were no grade 3 or higher late complications, with 8 patients having grade 1 or 2 toxicities, 1 had grade 2 gastrointestinal toxicity and 2 had grade 2 genitourinary toxicity. Four patients (15.4%) developed local recurrence, and 8 patients (30.8%) had distant metastases. The 2-year local control rate, progression-free survival rate and overall survival rate were 83.6%, 61.5% and 73.1%, respectively. There were no severe acute or late complications in this trial. Prophylactic extended-field C-ion RT for locally advanced squamous cell carcinoma of the uterine cervix was a safe treatment. Although the number of patients in this study was small, the results support further investigations to confirm the therapeutic efficacy and to avoid or reduce toxicity.

Radio-sensitization effect of an mTOR inhibitor, temsirolimus, on lung adenocarcinoma A549 cells under normoxic and hypoxic conditions.

The mammalian target of rapamycin (mTOR) correlates with cell survival under hypoxia and regulates hypoxia-inducible factor-1α (HIF-1α), a key protein in hypoxia-related events. However, the role of mTOR in radio-resistance has not been fully investigated. Therefore, the effect of mTOR on the radio-resistance of cancer cells under hypoxia was evaluated using the mTOR inhibitor temsirolimus. Clonogenic survival was examined in the A549 human lung adenocarcinoma cell line under normoxia or hypoxia, with or without temsirolimus. An oxygen enhancement ratio (OER) was calculated using the D(10) values, the doses giving 10% survival. Western blotting was performed to investigate the effect of temsirolimus on mTOR and the HIF-1α pathway under normoxia and hypoxia. A549 cells showed a radio-resistance of 5.1 and 14.2 Gy, as indicated by D(10) values under normoxia and hypoxia, respectively; the OER was 2.8. The cell survival rates under hypoxia and with temsirolimus remarkably decreased compared with those under normoxia. The D(10) values of the cells under normoxia and hypoxia were 4.8 and 5.4 Gy, respectively (OER = 1.1). mTOR expression was suppressed by temsirolimus under both normoxia and hypoxia. HIF-1α expression decreased under hypoxia in the presence of temsirolimus. These results suggest that temsirolimus can overcome the radio-resistance induced by hypoxia. When the fact that mTOR acts upstream of HIF-1α is considered, our data suggest that the restoration of radiation sensitivity by temsirolimus under hypoxia may be associated with the suppression of the HIF-1α pathway. Temsirolimus could therefore be used as a hypoxic cell radio-sensitizer.

Carbon-ion beams induce production of an immune mediator protein, high mobility group box 1, at levels comparable with X-ray irradiation.

X-ray radiotherapy activates tumor antigen-specific T-cell responses, and increases in the serum levels of high mobility group box 1 (HMGB1) induced by X-ray irradiation play a pivotal role in activating anti-tumor immunity. Here, we examined whether carbon-ion beams, as well as X-rays, can induce HMGB1 release from human cancer cell lines. The study examined five human cancer cell lines: TE2, KYSE70, A549, NCI-H460 and WiDr. The proportion of cells surviving X- or carbon-ion beam irradiation was assessed in a clonogenic assay. The D10, the dose at which 10% of cells survive, was calculated using a linear-quadratic model. HMGB1 levels in the culture supernatants were assessed by an ELISA. The D10 dose for X-rays in TE2, KYSE70, A549, NCI-H460 and WiDr cells was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, whereas that for carbon-ion beams was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. X-rays and carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of A549, NCI-H460 and WiDr cells at 72 h post-irradiation with a D10 dose. Furthermore, irradiation with X-rays or carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of all five cell lines at 96 h post-irradiation. There was no significant difference in the amount of HMGB1 induced by X-rays and carbon-ion beams at any time-point (except at 96 h for NCI-H460 cells); thus we conclude that comparable levels of HMGB1 were detected after irradiation with iso-survival doses of X-rays and carbon-ion beams.

Difference in distant failure site between locally advanced squamous cell carcinoma and adenocarcinoma of the uterine cervix after C-ion RT.

