Calhex231 Ameliorates Cardiac Hypertrophy by Inhibiting Cellular Autophagy in Vivo and in Vitro.

BACKGROUND/AIMS: Intracellular calcium concentration ([Ca2+]i) homeostasis, an initial factor of cardiac hypertrophy, is regulated by the calcium-sensing receptor (CaSR) and is associated with the formation of autolysosomes. The aim of this study was to investigate the role of Calhex231, a CaSR inhibitor, on the hypertrophic response via autophagy modulation. METHODS: Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in 40 male Wistar rats, while 10 rats underwent a sham operation and served as controls. Cardiac function was monitored by transthoracic echocardiography, and the hypertrophy index was calculated. Cardiac tissue was stained with hematoxylin and eosin (H&E) or Masson's trichrome reagent and examined by transmission electron microscopy. An angiotensin II (Ang II)-induced cardiomyocyte hypertrophy model was established and used to test the involvement of active molecules. Intracellular calcium concentration ([Ca2+]i) was determined by the introduction of Fluo-4/AM dye followed by confocal microscopy. The expression of various active proteins was analyzed by western blot. RESULTS: The rats with TAC-induced hypertrophy had an increased heart size, ratio of heart weight to body weight, myocardial fibrosis, and CaSR and autophagy levels, which were suppressed by Calhex231. Experimental results using Ang II-induced hypertrophic cardiomyocytes confirmed that Calhex231 suppressed CaSR expression and downregulated autophagy by inhibiting the Ca2+/calmodulin-dependent-protein kinase-kinase-ß (CaMKKß)­ AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway to ameliorate cardiomyocyte hypertrophy. CONCLUSIONS: Calhex231 ameliorates myocardial hypertrophy induced by pressure-overload or Ang II via inhibiting CaSR expression and autophagy. Our results may support the notion that Calhex231 can become a new therapeutic agent for the treatment of cardiac hypertrophy.

Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries.

Phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension (PAH). We had previously shown that calcium-sensing receptor (CaSR) is expressed in rat PASMCs. However, little is known about the role of CaSR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms. In this study, we investigated whether CaSR induces the proliferation of PASMCs in small pulmonary arteries from both rats and human with PAH. PAH was induced by exposing rats to hypoxia for 7-21 days. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVI), the percentage of medial wall thickness to the external diameter (WT %), and cross-sectional total vessel wall area to the total area (WA %) of small pulmonary arteries were determined by hematoxylin and eosin (HE), masson trichrome and Weigert's staining. The protein expressions of matrix metalloproteinase (MMP)-2 and MMP-9, the tissue inhibitors of metalloproteinase (TIMP)-3, CaSR, proliferating cell nuclear antigen (PCNA), phosphorylated extracellular signal-regulated kinase (p-ERK), and smooth muscle cell (SMC) phenotype marker proteins in rat small pulmonary arteries, including calponin, SMα-actin (SMAα), and osteopontin (OPN), were analyzed by immunohistochemistry and Western blotting, respectively. In addition, immunohistochemistry was applied to paraffin-embedded human tissues from lungs of normal human and PAH patients with chronic heart failure (PAH/CHF). Compared with the control group, mPAP, RVI, WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia. At the same time, the expressions of CaSR, MMP-2, MMP-9, TIMP-3, PCNA, OPN, and p-ERK were markedly increased, while the expressions of SMAα and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats. Neomycin (an agonist of CaSR) enhanced but NPS2390 (an antagonist of CaSR) weakened these hypoxic effects. We further found that the expression change of CaSR, PCNA, and SMC phenotypic marker proteins in PAH/CHF lungs was similar to those in PAH rats. Our data suggest that CaSR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.

RHBDF1 modulates cisplatin sensitivity of small cell lung cancer through YAP1/Smad2 signaling pathway.

