Acquired resistance to trastuzumab/pertuzumab or to T-DM1 in vivo can be overcome by HER2 kinase inhibition with TAS0728.

HER2-targeting antibodies (trastuzumab, pertuzumab) and a HER2-directed antibody-drug conjugate (trastuzumab emtansine: T-DM1) are used for the treatment of HER2-overexpressing breast cancer. However, these treatments eventually become ineffective due to acquired resistance and there is an urgent need for alternative therapies. TAS0728 is a small-molecule, irreversible selective HER2 kinase inhibitor. In the present study, we established new in vivo models of cancer resistance by continuous exposure to a combination of trastuzumab and pertuzumab or to T-DM1 for evaluating the effect of TAS0728 on HER2 antibody-resistant populations. Treatment with trastuzumab and pertuzumab or with T-DM1 initially induced tumor regression in NCI-N87 xenografts. However, tumor regrowth during treatment indicated loss of drug effectiveness. In tumors with acquired resistance to trastuzumab and pertuzumab or to T-DM1, HER2-HER3 phosphorylation was retained. Switching to TAS0728 resulted in a significant anti-tumor effect associated with HER2-HER3 signal inhibition. No alternative receptor tyrosine kinase activation was observed in these resistant tumors. Furthermore, in a patient-derived xenograft model derived from breast cancer refractory to both trastuzumab/pertuzumab and T-DM1, TAS0728 exerted a potent anti-tumor effect. These results suggest that tumors with acquired resistance to trastuzumab and pertuzumab and to T-DM1 are still dependent on oncogenic HER2-HER3 signaling and are vulnerable to HER2 signal inhibition by TAS0728. These results provide a rationale for TAS0728 therapy for breast cancers that are refractory to established anti-HER2 therapies.

TAS-116 inhibits oncogenic KIT signalling on the Golgi in both imatinib-naïve and imatinib-resistant gastrointestinal stromal tumours.

BACKGROUND: Despite the effectiveness of imatinib mesylate (IM), most gastrointestinal stromal tumours (GISTs) develop IM resistance, mainly due to the additional kinase-domain mutations accompanied by concomitant reactivation of KIT tyrosine kinase. Heat-shock protein 90 (HSP90) is one of the chaperone molecules required for appropriate folding of proteins such as KIT. METHODS: We used a novel HSP90 inhibitor, TAS-116, which showed specific binding to HSP90α/ß with low toxicity in animal models. The efficacy and mechanism of TAS-116 against IM-resistant GIST were evaluated by using IM-naïve and IM-resistant GIST cell lines. We also evaluated the effects of TAS-116 on the other HSP90 client protein, EGFR, by using lung cell lines. RESULTS: TAS-116 inhibited growth and induced apoptosis in both IM-naïve and IM-resistant GIST cell lines with KIT activation. We found KIT was activated mainly in intracellular compartments, such as trans-Golgi cisternae, and TAS-116 reduced autophosphorylated KIT in the Golgi apparatus. In IM-resistant GISTs in xenograft mouse models, TAS-116 caused tumour growth inhibition. We found that TAS-116 decreased phosphorylated EGFR levels and inhibited the growth of EGFR-mutated lung cancer cell lines. CONCLUSION: TAS-116 may be a novel promising drug to overcome tyrosine kinase inhibitor-resistance in both GIST and EGFR-mutated lung cancer.

Activity of TAS4464, a novel NEDD8 activating enzyme E1 inhibitor, against multiple myeloma via inactivation of nuclear factor κB pathways.