We investigated the first site of distant failure after carbon ion radiotherapy (C-ion RT) for locally advanced cervical cancer in three clinical trials. A total of 91 cases were enrolled in the three trials (Protocol 9702, 9704 and 9902). Histologically, 36 cases had squamous cell carcinoma (SqCC) and 55 cases had adenocarcinoma (AC), including 13 with adenosquamous cell carcinoma. The number of cases with Stage IIB, IIIB and IVA disease was 21, 59 and 11, respectively. Of the 91 cases, 42 had positive pelvic lymph nodes (PLNs). The median tumor size was 6.0 cm (range, 3.0–12.0 cm). The median follow-up duration for all cases was 40 months (range, 7–181 months). A total of 40 cases developed distant failure as the first site of failure: 13 of 36 (36.1%) SqCC cases had distant failure, with 9 of them with para-aortic lymph node (PALN) failure; 27 of 55 (44.0%) AC cases had distant failure, and 23 of them had distant failure excluding PALN metastasis. Distant failure rates of SqCC cases who had positive and negative PLNs before C-ion RT were 61.1% and 11.1%, respectively (P = 0.0045). Those of AC cases were 54.2% and 45.2%, respectively (P = 0.507). In conclusion, there were high rates of distant failure after C-ion RT in AC cases regardless of PLN status, and there were high rates of distant failure after C-ion RT, especially PALN failure, in SqCC cases with positive PLNs.

Can combined intracavitary/interstitial approach be an alternative to interstitial brachytherapy with the Martinez Universal Perineal Interstitial Template (MUPIT) in computed tomography-guided adaptive brachytherapy for bulky and/or irregularly shaped gynecological tumors?

Interstitial brachytherapy (ISBT) is an optional treatment for locally advanced gynecological tumours for which conventional intracavitary brachytherapy (ICBT) would result in suboptimal dose coverage. However, ISBT with Martinez Universal Perineal Interstitial Template (MUPIT), in which ~10-20 needles are usually applied, is more time-consuming and labor-intensive than ICBT alone, making it a burden on both practitioners and patients. Therefore, here we investigated the applicability of a combined intracavitary/interstitial (IC/IS) approach in image-guided adaptive brachytherapy for bulky and/or irregularly shaped gynecological tumours for which interstitial brachytherapy (ISBT) was performed.

HLA class I expression and its alteration by preoperative hyperthermo-chemoradiotherapy in patients with rectal cancer.

Enhancing immunologic responses, including human leukocyte antigen (HLA) class I expression on tumor cells and recognition and elimination of tumor cells by tumor-specific cytotoxic T lymphocyte (CTL), is considered a novel concept of radiotherapy. The present study examined patients who underwent preoperative hyperthermo-chemoradiotherapy (HCRT) for locally advanced rectal cancer to assess the correlation between HLA class I expression and clinical outcome.

Radiotherapy-induced anti-tumor immunity contributes to the therapeutic efficacy of irradiation and can be augmented by CTLA-4 blockade in a mouse model.

There is growing evidence that tumor-specific immune responses play an important role in anti-cancer therapy, including radiotherapy. Using mouse tumor models we demonstrate that irradiation-induced anti-tumor immunity is essential for the therapeutic efficacy of irradiation and can be augmented by modulation of cytotoxic T lymphocyte (CTL) activity.

Clinical outcomes of carbon ion radiotherapy for locally advanced adenocarcinoma of the uterine cervix in phase 1/2 clinical trial (protocol 9704).

This study sought to evaluate the toxicity and efficacy of carbon ion radiotherapy (C-ion RT) for locally advanced adenocarcinoma of the uterine cervix in a phase 1/2 clinical trial.

Dose-escalation study of carbon ion radiotherapy for locally advanced squamous cell carcinoma of the uterine cervix (9902).

The authors performed phase I/II clinical trial to evaluate the toxicity and efficacy of carbon ion radiotherapy (C-ion RT) for locally advanced squamous cell carcinoma of the uterine cervix.

