Tumor microenvironment confers mTOR inhibitor resistance in invasive intestinal adenocarcinoma.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is frequently activated in cancers and can be counteracted with the clinical mTORC1 inhibitors everolimus and temsirolimus. Although mTORC1 and dual mTORC1/2 inhibitors are currently under development to treat various malignancies, the emergence of drug resistance has proven to be a major complication. Using the cis-Apc/Smad4 mouse model of locally invasive intestinal adenocarcinoma, we show that administration of everolimus or the dual mTORC1/2 inhibitor AZD8055 significantly reduces the growth of intestinal tumors. In contrast, although everolimus treatment at earlier phase of tumor progression delayed invasion of the tumors, both inhibitors exhibited little effect on blocking invasion of the tumors when administered later in their progression. Biochemical and immunohistochemical analyses revealed that treatment of cis-Apc/Smad4 mice with everolimus or AZD8055 induced marked increases in epidermal growth factor receptor (EGFR) and MEK/ERK signaling in tumor epithelial and stromal cells, respectively. Notably, co-administration of AZD8055 and the EGFR inhibitor erlotinib or the MEK inhibitor trametinib was sufficient to suppress tumor invasion in cis-Apc/Smad4 mice. These data indicate that mTOR inhibitor resistance in invasive intestinal tumors involves feedback signaling from both cancer epithelial and stromal cells, highlighting the role of tumor microenvironment in drug resistance, and support that simultaneous inhibition of mTOR and EGFR or MEK may be more effective in treating colon cancer.

Development of spontaneous tumours and intestinal lesions in Fhit gene knockout mice.

The fragile histidine triad (FHIT) gene is frequently inactivated in various types of tumours. However, the system-wide pathology caused by FHIT inactivation has not been examined in detail. Here we demonstrate that Fhit gene knockout mice develop tumours in the lymphoid tissue, liver, uterus, testis, forestomach and small intestine, together with structural abnormalities in the small intestinal mucosa. These results suggest that Fhit plays important roles in systemic tumour suppression and in the integrity of mucosal structure of the intestines.

Cloning of differentially expressed sequence tags from nickel-transformed human embryonic lung cells.

Differential display polymerase chain reaction (DD-PCR) was used to analyze the differentially expressed genes from nickel-transformed human embryonic lung (HEL) cells (MRC-9 and IMR-90) and their control counterparts (non-treated). Two genes, MS515 and IC82, were confirmed by Northern blot analysis. MS515 was detected in control and nickel oxide (NiO)-transformed MRC-9 cells, as well as in non-small cell lung cancer (NSCLC) EBC-1 cells, while very weak expression was observed in nickel subsulfide (Ni(3)S(2))-transformed MRC-9 cells and small cell lung cancer (SCLC) SBC-2 cells. IC82 could not be detected in control IMR-90 cells, while it was expressed in EBC-1 cells and NiO- and Ni(3)S(2)-transformed IMR-90 cells. These findings indicate that individual nickel compounds have their own target gene(s) in inducing lung cancer. Sequencing analyses showed that the MS515 gene shared a high degree of homology (over 80%) with the gene Mena, which is involved in actin polymerization. IC82 showed 99% homology with human chromosome 4 clone C0440E08 and a coding sequence in the brain. The roles of these two genes in nickel carcinogenesis will be discussed.

[Long-term prognosis of asthmatic patients treated with low-dose beclomethasone dipropionate].

To clarify the prognosis of asthmatics treated with low-dose of inhaled beclomethasone dipropionate (BDP), we retrospectively assessed 43 patients treated with initial dose of 200 or 400 micrograms/day for 5 years, and obtained the following results. 1) 15 patients achieved step-down therapy (group A), 17 patients maintained initial dose of BDP (group B), and 11 patients required step-up therapy of BDP or daily use of oral prednisolone (group C). 2) There was no significant difference in age, sex, duration of disease, severity of disease, peripheral eosinophil counts, %FEV1 and histamine PC20 before BDP treatment among three groups. The percentage of atopic asthmatics was significantly higher in group C than in group A. 3) There was no significant difference in symptom and histamine PC20 between after 1 year state and after 5 years state in three groups. 4) After 1 year from the start of BDP treatment, only 18% patients got symptom free and neither patients exceeded 20,000 micrograms/ml of histamine PC20 in group C. Long-term treatment of low-dose BDP inhalation was effective on mild/moderate asthmatics. Patients requiring step-up therapy had not got sufficient improvement in bronchial hyperresponsiveness after one-year treatment.