The ubiquitin proteasome pathway is essential for the proliferation and survival of multiple myeloma (MM) cells. TAS4464, a novel highly potent inhibitor of NEDD8 activating enzyme, selectively inactivates cullin-RING ubiquitin E3 ligases, resulting in accumulation of their substrates. Here, we examined 14 MM cell lines treated with TAS4464. TAS4464 induced growth arrest and cell death in the MM cell lines even in the presence of bone marrow stromal cells. It also induced the accumulation of phospho-inhibitor of κBα and phospho-p100, impaired the activities of nuclear factor κB (NF-κB) transcription factors p65 and RelB, and decreased the expression of NF-κB target genes, suggesting that TAS4464 inhibits both the canonical and non-canonical NF-κB pathways. TAS4464 had similar effects in an in vivo human-MM xenograft mouse model in which it was also observed to have strong antitumor effects. TAS4464 synergistically enhanced the antitumor activities of the standard MM chemotherapies bortezomib, lenalidomide/dexamethasone, daratumumab and elotuzumab. Together, these results suggest that the anti-MM activity of TAS4464 occurs via inhibition of the NF-κB pathways, and that treatment with TAS4464 is a potential approach for treating MM by single and combination therapies.

[A Case of Metastatic Peripheral T-cell Lymphoma of the Central Nervous System During the Perinatal Period].

The diagnosis of malignant tumors during pregnancy is not uncommon; the incidence is one per six thousand pregnancies. However, the diagnosis of malignant lymphoma-especially T-cell lymphoma-during pregnancy is extremely rare. Thus, the early detection and management of T-cell lymphoma necessitates difficult decision-making. A 30-year-old woman developed consciousness disturbance on postpartum day three. Because brain MRI showed multiple edematous lesions in both hemispheres, vasculitis or encephalitis was initially suspected, and diagnostic therapy was initiated with the administration of steroids. One month later, the patient suddenly developed a subarachnoid hemorrhage followed by acute hydrocephalus. Emergent ventricular drainage and lesion biopsy were simultaneously performed. Based on the findings, the patient was diagnosed with peripheral T-cell lymphoma not otherwise specified(PTCL-NOS). Laboratory findings indicated Epstein-Barr virus(EBV)infection. Moreover, the same diagnosis was supported by breast and bone marrow biopsies. Thus, the brain lesions were presumed to be metastatic in nature. The prognosis of PTCL-NOS is severely poor in pregnant women as diagnosis is delayed owing to limitations of radiological examinations and because symptoms can be confused with those of other diseases or hyperemesis gravidarum. Additionally, the alteration of immunotolerance in association with pregnancy and EBV infection might have influenced the aggressive features of this case. When a pregnant woman presents with neurological symptoms, malignant lymphoma should be considered when making a differential diagnosis.

Antitumor Efficacy of Combination Therapy Consisting of S-1, Leucovorin, and Oxaliplatin against Human Gastric Cancer Xenografts.

BACKGROUND/AIM: A phase 3 trial of S-1, leucovorin (LV), and oxaliplatin for treating gastric cancer is now underway. However, the antitumor efficacy of the combination has not yet been examined in an in vivo preclinical study. This study examined the antitumor efficacy of combination therapy consisting of S-1, LV, and oxaliplatin against 4 human gastric cancer xenografts: NUGC-4, St-40, SC-2, and SC-4. METHODS: The antitumor efficacy was evaluated using human gastric cancer xenograft-bearing nude mice. S-1 and LV were administered orally once daily on days 1-7 at doses of 6.9 and 10 mg/kg, respectively. Oxaliplatin was administered intravenously at a dose of 8.3 mg/kg on day 1. The tumor volume was measured on day 15, and the relative tumor volume (RTV) was calculated. RESULTS: In all 4 xenograft models, S-1 alone and oxaliplatin alone, but not LV alone, had significant antitumor activities (p < 0.001). Combination therapy consisting of S-1 and LV resulted in a significantly smaller RTV than S-1 alone (p < 0.001). Combination therapy consisting of S-1 and oxaliplatin also resulted in a significantly smaller RTV than either S-1 alone (p < 0.001) or oxaliplatin alone (p < 0.001). Furthermore, combination therapy consisting of S-1, LV, and oxaliplatin resulted in the highest antitumor activity in these models (p < 0.001 vs. S-1 + LV; p < 0.001 or p = 0.003 vs. S-1 + oxaliplatin). CONCLUSION: Combination therapy consisting of S-1, LV, and oxaliplatin administered according to a 1-week-on/1-week-off schedule may be useful for the treatment of patients with gastric cancer.