Small cell lung cancer (SCLC) is a fatal tumor type that is prone to drug resistance. In our previous study, we showed that human rhomboid-5 homolog-1 (RHBDF1) was differentially expressed in 5 intrinsic cisplatin-resistant SCLC tissues compared with 5 intrinsic cisplatin-sensitive SCLC tissues by RNA sequencing, which intrigued us. We performed gain- and loss-of-function experiments to investigate RHBDF1 function, bioinformatics analysis, qRT-PCR, western blotting, and immunoprecipitation to elucidate the molecular mechanisms as well as detect RHBDF1 expression in SCLC by immunohistochemistry. We found that RHBDF1 knockdown promoted cell proliferation and cisplatin chemoresistance and inhibited apoptosis in vitro and in vivo. These effects could be reversed by overexpressing RHBDF1 in vitro. Mechanistically, RHBDF1 interacted with YAP1, which increased the phosphorylation of Smad2 and transported Smad2 to the nucleus. Among clinical specimens, the RHBDF1 was a low expression in SCLC and was associated with clinicopathological features and prognosis. We are the first to reveal that RHBDF1 inhibited cell proliferation and promoted cisplatin sensitivity in SCLC and elucidate a novel mechanism through RHBDF1/YAP1/Smad2 signaling pathway which played a crucial role in cisplatin chemosensitivity. Targeting this pathway can be a promising therapeutic strategy for chemotherapy resistance in SCLC.

Involvement of dopamine D2 receptors activation in ischemic post-conditioning-induced cardioprotection through promoting PKC-ε particulate translocation in isolated rat hearts.

Dopamine D2 receptors (DR2) are important regulators in many organs, including cardiac system. Protein kinase C (PKC) activation and translocation is associated with cardioprotection against ischemic post-conditioning (PC); however, the regulatory role of DR2 during this process has been unknown. This study hypothesized that the prevention of cardiomyocyte damage by DR2 activation is associated with PKC translocation to the cell membrane. In the present study, we found that the ischemia/reperfusion (I/R) increased the expressions of DR2 mRNA and protein, which were further enhanced by PC. Bromocriptine (DR2 agonist) up-regulated the PC-induced DR2 expressions, and Haloperidol (DR2 antagonist) reversed the increase of DR2 expressions by Bromocriptine. PC reduced I/R-induced cardiomyocytes damage, apoptosis and myocardial infarct size, and improved cardiac function. Compared with PC, Bromocriptine further enhanced the cardioprotective roles of PC, but Haloperidol canceled the protection effect of Bromocriptine. PC up-regulated PKC-ε translocation in the particulate fraction, which was further strengthened by Bromocriptine but canceled by Haloperidol. In the cytosolic fraction, the changes of the PKC-ε translocation were opposite to the particulate fraction. These findings suggest that DR2 activation provides cardioprotection via promoting PC-induced translocation of PKC-ε.

Involvement of calcium-sensing receptor in oxLDL-induced MMP-2 production in vascular smooth muscle cells via PI3K/Akt pathway.

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs) and up-regulated in atherosclerotic lesion by various stimuli, such as oxidized low-density lipoprotein (oxLDL). Calcium-sensing receptor (CaSR) is also expressed in VSMCs, but it remains unclear whether CaSR is associated with overproduction of MMP-2 in VSMCs. In this study, the expression of MMP-2 was detected by real-time PCR and Western blot analysis, and the gelatinolytic activity of MMP-2 was measured using gelatin zymography. Our results showed that oxLDL enhanced MMP-2 expression and activity in rat aortic VSMCs in a time- and dose-dependent manner. In addition, CaSR expression was up-regulated by oxLDL. Manipulating CaSR function in these cells by NPS2390 (an antagonist of CaSR) or GdCl(3) (an agonist of CaSR) affected the oxLDL-induced MMP-2 production. In VSMCs, oxLDL stimulated the rapid activation of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, as determined by Western blot analysis. Phosphorylation of Akt and MMP-2 production stimulated by oxLDL were attenuated by LY294002 (a specific inhibitor of PI3K). Activation of Akt was suppressed by NPS2390 but enhanced by GdCl(3). In contrast, oxLDL had no stimulatory effect on the phosphorylation of JNK, and pretreatment with SP600125 (an inhibitor of JNK) produced no significant effect on oxLDL-induced MMP-2 production. These results suggest that CaSR mediates oxLDL-induced MMP-2 production in VSMCs via PI3K/Akt signal pathway.

Calcium-sensing receptors induce apoptosis during simulated ischaemia-reperfusion in Buffalo rat liver cells.