Therapeutic effect of a new synthetic lipid A analog (ONO-4007) on a tumor implanted at different sites in rats.

The therapeutic effect of ONO-4007, a new lipid A analog, was examined on the 13762NF mammary adenocarcinoma in the F-344 rat system. Intravenous administration of ONO-4007 (2.5 mg/kg) had a very potent effect on subcutaneously (s.c.) inoculated 13762NF tumor cells, but none at all on intravenously (i.v.) or intraperitoneally (i.p.) inoculated ones. Also no beneficial therapeutic effect of ONO-4007 was observed on i.v. or i.p. inoculated tumor cells when it was combined with anticancer drugs such as carboplatin (CBDCA) or cyclophosphamide (CY). The therapeutic efficacy of ONO-4007 (2.5 mg/kg, 3 times a day for 6 days) for s.c. inoculated tumor cells was greater than that of CBDCA (20-30 mg/kg x 1), and it was also shown to stimulate spleen cells to produce tumor necrosis factor alpha (TNF-alpha) in vitro in a dose-dependent manner. These results suggest that enhanced production of TNF-alpha may be responsible in part for the beneficial effect of ONO-4007 against s.c. inoculated tumor cells. ONO-4007 may in the future become a useful modality for the treatment of human cancers.

Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: a platform for antiviral drug design.

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-pro penone, to 2.1-A resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Therapeutic effect of clarithromycin on a transplanted tumor in rats.

The therapeutic antitumor effect of clarithromycin (CAM) was examined with the 13762NF mammary adenocarcinoma and F-344 rat system. When CAM treatment at a dosage of 2 mg/kg of body weight orally for 21 days was commenced after inoculation of the tumor, no significant decrease in death rate was observed, although the loss in body weight was less than that in the untreated group. When tumor-bearing (TB) rats were treated with CAM in combination with carboplatin or cyclophosphamide, a significant decrease in the death rate was obtained, although neither treatment alone proved to be effective. A beneficial effect was also observed when CAM treatment was combined with surgical treatment. CAM showed no direct cytotoxicity to this tumor in vitro according to the MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Spleen cells obtained from TB rats receiving CAM treatment showed a stronger tumor-neutralizing activity than those from rats which had not received CAM treatment (Winn assay). Enhanced induction of cytotoxic cells to allogeneic tumor was also observed in rats immunized with allogeneic tumor cells together with CAM treatment (51Cr release assay). The 13762NF tumor produces transforming growth factor-beta (TGF-beta), tumor necrosis factor alpha, and matrix metalloproteinase-9, and treatment of tumor cells with CAM in vitro for 24 h significantly inhibited the expression of the genes coding for these proteins (reverse transcription-PCR). Levels of expression of the TGF-beta and interleukin-6 genes of spleen cells obtained from CAM-treated TB rats were both significantly lower than those of spleen cells from CAM-untreated TB rats. This study suggests that CAM has biological response modifier activities resulting in a beneficial therapeutic antitumor effect and might be useful for the treatment of human cancers.

Diuretic response to cyclophosphamide in rats bearing a matrix metalloproteinase-9-producing tumour.