Effective Sequential Combined Chemotherapy with Trifluridine/Tipiracil and Regorafenib in Human Colorectal Cancer Cells.

Salvage chemotherapy for refractory metastatic colorectal cancer using trifluridine/tipiracil (FTD/TPI) and regorafenib has shown survival benefits. We evaluated the antitumor effects of FTD or FTD/TPI combined with regorafenib in vitro and in vivo. SW620, HCT 116, and HT-29 human colorectal cancer cell lines were treated with FTD and regorafenib simultaneously and sequentially. Cell death, incorporation of FTD into DNA, and molecules related to FTD and regorafenib-associated cell death were investigated. The antitumor effects of FTD combined with regorafenib in SW620 and COLO205 xenografts were also evaluated. Cell death was greater after sequential treatment with FTD followed by regorafenib in SW620 cells, but not in HCT 116 and HT-29 cells, than after treatment with FTD alone, which was attributable to thymidylate synthase reduction with the induction of apoptosis. In contrast, simultaneous and sequential exposure to regorafenib followed by FTD, but not FTD alone, attenuated the cell death effect. Furthermore, combined FTD/TPI treatment followed by regorafenib had greater antitumor activity than either monotherapy in SW620 and COLO205 xenograft models. Treatment results following regorafenib administration subsequent to FTD or FTD/TPI suggest that sequential therapy with FTD/TPI prior to regorafenib may be effective in a clinical setting.

Cerebral tumor embolism from thyroid cancer treated by mechanical thrombectomy: illustrative case.

BACKGROUND: Development in mechanical thrombectomy is progressing dramatically. Tumor embolism has been rarely reported on the basis of pathological study of the retrieved thrombus. Herein, the authors report a case of cerebral tumor embolism from advanced thyroid cancer, which was successfully treated with mechanical thrombectomy. OBSERVATIONS: A 57-year-old man was diagnosed with thyroid cancer with multiple lung metastases and chemotherapy was planned. He experienced left hemiparesis and was bought to the emergency section of the authors' hospital. Magnetic resonance angiography revealed right internal carotid artery occlusion and endovascular treatment was performed. Using a combination of aspiration catheter and stent retriever, white jelly-like embolus was retrieved. The pathological study demonstrated thyroid cancer embolism. Pulmonary vein invasion following lung metastasis of thyroid cancer was most presumably the cause of the tumor embolism. LESSONS: Lung metastasis invading the pulmonary vein may be a cause of tumor embolism. Mechanical thrombectomy using a combination of stent retriever and aspiration catheter is effective in removing the tumor embolus and the pathological examination of the embolus is essential.

Oral fluoropyrimidine may augment the efficacy of aromatase inhibitor via the down-regulation of estrogen receptor in estrogen-responsive breast cancer xenografts.

The present preclinical study was designed to evaluate a new combination therapy comprised of the aromatase inhibitor anastrozole (ANA) and the oral fluoropyrimidines, UFT and S-1 against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7/Arom 14, which was stably transfected with the cDNA of human aromatase. MCF-7/Arom 14 cells showed a high aromatase activity and notably were able to grow in the presence of testosterone and estradiol (E(2)) in vitro. ANA and 5-fluorouracil (5-FU) inhibited cell growth at concentrations of 0.005-10 and 0.2-5 µM, respectively, and the combination of both drugs additively inhibited cell growth. The growth of MCF-7/Arom 14 tumors was significantly inhibited by ANA and S-1 or UFT in vivo. The combination of ANA with S-1 or UFT administered using a 21-day consecutive, metronomic-like regimen significantly enhanced the antitumor efficacy, suppressing tumor growth for 2-4 times longer than monotherapy. To investigate the mechanisms by which S-1 enhances the antitumor activity of ANA, the protein and mRNA expression levels of ER-α in tumor tissue after treatment with S-1, ANA, and the typical chemotherapeutic agents doxorubicin (ADM) or paclitaxel (TXL) were analyzed. The protein and mRNA expression levels of ER-α in the tumor tissue were markedly decreased after treatment with S-1 or S-1 + ANA, but not after treatment with either ADM or TXL. The reduced ER-α level after S-1 treatment might contribute to the increased antitumor activity of ANA by reducing ER-α-induced growth signaling in addition to the decrease in estrogen production induced by ANA. Based on these results, the combination of ANA and S-1 might yield a greater benefit than other chemotherapeutic agents in postmenopausal women with ER-positive breast cancer.