1. Calcium-sensing receptors (CaSR) exist in a variety of tissues. In 2010, we first identified its functional expression in Buffalo rat liver (BRL) cells and demonstrated that the activation of CaSR was involved in an increased intracellular calcium through the Gq subunit-phospholipase C-inositol triphosphate pathway. However, its role and related mechanism in hepatic ischaemia/reperfusion (I/R) injury is still unclear. 2. Therefore, in the present study, BRL cells were incubated in ischaemia-mimetic solution for 4 h, then reincubated in the normal culture medium for 10 h to establish a simulated I/R model. We assayed the apoptotic ratio of BRL cells by flow cytometry and Hoechst 33342 staining; analyzed the expression of CaSR, cytochrome c (Cyt-c), caspase-3, Bcl-2, Bax, extracellular signal-regulated protein kinase (ERK), and p38 by Western blotting; and measured the concentration of intracellular calcium by laser-scanning confocal microscopy. 3. The results showed that simulated I/R increased the expression of CaSR and induced apoptosis in BRL cells. GdCl(3), a specific activator of CaSR, further increased CaSR expression, intracellular calcium, and apoptosis in BRL cells during I/R. The activation of CaSR downregulated Bcl-2 expression, upregulated Cyt-c, caspase-3, and Bax expressions, and promoted p38 and ERK-1/2 phosphorylation. 4. In conclusion, increased CaSR expression plays a vital role in apoptosis induced by I/R injury, in which its mechanism is related with calcium overload and the activation of the mitochondrial and mitogen-activated protein kinase apoptotic pathways. The regulation of CaSR activity might serve as a novel pharmacological target to prevent and treat liver disease.

Downregulation of the ornithine decarboxylase/polyamine system inhibits angiotensin-induced hypertrophy of cardiomyocytes through the NO/cGMP-dependent protein kinase type-I pathway.

BACKGROUND: Polyamines and nitric oxide (NO) have been involved in the pathogenesis of cardiac hypertrophy. NO can regulate cardiac ion channels by direct actions on G-proteins and adenyl cyclase. The present study was undertaken to elucidate the molecular mechanism of interactions with polyamines and NO in cardiac hypertrophy. METHODS: Cardiaomyocyte hypertrophy was induced by angiotensinII (AngII). Hypertrophy was estimated by cell-surface area, atrial natriuretic peptide (ANP) mRNA expression, and the immunofluorescence of phalloidin. Pretreatment with alpha-difluoromethylornithine (DFMO) was done to deplete putrescine; KT5823 pretreatment was carried out to block the nitric oxide/cGMP-dependent protein kinase type-I (NO/PKG-I) pathway. Expressions of endothelial nitric oxide synthase (eNOS), PKG-I, c-fos and c-myc were analyzed by western blotting and immunofluorescence. The intracellular concentration of free calcium ([Ca2+](i)) was determined by confocal laser scanning microscopy. RESULTS: Hypertrophy of cardiomyocytes was induced by AngII, this caused an increase in putrescine, spermidine and total polyamine pool in association with a decreased level of NO. Expressions of eNOS and PKG-I were down-regulated, [Ca2+](i) was increased, and expressions of c-Fos and c-Myc upregulated. DFMO reversed these changes induced by AngII. CONCLUSIONS: Downregulation of polyamines inhibits cardiomyocyte hypertrophy, which is closely related to [Ca2+](i) and the NO/PKG-I pathway.

The functional expression of calcium-sensing receptor in the differentiated THP-1 cells.

The expression and function of calcium-sensing receptor (CaSR) in differentiated THP-1 (human acute monocytic leukemia cell line) cells are unknown currently. This study investigated above-mentioned issues using TRAP staining, immunofluorescence staining, Western blotting, ELISA, and Laser Confocal Scanning Microscopy techniques. We found that CaSR protein was expressed, and mainly located in the membrane and cytoplasm in differentiated THP-1 cells. Elevated extracellular calcium or GdCl(3) (an agonist of CaSR) raised intracellular calcium concentration. And this increase was inhibited or abolished by NPS2390 (an inhibitor of CaSR), U73122 (a specific inhibitor of phospholipase C, PLC) or thapsigargin (a Ca(2+)-ATPase inhibitor). The extracellular GdCl(3) elevation stimulated both of IL-1beta and TNFalpha release, and this effect of GdCl(3) was inhibited by NPS2390. In conclusion, CaSR is functionally expressed in differentiated THP-1 cells, and the activated CaSR contributes to intracellular calcium increment through Gq-PLC- inositol triphosphate (IP3) pathway and commits to cytokine secretion. These results suggest that CaSR might be involved in a variety of pathological processes mediated by activated monocyte-macrophages.

