Mechanisms of PTHrP-induced inhibition of smooth muscle contractility in the guinea pig gastric antrum.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) that causes hypercalcemia of malignancy appears to function as an endogenous smooth muscle relaxant. For example, PTHrP released upon bladder wall distension relaxes detrusor smooth muscle to accommodate urine. Here, we explored mechanisms underlying PTHrP-induced suppression of the smooth muscle contractility in the gastric antrum that also undergoes a passive distension. METHODS: Effects of PTHrP on phasic contractions and electrical slow waves in the antral smooth muscle of the guinea pig stomach were studied using isometric tension and intracellular microelectrode recordings, respectively. Fluorescent immunohistochemistry was also carried out to identify the distribution of PTH/PTHrP receptors. KEY RESULTS: Parathyroid hormone-related protein (1-100 nM) reduced the amplitude of phasic contractions and the basal tension. Nω -nitro-l-arginine (L-NA, 100 µM), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ, 10 µM), a guanylate cyclase inhibitor, diminished the PTHrP (10 nM)-induced reduction in the amplitude of phasic contractions. SQ22536 (300 µM), an adenylate cyclase inhibitor, attenuated the PTHrP-induced reduction in basal tension. The combination of ODQ (10 µM) and SQ22536 (300 µM) inhibited the PTHrP-induced reductions in both phasic contractions and basal tension. PTHrP (100 nM) had no inhibitory effect on the electrical slow waves in the antral smooth muscle. PTH/PTHrP receptors were expressed in cell bodies of PGP9.5-positive neurons in the myenteric plexus. CONCLUSIONS & INFERENCES: Parathyroid hormone-related protein exerts its inhibitory actions on the antral smooth muscle via both nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and cyclic adenosine monophosphate (AMP) pathways. Thus, PTHrP may act as an endogenous relaxant of the gastric antrum employing the two complementary signaling pathways to ensure the adaptive relaxation of stomach.

HDAC3 regulates DNMT1 expression in multiple myeloma: therapeutic implications.

Epigenetic signaling pathways are implicated in tumorigenesis and therefore histone deacetylases (HDACs) represent novel therapeutic targets for cancers, including multiple myeloma (MM). Although non-selective HDAC inhibitors show anti-MM activities, unfavorable side effects limit their clinical efficacy. Isoform- and/or class-selective HDAC inhibition offers the possibility to maintain clinical activity while avoiding adverse events attendant to broad non-selective HDAC inhibition. We have previously reported that HDAC3 inhibition, either by genetic knockdown or selective inhibitor BG45, abrogates MM cell proliferation. Here we show that knockdown of HDAC3, but not HDAC1 or HDAC2, as well as BG45, downregulate expression of DNA methyltransferase 1 (DNMT1) mediating MM cell proliferation. DNMT1 expression is regulated by c-Myc, and HDAC3 inhibition triggers degradation of c-Myc protein. Moreover, HDAC3 inhibition results in hyperacetylation of DNMT1, thereby reducing the stability of DNMT1 protein. Combined inhibition of HDAC3 and DNMT1 with BG45 and DNMT1 inhibitor 5-azacytidine (AZA), respectively, triggers synergistic downregulation of DNMT1, growth inhibition and apoptosis in both MM cell lines and patient MM cells. Efficacy of this combination treatment is confirmed in a murine xenograft MM model. Our results therefore provide the rationale for combination treatment using HDAC3 inhibitor with DNMT1 inhibitor to improve patient outcome in MM.

KDM6B modulates MAPK pathway mediating multiple myeloma cell growth and survival.

Recent studies have delineated cancer-type-specific roles of histone 3 lysine 27 (H3K27) demethylase KDM6B/JMJD3 depending on its H3K27 demethylase activity. Here we show that KDM6B is expressed in multiple myeloma (MM) cells; and that shRNA-mediated knockdown and CRISPR-mediated knockout of KDM6B abrogate MM cell growth and survival. Tumor necrosis factor-α or bone marrow stromal cell culture supernatants induce KDM6B, which is blocked by IKKß inhibitor MLN120B, suggesting that KDM6B is regulated by NF-κB signaling in MM cells. RNA-seq and subsequent ChIP-qPCR analyses reveal that KDM6B is recruited to the loci of genes encoding components of MAPK signaling pathway including ELK1 and FOS, and upregulates expression of these genes without affecting H3K27 methylation level. Overexpression of catalytically inactive KDM6B activates expression of MAPK pathway-related genes, confirming its function independent of demethylase activity. We further demonstrate that downstream targets of KDM6B, ELK1 and FOS, confer MM cell growth. Our study therefore delineates KDM6B function that links NF-κB and MAPK signaling pathway mediating MM cell growth and survival, and validates KDM6B as a novel therapeutic target in MM.