Erratum: Trifluridine/tipiracil increases survival rates in peritoneal dissemination mouse models of human colorectal and gastric cancer.

[This corrects the article DOI: 10.3892/ol.2017.6258.].

Frameless Free-Hand Navigation-Guided Biopsy for Brain Tumors: A Simpler Method with an Endoscope Holder.

CONTEXT/AIMS: Given the limitations of current navigation-guided brain biopsy methods, we aimed to introduce a novel method and validate its safety and accuracy. SETTING AND DESIGN: This was a retrospective study of twenty consecutive patients who underwent brain biopsy at Shimane University Hospital, Japan. SUBJECTS AND METHODS: Clinical records of 13 and 7 patients who underwent brain biopsy with the novel frameless free-hand navigation-guided biopsy (FFNB) method or a framed computed tomography-guided stereotactic biopsy (CTGB) method, respectively, were retrospectively reviewed. We compared age, sex, tumor location, histological diagnosis, maximum size of the tumor (target), depth from target to cortical surface on the same slice of CT or magnetic resonance imaging, operative position, anesthesia method, setup time for biopsy, incision-to-closure time, trial times for puncture, success rate, and complications in the two groups. STATISTICAL ANALYSIS: Fisher's exact test and the Wilcoxon rank-sum test were performed. RESULTS: Clinical characteristics and lesion size did not differ significantly between the FFNB and CTGB groups. The depth of the target lesion was significantly greater in the CTGB group (P < 0.05). All FFNB and CTGB procedures reached and obtained the target tissue. The number of punctures and the average incision-to-closure time did not differ between the FFNB and CTGB groups. However, the preoperative setup time was significantly shorter using FFNB (P = 0.0003). No complications were observed in either group. CONCLUSIONS: FFNB was comparable with CTGB in terms of safety, accuracy, and operative duration. The preoperative setup time was shorter using FFNB. Therefore, FFNB is a feasible method for brain tumor biopsy.

Aggressive Intraoperative Cisternal Clot Removal After Clipping Aneurismal Subarachnoid Hemorrhage in Elderly Patients.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (aSAH) in the elderly often has a poor prognosis even after surgical treatment in the acute phase. Additionally, subarachnoid clots are the strongest predictors of cerebral vasospasm and tend to be thicker and heavier due to cerebral atrophy. We aimed to compare the conventional surgical treatment in such patients and identify the independent predictors of a favorable outcome after aggressive surgical clot removal. METHODS: We included 40 patients with aSAH aged 70 or older. Each patient underwent aneurysmal clipping. We used the modified Rankin Scale to assess the primary outcome of neurologic status at discharge. We performed univariate analysis using the following factors: sex, age, neurologic, and general medical condition, radiographic data, aneurysm location, treatment approach, and timing of the aneurysm surgery. We divided the patients into irrigation and nonirrigation groups. We focused mainly on subarachnoid clots and analyzed them semiquantitatively using computed tomography. RESULTS: Clot removal was significantly greater in the irrigation group (n = 21) than in the nonirrigation group (n = 19). The period of intrathecal drainage was significantly shorter in the irrigation group (P = 0.002). The rate of occurrence of new low-density areas on CT scans was higher in the nonirrigation group. Outcomes were better in the irrigation group (P = 0.010). CONCLUSIONS: In elderly patients with aSAH in the acute phase, aggressive surgical clot removal after clipping showed favorable outcomes by facilitating early out-of-bed mobilization.

Takotsubo cardiomyopathy following mechanical thrombectomy for acute ischemic stroke: illustrative case.