The functional expression of calcium-sensing receptors in BRL cells and related signal transduction pathway responsible for intracellular calcium elevation.

The calcium-sensing receptors (CaSRs) exist in a variety of tissues and cells. In 2001, Canaff et al. first identified its expression in liver tissue and primary cultured hepatocytes, and demonstrated that GdCl(3) (a specific agonist of CaSR) can cause an increase in intracellular calcium and bile flow. However, authors did not elucidate its mechanisms. Therefore, this study sought to detect CaSR expression in BRL cell line, which is derived from buffalo rat liver, and to reveal the cellular signal transduction pathway by which the CaSR activation results in increased intracellular calcium by BRL cells. In this study, the expression and distribution of CaSR were detected by RT-PCR, Western blotting, and immunofluorescence, and the intracellular calcium concentration [Ca(2+)](i) was measured using LCSM. The results showed that CaSR mRNA and protein were expressed in BRL cells and mainly distributed in cell membrane and cytoplasm. Increased extracellular calcium or GdCl(3) could increase intracellular calcium concentration and CaSR expression. Moreover, this increase of [Ca(2+)](i) could be inhibited or even abolished by U73122 (a specific inhibitor of PLC), 2-APB (an inhibitor of IP(3) receptor), and thapsigargin (an inhibitor of endoplasmic reticulum calcium pump). In conclusion, CaSR is functionally expressed in BRL cells, and activation of CaSR involves in increased intracellular calcium through Gq-PLC-IP(3) pathway.

NPS2390, a Selective Calcium-sensing Receptor Antagonist Controls the Phenotypic Modulation of Hypoxic Human Pulmonary Arterial Smooth Muscle Cells by Regulating Autophagy.

BACKGROUND AND OBJECTIVES: Calcium-sensing receptor (CaSR) is known to regulate hypoxia-induced pulmonary hypertension (HPH) and vascular remodeling via the phenotypic modulation of pulmonary arterial smooth muscle cells (PASMCs) in small pulmonary arteries. Moreover, autophagy is an essential modulator of VSMC phenotype. But it is not clear whether CaSR can regulate autophagy involving the phenotypic modulation under hypoxia. METHODS: The viability of human PASMCs was detected by cell cycle and BrdU. The expressions of proliferation protein, phenotypic marker protein, and autophagy protein in human PASMCs were determined by western blot. RESULTS: Our results showed that hypoxia-induced autophagy was considerable at 24 h. The addition of NPS2390 decreased the expression of autophagy protein and synthetic phenotype marker protein osteopontin and increased the expression of contractile phenotype marker protein SMA-ɑ and calponin via suppressing downstream PI3K/Akt/mTOR signal pathways. CONCLUSIONS: Our study demonstrates that treatment of NPS2390 was conducive to inhibit the proliferation and reverse phenotypic modulation of PASMCs by regulating autophagy levels.

Involvement of calcium-sensing receptor in cardiac hypertrophy-induced by angiotensinII through calcineurin pathway in cultured neonatal rat cardiomyocytes.

Cardiac hypertrophy is a common pathological change accompanying cardiovascular disease. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiovascular tissue. However, the functional involvement of CaSR in cardiac hypertrophy remains unclear. Previous studies have shown that CaSR caused accumulation of inositol phosphate to increase the release of intracellular calcium. Moreover, Ca(2+)-dependent phosphatase calcineurin (CaN) played a vital role in the development of cardiac hypertrophy. Therefore, we investigated the expression of CaSR in cardiac hypertrophy-induced by angiotensin II (AngII) and the effects of CaSR activated by GdCl(3) on the related signaling transduction pathways. The results showed that AngII induced cardiac hypertrophy and up-regulated the expression of CaSR, meanwhile increased the intracellular calcium concentration ([Ca(2+)](i)) and activated CaN hypertrophic signaling pathway. Compared with AngII alone, the above changes were further obvious when adding GdCl(3). But the effects of GdCl(3) on the cardiac hypertrophy were attenuated by CsA, a specific inhibitor of CaN. In conclusion, these results suggest that CaSR is involved in cardiac hypertrophy-induced by AngII through CaN pathway in cultured neonatal rat cardiomyocytes.

Mammalian Eps15 homology domain 1 promotes metastasis in non-small cell lung cancer by inducing epithelial-mesenchymal transition.