When cyclophosphamide (CY) (100-120 mg kg(-1)) was administered intravenously (i.v.) to normal F-344 rats, oliguria occurred over the 5-day observation period. Conversely, in rats bearing matrix metalloproteinase-9 (MMP-9) producing 13762NF mammary adenocarcinoma (MTLn3 clone), polyuria occurred chiefly during the first 24 h after CY treatment. In parallel with urine volume, a decrease in the urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) was observed during the first 5 days after CY treatment in normal rats, but it increased in MTLn3-bearing rats. No elevation in blood urea nitrogen (BUN) or serum creatinine (Cr) values was observed for either group. Both urine volume and urinary excretion of NAG after CY treatment were lower in rats bearing the MTC clone (lower production of MMP-9) than for those bearing the MTLn3 clone. In the case of treatment with cisplatin (CDDP, 4-6 mg kg(-1)), urine volume, urinary NAG excretion and BUN and serum Cr values all increased in normal rats and were all found to be higher in MTLn3-bearing rats than in normal rats. The diuretic response to these drugs in tumour-bearing (TB) rats may be associated with MMP-9 produced by the tumour cells. This report suggests that the nephrotoxicity due to anti-cancer drugs may change when the drugs are used for the treatment of patients bearing a MMP-9-producing tumour.

Coincidental alterations of p16INK4A/CDKN2 and other genes in human lung cancer cell lines.

Cyclin-dependent kinase (CDK) inhibitor genes have recently been proposed as new tumor suppressor genes. To define the possible participation of CDK inhibitor genes in lung carcinogenesis, we investigated the alterations of p15INK4B, p16INK4A, p21Waf1, and p27Kip1 genes in 34 human lung cancer cell lines using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing, and southern dot blot methods. Among the four CDK inhibitor genes, alterations of only the p16INK4A gene were found in 8 out of 34 (24%) cell lines, and all eight cell lines having a p16INK4A gene alteration had an alteration of either the K-ras of p53 gene. Conversely, p16INK4A gene alterations were found in none of the 3 cell lines having Rb gene alterations and none of the 3 cell lines having amplification of the N-myc gene. Polymorphism was found in both p21Waf1 and p27Kip1 genes, but no association was found between the polymorphism and alterations of other genes. These results suggest that p16INK4A gene alterations may play a certain role for lung carcinogenesis in co-operation with either K-ras or p53 gene alterations.

[A fundamental study on clinical significance of measuring eosinophil protein X in spot urine in asthmatics].

To define the clinical significance of measuring eosinophil protein X (EPX) in spot urine in asthmatics, we undertook the fundamental study in 32 stable asthmatics having anti-asthmatic agents and 10 normal healthy controls, and obtained the following results. 1) Peripheral eosinophil counts, urinary EPX (u-EPX), urinary leukotriene E4 (u-LTE4), and serum eosinophil cationic protein (s-ECP) values were significantly higher in asthmatics than those in the controls. 2) U-EPX values were not associated with the type of asthma and severity of the disease. 3) A significant correlation was observed between u-EPX values and peripheral eosinophil counts, but not between s-ECP values and peripheral eosinophil counts. Furthermore, no correlation was observed between u-EPX and s-ECP values. 4) U-EPX values did not correlate with either u-LTE4, %FEV1, or histamine PC20 values. Accordingly, EPX value in spot urine may be a useful maker to assess the activation of eosinophils in asthmatics.

Sarcoidosis accompanied by pleural effusion and multiple bronchial stenoses.

Diffuse parenchymal infiltrates in lower lung fields, right pleural effusion, multiple bronchial stenoses and hepatosplenomegaly were observed in a 30-year-old woman with iritis. Pleural effusion was a yellow exudate which was predominantly lymphocytic. A pulmonary function test showed an obstructive pattern, and multiple bronchial stenoses were observed on bronchoscopy. Both transbronchial lung biopsy (TBLB) and bronchial mucosal biopsy failed in demonstrating non-caseating granulomas, but liver biopsy revealed granulomas consistent with sarcoidosis. This is a very rare case, in which pleural effusion and bronchial stenoses occurred in the same patient.

[Allergic bronchopulmonary aspergillosis in a patient with no symptoms of asthma until after bronchial lavage].