BACKGROUND: Excess neurological stress by hemorrhagic stoke induces cardiomyopathy, namely takotsubo cardiomyopathy. Here, the authors report a case of takotsubo myopathy following mechanical thrombectomy for acute large vessel occlusion. OBSERVATIONS: A 73-year-old man was emergently brought to the authors' hospital because of left hemiparesis and consciousness disturbance. An ischemic lesion of the right cerebral hemisphere and the right internal carotid artery occlusion was revealed. Emergently, endovascular treatment was performed, and occlusion of the artery was reanalyzed. However, he suffered from hypotension with electrocardiogram abnormality. Subsequently, coronary angiography was performed, but the arteries were patent. The authors made a diagnosis of takotsubo cardiomyopathy. LESSONS: Endovascular recanalization for large cerebral artery occlusion is so effective that it is becoming widely used. Even in the successful recanalization, we need to care for the takotsubo cardiomyopathy.

Modification of TRPV4 activity by acetaminophen.

N-Acetyl-p-aminophenol (APAP/acetaminophen) is a widely used analgesic/antipyretic with weaker inhibitory effects on cyclooxygenase compared to those of non-steroidal anti-inflammatory drugs. The effect of APAP is mediated by its metabolites, N-arachidonoyl-phenolamine and N-acetyl-p-benzoquinone imine, which activate transient receptor potential (TRP) channels, including TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or cannabinoid receptor type 1. However, the exact molecular mechanism underlying the cellular actions of APAP remains unclear. Recently, we observed that APAP promotes cell migration through TRPV4; in this study, we examined the effect of APAP on Ca2+-channel activity of TRPV4. In the rat cell line PC12 expressing TRPV4, GSK1016790A (GSK), a TRPV4 agonist, stimulated an increase in [Ca2+]i; these effects were abrogated by HC-067047 treatment. This GSK-induced Ca2+ entry through TRPV4 was inhibited by APAP in a dose-dependent manner, whereas APAP alone did not affect [Ca2+]i. The specificity of the effect of APAP on TRPV4 was further confirmed using HeLa cells, which lack endogenous TRPV4 but stably express exogenous TRPV4 (HeLa-mTRPV4). GSK-induced [Ca2+]i elevation was only observed in HeLa-mTRPV4 cells compared to that in the control HeLa cells, indicating the specific action of GSK on TRPV4. APAP dose-dependently suppressed this GSK-induced Ca2+ entry in HeLa-mTRPV4. However, it is unlikely that the metabolites of APAP were involved in these effects as the reaction in this study was rapid. The results suggest that APAP suppresses the newly identified target TRPV4 without being metabolized and exerts antipyretic/analgesic and/or other effects on TRPV4-related phenomena in the body. The effect of APAP on TRPV4 was opposite to that on TRPV1 or TRPA1, as the latter is activated by APAP.

Schlafen11 Expression Is Associated With the Antitumor Activity of Trabectedin in Human Sarcoma Cell Lines.

BACKGROUND/AIM: Trabectedin is a DNA-damaging agent and has been approved for the treatment of patients with advanced soft tissue sarcoma. Schlafen 11 (SLFN11) was identified as a dominant determinant of the response to DNA-damaging agents. The aim of the study was to clarify the association between SLFN11 expression and the antitumor activity of trabectedin. MATERIALS AND METHODS: The antitumor activity of trabectedin was evaluated under different expression levels of SLFN11 regulated by RNA interference and CRISPR-Cas9 systems, and the combined antitumor activity of ataxia telangiectasia and Rad3-related protein kinase (ATR) inhibitor and trabectedin in sarcoma cell lines using in vitro a cell viability assay and in vivo xenograft models. RESULTS: SLFN11-knockdown cell lines had a lower sensitivity to trabectedin, compared to parental cells. ATR inhibitor enhanced the antitumor activity of trabectedin in SLFN11-knockdown cells and in a SLFN11-knockout xenograft model. CONCLUSION: SLFN11 expression might be a key factor in the antitumor activity of trabectedin.

TAS4464, A Highly Potent and Selective Inhibitor of NEDD8-Activating Enzyme, Suppresses Neddylation and Shows Antitumor Activity in Diverse Cancer Models.