Changes in bone mineral density in uterine cervical cancer patients after radiation therapy.

To prospectively investigate the changes in bone mineral density (BMD) after pelvic radiation therapy in patients with uterine cervical cancer.

Impact of boost irradiation on pelvic lymph node control in patients with cervical cancer.

Radiation therapy (RT) for metastatic pelvic lymph nodes (PLNs) is not well established in cervical cancer. In this study the correlation between size of lymph nodes and control doses of RT was analyzed. Between January 2002 and December 2007, 245 patients with squamous cell carcinoma of the cervix treated with a combination of external beam irradiation with or without boost irradiation and high-dose rate brachytherapy were investigated. Size of lymph node was measured by computed tomography before RT and just after 50 Gy RT. Of the 245 patients, 78 had PLN metastases, and a total of 129 had enlarged PLNs diagnosed as metastases; 22 patients had PLN failure. The PLN control rate at 5 years was 79.5% for positive cases and 95.8% for negative cases. In cases with positive PLNs, 12 of 129 nodes (9.3%) developed recurrences. There was significant correlation between PLN control rate and size of PLN after 50 Gy (<10 mm: 96.7%, ≥ 10 mm: 75.7 % (P<0.001)). In addition, the recurrence in these poor-response nodes was significantly correlated with dose of RT. Nine of 16 nodes receiving ≤ 58 Gy had recurrence, but none of 21 nodes receiving > 58 Gy had recurrence (P = 0.0003). These results suggested that the response of lymph nodes after RT was a more significant predictive factor for recurrence than size of lymph node before RT, and poor-response lymph nodes might require boost irradiation at a total dose of > 58 Gy.

Carbon-ion radiotherapy for marginal lymph node recurrences of cervical cancer after definitive radiotherapy: a case report.

Recurrences of cervical cancer after definitive radiotherapy often occur at common iliac or para-aortic lymph nodes as marginal lymph node recurrences. Patients with these recurrences have a chance of long-term survival by optimal re-treatment with radiotherapy. However, the re-irradiation often overlaps the initial and the secondary radiotherapy fields and can result in increased normal tissue toxicities in the bowels or the stomach. Carbon-ion radiotherapy, a form of particle beam radiotherapy using accelerated carbon ions, offers more conformal and sharp dose distribution than X-ray radiotherapy. Therefore, this approach enables the delivery of high radiation doses to the target while sparing its surrounding normal tissues. Marginal lymph node recurrences in common iliac lymph nodes after radiotherapy were treated successfully by carbon-ion radiotherapy in two patients. These two patients were initially treated with a combination of external beam radiotherapy and intracavitary and interstitial brachytherapy. However, the diseases recurred in the lymph nodes near the border of the initial radiotherapy fields after 22 months and 23 months. Because re-irradiation with X-ray radiotherapy may deliver high doses to a section of the bowels, carbon-ion radiotherapy was selected to treat the lymph node recurrences. A total dose of 48 Gy (RBE) in 12 fractions over 3 weeks was given to the lymph node recurrences, and the tumors disappeared completely with no severe acute toxicities. The two patients showed no evidence of disease for 75 months and 63 months after the initial radiotherapy and for 50 months and 37 months after the carbon-ion radiotherapy, respectively. No severe late adverse effects are observed in these patients. The two presented cases suggest that the highly conformal dose distribution of carbon-ion radiotherapy may be beneficial in the treatment of marginal lymph node recurrences after radiotherapy. In addition, the higher biological effect of carbon-ion radiotherapy and its superior dose distribution may provide more effective tumor control in treatment for re-irradiation of the marginal recurrences in radiation resistant tumors other than cervical cancer.

Comparison of hematological toxicities between innovator and generic cisplatin formulations in cervical cancer patients treated with concurrent chemoradiotherapy.