The identification of the earliest molecular events responsible for the metastatic dissemination of non-small cell lung cancer (NSCLC) remains critical for early detection, prevention, and treatment interventions. In this study, we hypothesized that Mammalian Eps15 homology domain 1 (EHD1) might be responsible for the metastatic behavior of cells in NSCLC. We demonstrated that upregulation of EHD1 is associated with lymph nodes metastasis and unfavorable survival in patients with NSCLC. EHD1 knockdown inhibited the invasion and migration of human NSCLC cells, and overexpression of EHD1 increased the metastatic potential of lung cancer cells. Using the Affymetrix Human Gene 1.0 ST platform, microarray analysis revealed that an association between EHD1 and epithelial-mesenchymal transition (EMT), supported by downregulation of mesenchymal markers and upregulation of epithelial markers following knockdown of EHD1 in cell lines. Moreover, overexpression of EHD1 induced the EMT and increased the metastatic potential of lung cancer cells in vitro and in vivo. These results provide a model to illustrate the relationship between EHD1 expression and lung cancer metastasis, opening up new avenues for the prognosis and therapy of lung cancer.

Distant Metastasis and Survival Outcomes after Computed Tomography-Guided Needle Biopsy in Resected Stage I-III Non-Small Cell Lung Cancer.

Background and objective Several reports describe the needle-tract implantation after percutaneous needle lung biopsy. The present study evaluated whether preoperative computed tomography-guided needle biopsy (CTNB) affected the distant metastasis and overall survival in patients with early non-small cell lung cancer (NSCLC). Methods A total of 1667 patients with pathological stage I-III NSCLC were assimilated. Of these, 168 patients received preoperative CTNB, whereas 1499 patients were not subjected to any biopsy before surgical resection. Propensity score matching method was adopted to balance the observed covariates between the two groups. Cox regression analysis and Kaplan-Meier estimations were used for survival analysis. Subset analysis was performed in the p-stage ≤ II cases. Results The distant metastasis and mortality were not significantly increased for all patients with preoperative CTNB (P = 0.142 and P = 0.125, respectively). The subset analysis of p-stage ≤ II cases showed that CTNB increased the risk of distant metastasis (P = 0.032) while not increasing the risk of mortality (P = 0.086). Conclusion CTNB can increase the risk of distant metastasis in the p-stage ≤ II patients.

[Enhancement of HER-2 degradation by carbamazepine in breast cancer SKBR-3 cell line].

OBJECTIVE: To investigate the anti-tumor effect and the mechanism of down-regulation of HER - 2 by cabamazepine in SKBR - 3 cells , a breast cancer cell line with HER - 2 over - expression. METHODS: Western blotting was performed to evaluate the Her-2 expression level. The mRNA level of HER-2 was detected by RT-PCR. Immunoprecipitation was applied to detect the chaperon function and acetylation level of HSP90. The viability of cells was tested by MTT assay. RESULTS: Cabamazepine treatment down-regulated HER-2 expression. Only HER-2 protein level decrease was observed with 10 micromol/L cabamazepine treatment, but both protein and mRNA expressions were inhibited by 100 micromol/L cabamazepine. Cabamazepine treatment could induce a higher acetylation level of HSP90 and destroy its chaperon function. Cabamazepine exerted synergism with Herceptin in promoting HER-2 protein degradation and synergism or potentiation with Herceptin or 17-AAG in inhibition of proliferation. CONCLUSION: Cabamazepine can reduce the expression of HER-2 and show a synergistic effect with Herceptin or 17-AAG. There may be potential benefits of carbamazepine for cancer therapy in future. HER-2;

Suppression of calcium­sensing receptor ameliorates cardiac hypertrophy through inhibition of autophagy.