NEDD8-activating enzyme (NAE) is an essential E1 enzyme of the NEDD8 conjugation (neddylation) pathway, which controls cancer cell growth and survival through activation of cullin-RING ubiquitin ligase complexes (CRL). In this study, we describe the preclinical profile of a novel, highly potent, and selective NAE inhibitor, TAS4464. TAS4464 selectively inhibited NAE relative to the other E1s UAE and SAE. TAS4464 treatment inhibited cullin neddylation and subsequently induced the accumulation of CRL substrates such as CDT1, p27, and phosphorylated IκBα in human cancer cell lines. TAS4464 showed greater inhibitory effects than those of the known NAE inhibitor MLN4924 both in enzyme assay and in cells. Cytotoxicity profiling revealed that TAS4464 is highly potent with widespread antiproliferative activity not only for cancer cell lines, but also patient-derived tumor cells. TAS4464 showed prolonged target inhibition in human tumor xenograft mouse models; weekly or twice a week TAS4464 administration led to prominent antitumor activity in multiple human tumor xenograft mouse models including both hematologic and solid tumors without marked weight loss. As a conclusion, TAS4464 is the most potent and highly selective NAE inhibitor reported to date, showing superior antitumor activity with prolonged target inhibition. It is, therefore, a promising agent for the treatment of a variety of tumors including both hematologic and solid tumors. These results support the clinical evaluation of TAS4464 in hematologic and solid tumors.

Trifluridine/tipiracil overcomes the resistance of human gastric 5-fluorouracil-refractory cells with high thymidylate synthase expression.

Trifluridine/tipiracil (FTD/TPI or TFTD, also known as TAS-102) is a combination of the antineoplastic thymidine analog, FTD, and thymidine phosphorylase inhibitor, TPI (molar ratio 1:0.5). FTD/TPI was approved in Japan, the United States, and the European Union for the treatment of unresectable advanced or recurrent colorectal cancer. We evaluated the in vitro and in vivo efficacy and mechanisms of action of FTD and FTD/TPI against 5-fluorouracil (5-FU)-resistant MKN45/5FU, MKN74/5FU, and KATOIII/5FU human gastric cancer cells overexpressing thymidylate synthase (TS) and their respective parent cell lines. MKN45/5FU and KATOIII/5FU cells were not cross-resistant to FTD, whereas MKN45/5FU cells were 3.7-fold more resistant than the parental cells in vitro. FTD was also incorporated into genomic DNA in a concentration-dependent manner in 5-FU-resistant and parental cells. Additionally, deoxyuridine monophosphate levels in MKN45/5FU cells after 24-h FTD treatment were 3.0-fold higher than those in parental cells, and FTD treatment for 72 h induced G2/M arrest in MKN45/5FU cells, unlike the S phase arrest in MKN45 cells. Thus, TS-overexpressing MKN45/5FU cells, but not MKN74/5FU and KATOIII/5FU cells, showed partial cross-resistance to FTD. However, FTD/TPI (administered orally twice a day) exhibited antitumor activity to the same extent in MKN45 and MKN45/5FU xenograft mouse models, overcoming in vitro cross-resistance to FTD. DNA incorporation rather than TS inhibition seems to be the main action of FTD under these in vivo conditions. Thus, FTD/TPI is a promising chemotherapeutic agent against gastric cancers recurring following 5-FU therapy.

Pyothorax-associated Angiosarcoma Metastasized to the Brain with Multiple and Progressively Expanding Hematomas: Case Report and Literature Review.