An asymptomatic 56-year-old man was admitted to our hospital because of an abnormal shadow on a chest X-ray film. Allergic bronchopulmonary aspergillosis was diagnosed on the basis of five findings: eosinophilia, immediate skin reactivity to Aspergillus antigen, the presence of precipitating antibodies against Aspergillus antigen, a high concentration of IgE in serum, and central bronchiectasis. He had no symptoms of asthma at the time of diagnosis, but did a few days after he underwent bronchial lavage. We speculate that the asthma attack was related to the bronchial Lavage as follows: First, drainage of mucus plugs by bronchial lavage may have exposed the bronchial epithelium, which had already been sensitized, to aspergillus antigens. Second, the scattered antigen may have dose-dependently stimulated the bronchi. Third, the infection may have increased bronchial responsiveness to the antigen. Symptoms of bronchial asthma are not necessary for the diagnosis of allergic bronchopulmonary aspergillosis.

Grouping of anticancer drugs based on administration method-related toxicity patterns in rats.

As part of the study of the appropriate usage of anticancer drugs, and the effects of the administration method (single dose, or divided doses) on the death rate from toxicity, five anticancer drugs (nimustine hydrochloride = ACNU, cyclophosphamide = CY, carboplatine = CBDCA, mitomycin C = MMC, vindesine = VDS) were studied in F-344 rats. A reduced death rate from toxicity was achieved by divided-dose administration of ACNU and CBDCA (group 1), but it was higher for CY and VDS (group 2), and there was no significant difference with MMC (group 3). In conclusion, the drugs were divided into three groups according to the administration method-related toxicity patterns. Improved knowledge of the properties of anticancer drugs is important for the success of chemotherapy.

Synthesis and structure--activity relationships of fused imidazopyridines: a new series of benzodiazepine receptor ligands.

2-Arylimidazo[4,5-c]quinolines and analogous fused imidazopyridines were synthesized and evaluated as benzodiazepine receptor ligands. Affinity to the receptors was greatly affected by the bulkiness of the aryl group at the 2-position, compared to the pyrazoloquinolines such as CGS-9896. Derivatives with an isoxazole moiety at the 2-position showed high binding affinity and in vivo activity. In the imidazo[4,5-c]quinoline series, substitution at the 6-position decreased or abolished activity. Most derivatives with an unsubstituted isoxazolyl group showed antagonist or inverse agonist activity except for the 7-halo analogues, which exhibited agonist activity. On the other hand, 5-methylisoxazol-3-yl or 3-methylisoxazol-5-yl derivatives generally exhibited agonist activity. A similar substitution effect on the isoxazole moiety was observed in the imidazopyridines fused with a nonaromatic ring. From the detailed pharmacological evaluation, S-8510, 2-(3-isoxazolyl)-3,6,7,9-tetrahydroimidazo[4,5-d]pyrano++ +[4,3-b]pyridine monophosphate, possessing weak inverse agonist activity was selected as a therapeutic candidate for the treatment of some symptoms of senile dementia.

A case of small cell carcinoma of the lung associated with hypertrophic pulmonary osteoarthropathy.

A 73-year-old man had complained of painful swelling in the upper and lower extremities for several months before detection of small cell lung carcinoma. A bone scan showed abnormal symmetrical accumulations in the extremities, but periosteal proliferation was not demonstrated by X-ray. When a complete remission was obtained by chemoradiotherapy, the joint manifestation also subsided completely. In almost all reported cases, hypertrophic pulmonary osteoarthropathy was observed in all patients with non-small cell lung carcinoma. To our knowledge, this is the first case report of hypertrophic pulmonary osteoarthropathy associated with small cell lung carcinoma published in the English literature.

Evaluation of a polymerase chain reaction diagnostic method and risk factors for cytomegalovirus pneumonia.

In order to evaluate the diagnostic methods and risk factors for cytomegalovirus (CMV) pneumonia, 22 patients with suspected CMV pneumonia were examined by means of the polymerase chain reaction (PCR) and serological (Ab) methods (complement fixation test = CF and enzyme-immunoassay = EIA: IgG, IgM). Thirteen patients showed positive for either the PCR or serological method; the PCR method was positive in 77% (10/13) (sputum = 8/10, bronchoalveolar lavage fluid = 3/4, peripheral blood = 3/12, urine = 2/11) and the serological method in 64%(7/11)(CF = 5/10, IgG = 3/11, IgM = 0/11). All patients diagnosed as having CMV pneumonia had underlying disease, most of which were idiopathic interstitial pneumonia and malignancies. Twelve out of 13 patients had been treated with steroids for long- or short (pulse)-periods. The cure rate was high for these patients whose PCR results became negative following anti-CMV treatments. These results indicate that a) the steroid treatment acts as a risk factor for the CMV pneumonia, b) the PCR method is a useful tool not only for early diagnosis but also for assessment of the therapeutic effect on CMV pneumonia, and c) concomitant assays of PCR and Ab may be preferable in making a conclusive diagnosis of CMV pneumonia.