The calcium-sensing receptor (CaSR) releases intracellular calcium ([Ca2+]i) by accumulating inositol phosphate. Changes in [Ca2+]i initiate myocardial hypertrophy. Furthermore, autophagy associated with [Ca2+]i. Autophagy has previously been demonstrated to participate in the hypertrophic process. The current study investigated whether suppression of CaSR affects the hypertrophic response via modulating autophagy. Isoproterenol (ISO) was used to induce cardiac hypertrophy in Wistar rats. Hypertrophic status was determined by echocardiographic assessment, hematoxylin and eosin, and Masson's staining. The protein expression levels of CaSR and autophagy level were observed. Changes of hypertrophy and autophagy indicators were observed following intravenous injection of a CaSR inhibitor. An ISO­induced cardiomyocyte hypertrophy model was established and used determine the involvement of GdCl3. [Ca2+]i was determined using Fluo­4/AM dye followed by confocal microscopy. The expression levels of various active proteins were analyzed by western blotting. The size of the heart, expression levels of CaSR and autophagy level were markedly increased in hypertrophic myocardium. In addition, the present study demonstrated that the indicators of hypertrophy and autophagy were effectively suppressed by CaSR inhibitor. Furthermore, similar effects were demonstrated in neonatal rat hypertrophic cardiomyocytes treated with ISO. It was also observed that CaSR regulates the Ca2+/calmodulin­dependent protein kinase kinase ß (CaMKKß)­AMP­activated protein kinase (AMPK)­mammalian target of rapamycin (mTOR) signaling pathway induced by ISO in cardiomyocytes. Furthermore, the AMPK inhibition significantly reduced the autophagy level following CaSR stimulation (P<0.05). The results of the present demonstrated that inhibition of CaSR may ameliorate cardiac hypertrophy induced by ISO and the effect may be associated with the inhibition of autophagy and suppression of the CaMKKß­AMPK­mTOR signaling pathway.

Direct interaction and cooperative role of tumor suppressor p16 with band 3 (AE1).

By using the C-terminal 112-residue of band 3 to screen the K562 cDNA library, we find that the p16 interacts with band 3, which was confirmed both in yeast and in mammalian cells. Functional experiments show that p16 facilitates the movement of band 3 to plasma membrane with increased anion transport activity in 293t cells. Moreover, expression of endogenous p16 in 293t cells was increased at 24 and 36 h after transfection with band 3. Our findings provide a novel regulation pathway for both band 3 and p16.

As2O3 enhances the anion transport activity of band 3 and the action is related with the C-terminal 16 residues of the protein.

Successful application of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL) has been attracting worldwide interest, but the exact mechanism for the action of As2O3 remains somewhat obscure. In the present work, we show for the first time that As2O3 facilitates the DIDS-sensitive anion transport activity of band 3 protein in red blood cells (RBCs) isolated from normal adults and APL patients. To elucidate the effect of As2O3 on band 3 protein, constructs encoding the full length of the band 3 transmembrane domain (mdb3) and its C-terminal deletion forms were transfected into yeast cells by a yeast display system. The results demonstrate that deletion of the C-terminal 16 residues of mdb3 (mdb3-d16) does not affect anion transport activity of mdb3 or its sensitivity to DIDS, but decreases its sensitivity to As2O3 in the yeast cell. More intriguingly, the forced expression of intact mdb3 by transfection significantly induces cell apoptosis in HeLa cells, to a higher degree than in cells transfected with mdb3-d16 or empty vector. Expression of activated caspase 3 in HeLa cells also indicates that the C-terminal 16 residues are important for mdb3-mediated apoptosis in cells treated with As2O3. Our results provide the first evidence that As2O3 enhances the anion transport activity of band 3 and the action is related with the C-terminal 16 residues of the protein.

Expression of calcium sensing receptor and E-cadherin correlated with survival of lung adenocarcinoma.

BACKGROUND: It has been reported that the calcium sensing receptor (CaSR), a widely expressed G protein-coupled receptor, can stimulate cell differentiation and proliferation. However, in malignant tumors, loss of CaSR expression has been associated with tumorigenesis, metastasis, and progression. Recent studies have indicated that the CaSR could promote the expression of E-cadherin, which was considered a tumor suppressor. However, in human lung adenocarcinoma, the importance of the CaSR and E-cadherin has not been sufficiently investigated. METHODS: Expression levels of CaSR and E-cadherin in paraffin sections from 117 resected lung adenocarcinoma patients were evaluated by immunohistochemistry. We analyzed the correlation between our target proteins and clinical variables. Clinical significance was analyzed by multivariate Cox regression analysis, Kaplan-Meier curve, and log-rank test. RESULTS: Expression of the CaSR in lung adenocarcinoma tissue was significantly lower than in the normal sample (P = 0.003). Kendall tau-b analysis showed that, in a lung adenocarcinoma sample, the expression of CaSR positively correlated with a high level of E-cadherin (P < 0.001). Lung adenocarcinoma patients with a strong expression of CaSR (P = 0.034) or E-cadherin (P = 0.001) had longer overall survival. Multivariate Cox proportional hazards model analysis showed that the combined marker was an independent prognostic indicator of overall survival (hazard ratio = 0.440, confidence interval = 0.249-0.779, P = 0.005). CONCLUSIONS: We identified the CaSR as a new prognostic biomarker in lung adenocarcinoma. These results also suggested that the CaSR may become a new therapeutic target of lung adenocarcinoma.