To compare the incidence and degree of hematological toxicity between innovator and generic cisplatin formulations, decreases in white blood cell (WBC) count (leukopenia) and platelet counts (thrombocytopenia) were retrospectively examined, using the Common Toxicity Criteria for Adverse Events ver. 4.0, in patients with uterine cervical cancer treated with concurrent chemoradiotherapy using innovator (innovator group, n = 22) or generic (generic group, n = 22) cisplatin formulations. There were no significant differences in patient characteristics except in the technique of external irradiation; larger numbers of patients in the innovator and generic groups were irradiated using the parallel-opposed two-field technique and the four-field box technique, respectively (P = 0.00012), which is in line with the historical progress of external beam radiation therapy. The numbers of patients showing Grade 1, 2, 3 and 4 leukopenia were 1 (4.5%), 14 (64%), 7 (32%) and 0 (0.0%) in the innovator group, and 1 (4.5%), 6 (27%), 13 (59%) and 2 (9.0%) in the generic group, respectively. The number of patients showing Grade 3-4 leukopenia was significantly greater in the generic group than in the innovator group (P = 0.034). There was no significant relationship between the incidence of Grade 3-4 leukopenia and the technique of external irradiation. There were no significant differences in the incidence and degree of thrombocytopenia between the two groups. These results indicate the possibility that the generic cisplatin formulation may have a different toxicity profile compared to the innovator formulation in terms of the incidence of leukopenia.

Adaptive radiotherapy based on the daily regression of a tumor in carbon-ion beam irradiation.

We propose a new application of adaptive radiotherapy using a scanning beam, taking into account the daily regression of a tumor. No patient-specific hardware (such as collimators and compensating filters) is needed for the scanning technique; thus, it allows re-planning immediately before each fractional irradiation using the sophisticated conformations of dose distributions. We retrospectively modeled the tumor volume regression curves as a function of the dose from the CT images which were taken three times during the treatment course. The daily shape of the tumor was mathematically interpolated assuming constant continuity of the tumor deformation. We simulated the adaptive radiotherapy by optimizing the dose distribution on the estimated daily tumor volume for every fraction. The dose-volume histogram (DVH) for the organ at risk in the adaptive radiotherapy was compared with that of the current clinical protocol. We performed analysis using the CT images of cervical cancer patients who received carbon-ion radiotherapy in broad-beam irradiation. The DVH for the rectum and the sigmoid colon was improved by adaptive radiotherapy considering the inter-fractional tumor regression. The result shows that this approach has possible advantages.

Critical role of farnesoid X receptor for hepatocellular carcinoma cell proliferation.

Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.

Role of FGD1, a Cdc42 guanine nucleotide exchange factor, in epidermal growth factor-stimulated c-Jun NH2-terminal kinase activation and cell migration.

FGD1 encodes a guanine nucleotide exchange factor for Cdc42. Mutations in the FGD1 gene are responsible for an X-linked disorder known as Aarskog-Scott syndrome (AAS). While most mutations were found in the catalytic region, which consists of Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, a missense mutation in the proline-rich domain is also found in a patient with typical clinical features as AAS. In this mutant FGD1, the serine residue at 205 is replaced with isoleucine. We recently demonstrated that FGD1 translocated to the membrane in response to extracellular stimuli such as epidermal growth factor (EGF) whereas FGD1 with S(205)/I substitution did not. Here we show that the proline-rich domain is critical for FGD1-induced directionally persistent cell migration. When inducibly expressed in HeLa Tet-Off cells, FGD1 stimulates directional migration whereas FGD1 with S(205)/I substitution does not affect it. We further demonstrate that FGD1 augments EGF-stimulated c-Jun NH(2)-terminal kinase (JNK) activation. In the presence of JNK inhibitor SP600125, motility of FGD1-expressing cells is significantly impaired, indicating a critical role of JNK in cell migration. However, FGD3, an FGD1 homologue lacking the proline-rich domain, and FGD1 with S(205)/I substitution augment EGF-stimulated JNK activation similarly to FGD1, suggesting that the proline-rich domain is not involved in the regulation of JNK. Finally, we show that FGD1, but not FGD1 with S(205)/I substitution, is phosphorylated in response to EGF, suggesting that the phosphorylation of S(205) may trigger the FGD1 translocation to the leading edge membrane and enable cells to undergo directional migration.