Novel angiotensin II receptor antagonists. Design, synthesis, and in vitro evaluation of dibenzo[a,d]cycloheptene and dibenzo[b,f]oxepin derivatives. Searching for bioisosteres of biphenylyltetrazole using a three-dimensional search technique.

Three-dimensional substructure searching (3D search), using the program MACCS-3D, was utilized for designing novel angiotensin II receptor antagonists which contain a bioisostere of the biphenylyltetrazole moiety of DuP 753. A 3D query was prepared from an overlay model of substructures of several potent AII antagonists. The search system retrieved 139 compounds from the database MDDR-3D, which consisted of 29,400 medicinal patent compounds. A tricyclic compound was selected from the retrieved compounds and then evolved by considering steric fitness to the overlay model and synthetic feasibility. Finally, various novel AII antagonists having dibenzo[a,d]cycloheptene or dibenzo[b,f]oxepin were designed and synthesized. The receptor binding activity (Ki) for several members of this series was in the 10(-10) M range, demonstrating the ability of 3D search technique to explore new lead structures.

Synthesis and evaluation of novel pyrazolo[1,5-alpha]pyrimidine derivatives as nonpeptide angiotensin II receptor antagonists.

A novel series of 6-alkyl-7-oxo-4,7-dihydropyrazolo[1,5-alpha]pyrimidine-3-carboxyli c acid derivatives was prepared as angiotensin II (AII) receptor antagonists. When evaluated in an in vitro binding assay using COS cells transfected with a cDNA encoding a human AT1 angiotensin II receptor, the compounds in this series showed Ki values in the range of 0.4-4.0 nM. In anesthetized spontaneously hypertensive rats (SHRs), administration of the 6-propyl derivative 4d (1 mg/kg, i.v.) reduced the mean blood pressure (MBP) by a maximum of more than 30 mmHg from the normal value.

Kinetic studies on the interaction of nonlabeled antagonists with the angiotensin II receptor.

Angiotensin AT1 receptor antagonists are divided into two types, surmountable and insurmountable, based on the way they inhibit angiotensin II-induced vasoconstriction. To elucidate what causes the difference, we studied how antagonists associate with and dissociate from AT1 receptor sites in rat liver membranes. Three antagonists, 6-propyl-7-oxo-4[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-4,7- dihydropyrazolo[1,5-a]pyrimidine-3-carboxylic acid (SRL1080227), 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylic acid (CV-11974) and 2-butyl-3-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo- [4,5-b]pyridine (FK739), showed competitive antagonism when they were added simultaneously with [125I]angiotensin II, but CV11974 and SRL1080227 showed apparently noncompetitive antagonism when membranes were preincubated with each antagonist. The longer the preincubation time with CV11974 or SRL1080227 was, the more effectively the antagonist inhibited [125I]angiotensin II binding, while the inhibition by FK739 did not change with the preincubation time. To estimate their dissociation rate from the receptor binding site, we studied [125I]angiotensin II binding to membranes which had been preincubated with each antagonist and washed twice. Membranes pretreated with FK739 completely recovered the ability to bind [125I]angiotensin II with a period of 60 min, while membranes preincubated with CV11974 did not read this level of recovery. [125I]angiotensin II binding to membranes preincubated with SRL1080227 increased gradually, but did not reach the control level during the experiment. The kinetic properties of SRL1080227, CV11974 and FK739 were consistent with their characteristic modes in inhibiting angiotensin II-induced contraction of isolated rabbit aorta and decreasing blood pressure of spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)