L-arginine inhibits isoproterenol-induced cardiac hypertrophy through nitric oxide and polyamine pathways.

Polyamines (putrescine, spermidine and spermine) are essential for cell growth and differentiation. Nitric oxide exhibits antihypertrophic functions and inhibits cardiac remodelling. However, the metabolism of polyamines and the potential interactions with nitric oxide in cardiac hypertrophy remain unclear. We randomly divided Wistar rats into four treatment groups: controls, isoproterenol (ISO), ISO and L-arginine, and L-arginine. Isoproterenol (5 mg/kg/day, subcutaneously) and/or L-arginine (800 mg/kg/day, intraperitoneally) was administered once daily for 7 days. The expression of atrial natriuretic peptide mRNA was determined by reverse transcription-polymerase chain reaction, and fibrogenesis of heart was assessed by Van Gieson staining. Polyamines were measured with high-performance liquid chromatography, and plasma nitric oxide content and lactate dehydrogenase (LDH) activity were determined with a spectrophotometer. The expression levels of ornithine decarboxylase, spermidine/spermine N1-acetyltransferase (SSAT), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were analysed by Western blot. Heart-to-body weight ratio, left ventricle-to-body weight ratio, atrial natriuretic peptide mRNA expression, collagen fibres and LDH activity were elevated, both ornithine decarboxylase and SSAT proteins were up-regulated, and total polyamines were increased in the group treated with ISO. Additionally, the expression of iNOS was up-regulated, eNOS was down-regulated, and nitric oxide levels were low. Notably, cotreatment with L-arginine reversed most of these changes except for SSAT expression,which was further up-regulated. We propose that increased polyamines and decreased nitric oxide are involved in cardiac hypertrophy induced by ISO and suggest that L-arginine pre-treatment can attenuate cardiac hypertrophy through the regulation of key enzymes of the polyamine and nitric oxide pathways.

[Inhibitory effect of carbamazepine on proliferation of estrogen-dependent breast cancer cells].

BACKGROUND & OBJECTIVE: Carbamazepine, which has been used as an anti-epileptic drug in clinic for many years, is currently recognized as a histone deacetylase inhibitor (HDI), most of which showed anti-tumor characteristics. This study was to investigate the inhibitory effect of carbamazepine on estrogen dependent breast cancer cell lines with estrogen receptor alpha (ERalpha) expression and further explore the underlying mechanisms. METHODS: Sulforhodamine B viability assay was used to evaluate the viability of various cells treated with different drugs. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect the protein and mRNA expression of ERalpha and Cyclin D1. Immunofluorescence assay was employed to observe HER-2 expression in MCF-7RT cells, which were resistant to tamoxifen. Immunoprecipitation was performed to detect the chaperon function and acetylation level of Hsp90. RESULTS: Carbamazepine treatment could inhibit the proliferation of MCF-7 and T47D cells stimulated by estradiol (P<0.01). Carbamazepine and 4-hydroxytamoxifen (4-OHT) demonstrated a synergic effect on the inhibition of proliferation of MCF-7 cells stimulated by estradiol (q=1.00). Cabamazepine reversed the proliferation of MCF-7RT cells stimulated by 4-hydroxytamoxifen (P<0.01). Carbamazepine treatment could decrease the expression of ERalpha and Cyclin D1 at protein and mRNA level in ERalpha-positive cells and could reduce HER-2 expression in MCF-7RT cells. The decrease of ERalpha and Cyclin D1 expression was inhibited by MG132, an inhibitor of 26S proteosome. Carbamazepine treatment elevated the acetylation level of Hsp90 and disrupted its chaperon function. CONCLUSIONS: Carbamazepine shows significant anti-proliferation effect in ERalpha-positive breast cancer cell lines and this might be due to the enhancement of proteosome-mediated degradation of ERalpha and Cyclin D1 by carbamazepine. Furthermore, carbamazepine could reverse HER-2 dependent drug resistance to 4-OHT by reducing HER-2 expression.