Rational combination treatment with histone deacetylase inhibitors and immunomodulatory drugs in multiple myeloma.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.

Class IIa HDAC inhibition enhances ER stress-mediated cell death in multiple myeloma.

Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.

Histone deacetylase 3 as a novel therapeutic target in multiple myeloma.

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

Small-molecule multi-targeted kinase inhibitor RGB-286638 triggers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs.

Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

Myeloablative cord blood transplantation for adults with hematological malignancies using tacrolimus and short-term methotrexate for graft-versus-host disease prophylaxis: single-institution analysis.

We studied clinical outcomes of 25 adult patients with hematological malignancies who underwent cord blood transplantation (CBT) after a myeloablative conditioning regimen, including high-dose cytosine arabinoside (CA) (8 g/m(2)), cyclophosphamide (CY) (120 mg/kg), and total-body irradiation (TBI) (12 Gy). For graft-versus-host disease (GVHD) prophylaxis, all patients received a combination of tacrolimus and short-term methotrexate (sMTX). Neutrophil engraftment was achieved in 20 of 25 patients. Of the 22 evaluable patients, 2 and 7 had grades I and II acute GVHD, respectively, and only 1 developed grade III acute GVHD after discontinuation of tacrolimus due to encephalopathy. Chronic GVHD developed in 13 of 19 evaluable patients, including 4 with the extensive type. However, the Karnofsky scores of survivors at 1 year after CBT were 90% or 100%. Eight of 25 patients died of nonrelapse causes (n = 4) and relapse/progressive disease (n = 4); 17 patients are currently alive with 15 free of disease at the present time (median follow-up, 24 months). The probability of disease-free survival at 2 years among patients with standard risk was 89% and that of high-risk patients was 30%. Transplantation-related mortality within 100 days was 12%. These results suggested that the CA/CY/TBI combination is a promising conditioning regimen for myeloablative CBT. Furthermore, tacrolimus and sMTX seemed to have suppressed severe acute GVHD and chronic GVHD, which may also contribute to the favorable results.

Study of an improved Allyl Di-Glycol carbonate sheet for high energy proton detection.

An allyl di-glycol carbonate (ADC) sheet which has been utilised as a neutron detector for personal dosimetry has recently been studied for its application as a device for radiation exposure control for astronauts in space, where protons are the dominant radiation. It is known that the fabrication process, modified by adding some kind of antioxidant to improve the sensitivity of ADC to high energy protons, causes a substantial increase in false tracks, which disturb the automatic counting of proton tracks using the auto-image analyser. This made clear the difficulty of fabricating ADC sheets which have sufficient sensitivity to high energy protons, while maintaining a good surface. In this study, we have tried to modify the fabrication process to improve the sensitivity to high energy protons without causing a deterioration of the surface condition of ADC sheets. We have successfully created fairly good products.

Relationship between large conductance calcium-activated potassium channel and bursting activity.

To elucidate the role of the large conductance calcium-activated potassium channel (BK(Ca) channel) in the production of bursting activity, which is characteristic of convulsions, effects of iberiotoxin (IbTX), a selective blocker of the BK(Ca) channel, on bursting activity, induced by various procedures were examined using primary cultured neurons from the cerebral cortex of mice. IbTX completely inhibited bursting activity induced by pentylenetetrazol (PTZ), caffeine, 1,4,5-inositol triphosphate (IP3) and direct forced increase of intracellular calcium. Inherent spontaneous bursting activity in the cerebral cortical neurons of the El mouse, which shows a high susceptibility to convulsions was also completely inhibited by IbTX. Apamin, a specific blocker of the small conductance calcium-activated potassium channel (SK(Ca) channel) showed no inhibition of bursting activity. These findings suggest that the BK(Ca) channel is essential for the production of bursting activity, and also suggest the possibility of clinical use of blocking agents of the BK(Ca) channel against intractable epilepsy.

Gene mapping of SEZ group genes and determination of pentylenetetrazol susceptible quantitative trait loci in the mouse chromosome.