The brain metastasis of angiosarcoma is very rare, and little is known about its clinical features or therapeutic strategy. A 74-year-old male was hospitalized for disturbance of consciousness. Radiological examination revealed multiple cerebral hematomas. Gadolinium contrast-enhanced magnetic resonance imaging showed no significant enhancement at any of the lesions. To detect a suspected metastatic brain tumor or abscess, a full-body scan was performed but revealed only a poorly enhanced mass in the removal cavity caused by thoracoplasty in the left upper chest. After admission, a cascade of expansion of those hematomas occurred in the right frontal, left parietal, and right temporal lobes, and each lesion thus had to be sequentially removed by craniotomies. The pathological diagnosis of the right frontal lesion was an abscess with hematoma. However, a malignant vascular tumor was highly suspected because of many CD31(+)/Ki-67(+) cells in the left parietal lesion. A mass in the scar caused by thoracoplasty was suspected to be the primary lesion, and brain metastasis of angiosarcoma was finally diagnosed. Whole-brain irradiation and systemic paclitaxel administration were performed, and a complete response for the brain lesions was obtained for 22 months; the patient then died of an intratracheal hemorrhage. This case represents the first report of multiple brain metastases from pyothorax-associated angiosarcoma accompanied by sequentially and gradually expanding hematomas, as well as the first case with the control of metastatic brain lesions for over 1 year after the onset of neurological symptoms. Control of the lesions could be achieved by their total removal with complete hemostasis, as well as additional radio- and chemotherapy.

Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

BACKGROUND: N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. METHODS: Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. RESULTS: APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. CONCLUSION: APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions.

Verification procedure for isocentric alignment of proton beams.

We present a technique--based on the Lutz, Winston, and Maleki test used in stereotactic linear accelerator radiosurgery--for verifying whether proton beams are being delivered within the required spatial coincidence with the gantry mechanical isocenter. Our procedure uses a proton beam that is collimated by a circular aperture at its central axis and is then intercepted by a small steel sphere rigidly supported by the patient couch. A laser tracker measurement system and a correction algorithm for couch position assures precise positioning of the steel sphere at the mechanical isocenter of the gantry. A film-based radiation dosimetry technique, chosen for the good spatial resolution it achieves, records the proton dose distribution for optical image analysis. The optical image obtained presents a circular high-dose region surrounding a lower-dose area corresponding to the proton beam absorption by the steel sphere, thereby providing a measure of the beam alignment with the mechanical isocenter. We found the self-developing Gafchromic EBT film (International Specialty Products, Wayne, NJ) and commercial Epson 10000 XL flatbed scanner (Epson America, Long Beach, CA) to be accurate and efficient tools. The positions of the gantry mechanical and proton beam isocenters, as recorded on film, were clearly identifiable within the scanning resolution used for routine alignment testing (0.17 mm per pixel). The mean displacement of the collimated proton beam from the gantry mechanical isocenter was 0.22 +/- 0.1 mm for the gantry positions tested, which was well within the maximum deviation of 0.50 mm accepted at the Proton Therapy Center in Houston.

[Benefits of TS-1 plus leucovorin combination therapy for colorectal cancer: evaluation of therapeutic effect of TS-1 and leucovorin combination therapy using rodent model fed a low folate diet].

We evaluated the therapeutic effect of TS-1, a novel oral fluoropyrimidine, in combination with leucovorin with in vivo experiments using a murine tumor xenograft model fed a low folate diet. The reduced folate levels in the tumors of mice fed a low folate diet were significantly lower than those in the tumors of mice fed a normal diet. In addition, the basal reduced folate levels in the tumors of mice fed a low folate diet were comparable to those in human colorectal cancer tissues. Furthermore, when leucovorin was orally administered, the reduced folate levels in the tumors of mice fed a low folate diet were more than two-fold higher than those of mice fed the normal diet. The leucovorin-induced potentiation of TS-1 antitumor activity was obviously higher in COL-1 tumor-bearing mice fed a low folate diet than in mice fed a normal diet. The remarkable increase in reduced folate levels following the administration of leucovorin contributed to the leucovorin-induced potentiation of TS-1 antitumor activity in this low-folate-diet animal model. These results suggest that rodent models fed a low folate diet are suitable for in vivo evaluation of reduced folate drugs like leucovorin. In this model, the combination of leucovorin with TS-1 resulted in a higher antitumor efficacy than TS-1 alone or the combination of UFT and leucovorin, suggesting that TS-1 and leucovorin combination therapy may be an effective regimen for patients with colorectal cancer.