Human lactoferrin activates NF-kappaB through the Toll-like receptor 4 pathway while it interferes with the lipopolysaccharide-stimulated TLR4 signaling.

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)-triggered nuclear factor-kappaB (NF-kappaB) activation. Endotoxin-depleted hLF induces NF-kappaB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-kappaB activation at a level comparable to that seen in wild-type MEFs, whereas TRAF6-deficient MEFs show significantly impaired NF-kappaB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF-kappaB activation in myeloid differentiating factor 88 (MyD88)-dependent signaling pathways, while the role of TRAF6 in the MyD88-independent signaling pathway has not been clarified extensively. When we examined the hLF-dependent NF-kappaB activation in MyD88-deficient MEFs, delayed, but remarkable, NF-kappaB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88-dependent and MyD88-independent pathways are involved. Indeed, hLF fails to activate NF-kappaB in MEFs lacking Toll-like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88-depependent and MyD88-independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide-induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4-mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide-dependent TLR4 activation, probably through its polypeptide moiety.

Proline-rich domain plays a crucial role in extracellular stimuli-responsive translocation of a Cdc42 guanine nucleotide exchange factor, FGD1.

We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, and its homologue FGD3 are targeted by the ubiquitin ligase SCF(FWD1) upon phosphorylation of two serine residues in their DSGIDS motif and subsequently degraded by the proteasome. FGD1 and FGD3 share highly homologous Dbl homology (DH) and adjacent pleckstrin homology (PH) domains, both of which are responsible for GEF activity. However, their function and regulation are remarkably different. Here we demonstrate extracellular signal-responsive translocation of FGD1, but not FGD3. During the wound-healing process, translocation of FGD1 to the leading edge membrane occurs in cells facing to the wound. Furthermore, epidermal growth factor (EGF) stimulates the membrane translocation of FGD1, but not FGD3. As the most striking difference, FGD3 lacks the N-terminal proline-rich domain that is conserved in FGD1, indicating that proline-rich domain may play a crucial role in signal-responsive translocation of FGD1. Indeed, there is a faciogenital dysplasia patient who has a missense mutation in proline-rich domain of FGD1, by which the serine residue at position 205 is substituted with isoleucine. When expressed in cells, the mutant FGD1 with S(205)/I substitution fails to translocate to the membrane in response to the mitogenic stimuli. Thus we present a novel mechanism by which the activity of FGD1, a GEF for Cdc42, is temporally and spatially regulated in cells.

Hypoxia downregulates farnesoid X receptor via a hypoxia-inducible factor-independent but p38 mitogen-activated protein kinase-dependent pathway.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been shown to play pivotal roles in bile acid homeostasis by regulating the biosynthesis, conjugation, secretion and absorption of bile acids. Accumulating data suggest that FXR signaling is involved in the pathogenesis of liver and metabolic disorders. Here we show that FXR expression is significantly suppressed in HepG2 cells exposed to hypoxia. Concomitantly, the expression of the bile salt export pump, known as an FXR target gene product and responsible for the excretion of bile acids from the liver, is also decreased under hypoxia. Overexpression of hypoxia-inducible factor (HIF)-1alpha does not mimic the suppressive effect of hypoxia on FXR expression. Furthermore, simultaneous knockdown of HIF-1alpha, HIF-2alpha and HIF-3alpha fails to restore the FXR expression level under hypoxia, indicating that HIF is not involved in hypoxia-evoked FXR downregulation. Instead, we demonstrate that p38 mitogen-activated protein kinase is an indispensable factor for FXR downregulation under hypoxia. Thus, we propose a novel liver disorder model in which two signaling molecules, p38 mitogen-activated protein kinase and FXR, may contribute to the linkage of two pathogenic conditions, i.e. ischemia, a condition accompanying hypoxia, and cholestasis, a condition with intrahepatic accumulation of cytotoxic bile acids.