Gene mapping of the newly discovered SEZ genes (seizure-related genes) in the mouse was performed by linkage analysis. SEZ6 was on chromosome 11, SEZ12 on chromosome 16, SEZ15 on chromosome 3 and SEZ17 (PTZ17) on chromosome 18. The mouse chromosomal locus related to high susceptibility to pentylenetetrazol (PTZ) was also determined by linkage analysis using the recombinant inbred mouse, BXD (C57BLxDBA). A significant level of PTZ susceptibility was found on chromosome 2. Chromosomal loci of the newly discovered SEZ genes were not coincident with the significant chromosomal loci to PTZ susceptibility. Since epilepsy is assumed to be a disease syndrome which is probably manifested by abnormal expression of multifocal genes, determination of the role of each chromosomal locus in the provocation of seizure activity is important.

Regulation of gene expression by herbal medicines--a new paradigm of gene therapy for multifocal abnormalities of genes.

Japanese herbal medicine has long been considered as only supplementary therapy to Western medicine. However, we discovered that an herbal mixture, Saiko-keishi-to-ka shakuyaku (SK, TJ-960), showed regulatory function of gene expression such as increased expression of seizure-related gene PTZ-17, proto-oncogene c-fos and heat shock protein HSP 72. These results provide a scientific basis for an important ancient concept and usage of herbal mixtures as a "therapy against diseases which will be suffered in the future". Our results also give an adequate provide break-throughs for therapy and even prevention of intractable epilepsy, Alzheimer's disease, developmental disorders during pregnancy and the postnatal period, and also probably for prevention of metastasis or relapse of various cancers.

Protective effects of peony root extract and its components on neuron damage in the hippocampus induced by the cobalt focus epilepsy model.

Protective effects of peony root extract and its components on neuron damage in the CA1 area of the hippocampus induced by the cobalt focus epilepsy model were examined. Neuron damage in the CA1 area of the hippocampus and frequent spike discharges induced by application of metallic cobalt to the cerebral cortex of rats were completely prevented when peony root extract was continuously administered orally at 1 g/kg/day for 30 days prior to cobalt application. Component crude gallotannin fraction showed marked but incomplete protective action. A combination of crude gallotannin fraction and paeoniflorin showed complete protective action in the same way as peony root extract against neuron damage although use of paeoniflorin alone had no effect. These findings together with our previous reports indicate that peony root extract and its component, gallotannin, have excellent protective effects on neuron damage in addition to anticonvulsant action by prior oral administration.

[Marked increase of atmospheric pollen dispersion of ragweed (Ambrosia spp.)--annual changes in atmospheric pollen counts of major allergen plants in autumn in Saitama Prefecture].

An annual atmospheric pollen survey was performed for 14 consecutive years in the autumn at Sakado city, Saitama prefecture. The survey was performed on the transition of pollen dispersion of major allergen plants: ragweed (Ambrosia spp.), Humulus japonicus, Artemisia spp. and Gramineae. 1. Annual total pollen count of ragweed showed marked increases beginning from 1991. Total pollen count in 1991 was 8.8 times and that in 1996 was 18.6 times that in 1983. This increase is probably caused by marked proliferation of giant ragweed which is left without mowing as it is on a dry riverbed, and consequently produces much more pollen than short ragweed. 2. Annual increases in total pollen counts of other major plants which disperse their pollen in the same season as ragweed were 0.95 times in 1991 and 0.5 times in 1996 that in 1983 for Humulus japonicus, 0.68 times in 1991 and 1.5 times in 1996 that in 1983 for Artemisia spp. and 1.3 times in 1991 and 1.4 times in 1996 that in 1983 for Gramineae. None of these species showed a marked increase of pollen dispersion although they showed some annual variation. The above findings suggest that changes in the proliferous state of various allergenic plants due to environmental change should be considered with respect to characteristics of pollen allergy.

[Pollen dispersion of Cryptomeria japonica in autumn].

To clarify the significance of pollen dispersion of Cryptomeria japonica in autumn, the distribution pattern of pollen dispersion using specimens collected from 1987 to 1995 by Durhum collector was examined. After the dispersion period in the spring, the pollen count was suddenly decreased. The total pollen count from October to December showed no relation to the total pollen count for that of in spring of the same year; but it was closely correlated (r = 0.877) with the total pollen count during the dispersion period in the spring of the next year. In particular, accurate correlation between the total pollen count during November and that in January to May of the following year was observed with the correlation index of r = 0.909. Patients with a pollinosis probably for Cryptomeria japonica were also observed in the autumn. These results suggest that we can forecast the degree of pollen dispersion of Cryptomeria japonica in the coming year from the total pollen count during November of the year before.

Septate-like junctions in the normal male rat pituitary gland.

Septate-like junctions were observed in the rat anterior pituitary gland of the adult male solely between adjacent folliculo-stellate cells. Considering their location, it is presumed that their function is cellular adhesion and mechanical support for the hypophyseal follicles.