Novel insights into FGD3, a putative GEF for Cdc42, that undergoes SCF(FWD1/beta-TrCP)-mediated proteasomal degradation analogous to that of its homologue FGD1 but regulates cell morphology and motility differently from FGD1.

We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, is targeted by the ubiquitin ligase SCF(FWD1/beta-TrCP) upon phosphorylation of two serine residues in its DSGIDS motif and subsequently degraded by the proteasome. Here we show that FGD3, which was identified as a homologue of FGD1 but has been poorly characterized, has conserved the same motif and is down-regulated similarly by SCF(FWD1/beta-TrCP). Although FGD3 and FGD1 share strikingly similar Dbl homology (DH) domains and adjacent pleckstrin homology (PH) domains, both of which are responsible for guanine nucleotide exchange, there also exist remarkable differences in their structures. Indeed, FGD1 and FGD3 induced significantly different morphological changes in HeLa Tet-Off cells: whereas FGD1 induced long finger-like protrusions, FGD3 induced broad sheet-like protrusions when the level of GTP-bound Cdc42 was significantly increased by the inducible expression of FGD3. Furthermore, FGD1 and FGD3 reciprocally regulated cell motility: when inducibly expressed in HeLa Tet-Off cells, FGD1 stimulated cell migration whereas FGD3 inhibited it. Thus we demonstrate that the highly homologous GEFs, FGD1 and FGD3 play different roles to regulate cellular functions but that their intracellular levels are tightly controlled by the same destruction pathway through SCF(FWD1/beta-TrCP).

Coordination of oxidized protein hydrolase and the proteasome in the clearance of cytotoxic denatured proteins.

Intracellular accumulation of denatured proteins impairs cellular function. The proteasome is recognized as an enzyme responsible for the effective clearance of those cytotoxic denatured proteins. As another enzyme that participates in the destruction of damaged proteins, we have identified oxidized protein hydrolase (OPH) and found that OPH confers cellular resistance to various kinds of oxidative stress. In this study, we demonstrate the roles of the proteasome and OPH in the clearance of denatured proteins. The inhibition of proteasome activity results in the elevation of protein carbonyls in cells under oxidative stress. On the other hand, cells overexpressing OPH retain higher resistance to oxidative stress, even though the proteasome activity is inhibited. Furthermore, upon inhibition of the proteasome activity, OPH is recruited to a novel organelle termed the aggresome where misfolded or denatured proteins are processed. Thus, OPH and the proteasome coordinately contribute to the clearance of cytotoxic denatured proteins.

Protective effect of supplementation of fish oil with high n-3 polyunsaturated fatty acids against oxidative stress-induced DNA damage of rat liver in vivo.

The present study was undertaken to know the effect of supplementation of fish oil with high n-3 polyunsaturated fatty acids (PUFA) on oxidative stress-induced DNA damage of rat liver in vivo. Male Wistar rats were fed a diet containing fish oil or safflower oil with high n-6 PUFA at 50 g/kg of diet and an equal amount of vitamin E at 59 mg/kg of diet for 6 weeks. Livers of rats fed fish oil were rich in n-3 PUFA, whereas those of rats fed safflower oil were rich in n-6 PUFA. Ferric nitrilotriacetate was intraperitoneally injected to induce oxidative stress. The degree of lipid peroxidation of the liver was assessed by the levels of phospholipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS), and the degree of oxidative DNA damage was assessed by comet type characterization in alkaline single-cell gel electrophoresis and 8-hydroxy-2'-deoxyguanosine levels. The levels of TBARS of the livers of the fish oil diet group increased to a greater extent than those of the safflower oil diet group, whereas the levels of the hydroperoxides of the livers of both diet groups increased to a similar extent. The vitamin E level of livers of the fish oil diet group was remarkably decreased. The degree of DNA damage of both diet groups was increased, but the increased level of the fish oil diet group was remarkably lower than that of the safflower oil diet group. The above results indicate that fish oil supplementation does not enhance but appears to protect against oxidative stress-induced DNA damage and suggest that lipid peroxidation does not enhance but lowers the DNA damage.