Prostaglandin D2 synthase inhibits the exaggerated growth phenotype of spontaneously hypertensive rat vascular smooth muscle cells

Lipocalin-type prostaglandin D2 synthase (L-PGDS) has recently been linked to a variety of pathophysiological cardiovascular conditions including hypertension and diabetes. In this study, we report on the 50% increase in L-PGDS protein expression observed in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). L-PGDS expression also increased 50% upon the differentiation of normotensive control cells (WKY, from Wistar-Kyoto rats). In addition, we demonstrate differential effects of L-PGDS treatment on cell proliferation and apoptosis in VSMCs isolated from SHR versus WKY controls. L-PGDS (50 microg/ml) was able to significantly inhibit VSMC proliferation and DNA synthesis and induce the apoptotic genes bax, bcl-x, and ei24 in SHR but had no effect on WKY cells. Hyperglycemic conditions also had opposite effects, in which increased glucose concentrations (20 mm) resulted in decreased L-PGDS expression in control cells but actually stimulated L-PGDS expression in SHR. Furthermore, we examined the effect of L-PGDS incubation on insulin-stimulated Akt, glycogen synthase kinase-3beta (GSK-3beta), and ERK phosphorylation. Unexpectedly, we found that when WKY cells were pretreated with L-PGDS, insulin could actually induce apoptosis and failed to stimulate Akt/GSK-3beta phosphorylation. Insulin-stimulated ERK phosphorylation was unaffected by L-PGDS pretreatment in both cell lines. We propose that L-PGDS is involved in the balance of VSMC proliferation and apoptosis and in the increased expression observed in the hypertensive state is an attempt to maintain a proper equilibrium between the two processes via the induction of apoptosis and inhibition of cell proliferation.     

Primary versus secondary events in hypertension

Functional modifications, such as a reduction in hormonal response, which occur in the cardiovascular system in hypertension, are reflected at the cellular level by anomalies in cyclic nucleotide and other messenger systems. To distinguish between primary and adaptive abnormalities, we pursued three research strategies. (i) Investigations on various models of hypertension. To be considered a primary defect, an abnormality should also be present in other genetically hypertensive models. Indeed, we have confirmed the occurrence of cellular hyperplasia in the heart of spontaneously hypertensive rats (SHR) as well as in spontaneously hypertensive mice (SHM). An increase of calmodulin levels in the heart and kidney is also observed in both the SHR and SHM. (ii) Studies on the evolution of hypertension with age. In humans, a decrease of cAMP levels in response to beta-adrenergic stimulation in older patients is contrasted with an excess in younger subjects. In the SHR, protein kinase activity of the heart is lower in the prehypertensive stages, whereas this defect appears much later in the aorta. (iii) Experiments on anomalies in newborns and cultured cells. The heart and kidney in the SHR exhibit significant hyperplasia at birth, and an abnormal growth continues in tissue culture. We hope that these strategies will eventually help to provide biochemical and functional markers for genetic analysis of factors which may be involved in the pathogenesis of hypertension.     

Apoptosis enzyme-linked immunosorbent assay distinguishes anticancer drugs from toxic chemicals and predicts drug synergism

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

Here, we report that LMK235, a class I and histone deacetylase (HDAC6)‐preferential HDAC inhibitor, reduces hypertension via inhibition of vascular contraction and vessel hypertrophy. Angiotensin II‐infusion mice and spontaneously hypertensive rats (SHRs) were used to test the anti‐hypertensive effect of LMK235. Daily injection of LMK235 lowered angiotensin II‐induced systolic blood pressure (BP). A reduction in systolic BP in SHRs was observed on the second day when SHRs were treated with 3 mg/kg LMK235 every 3 days. However, LMK235 treatment did not affect angiotensin‐converting enzyme 1 and angiotensin II receptor mRNA expression in either hypertensive model. LMK235, acting via the nitric oxide pathway, facilitated the relaxing of vascular contractions induced by a thromboxane A2 agonist in the rat aortic and mesenteric artery ring test. In addition, LMK235 increased nitric oxide production in HUVECs and inhibited the increasing of aortic wall thickness in both animal hypertensive models. LMK235 decreased the enhanced cell cycle‐related genes cyclin D1 and E2F3 in angiotensin II‐infusion mice and restored the decreased p21 expression. In addition, LMK235 suppressed calcium calmodulin‐dependent protein kinase II (CaMKII) α, which is related to vascular smooth muscle cell proliferation. Inhibition or knockdown of HDAC5 blocked the CaMKIIα‐induced cell cycle gene expression. Immunoprecipitation demonstrated that class I HDACs were involved in the inhibition of CaMKII α‐induced HDAC4/5 by LMK235. We suggest that LMK235 should be further investigated for its use in the development of new therapeutic options to treat hypertension via reducing vascular hyperplasia or vasoconstriction.     

Cyclin-dependent kinase inhibitors sensitize tumor cells to nutlin-induced apoptosis: a potent drug combination

Current chemotherapy focuses on the use of genotoxic drugs that may induce general DNA damage in cancer cells but also high levels of toxicity in normal tissues. Nongenotoxic activation of p53 by targeting specific molecular pathways therefore provides an attractive therapeutic strategy in cancers with wild-type p53. Here, we explored the antitumor potential of cyclin-dependent kinase (CDK) inhibitors in combination with a small molecule inhibitor of p53-murine double minute 2 (MDM2) interaction. We show that low doses of CDK inhibitors roscovitine and DRB synergize with the MDM2 antagonist nutlin-3a in the induction of p53 activity and promote p53-dependent apoptosis in a dose- and time-dependent manner. Statistical measurement of the combination effects shows that the drug combination is additive on the reduction of cell viability and synergistic on inducing apoptosis, a critical end point of cytotoxic drugs. The degree of apoptosis observed 24 to 48 h after drug treatment correlated with the accumulation of p53 protein and concomitant induction of proapoptotic proteins Puma and PIG3. The antiproliferative and cytotoxic effects of this drug combination are validated in a range of tumor-derived cells including melanoma, colon carcinoma, breast adenocarcinoma, and hepatocarcinoma cells. Furthermore, this drug combination does not induce phosphorylation of Ser(15) on p53 and does not induce genotoxic stress in the cell. Given that many cytotoxic drugs rely on their ability to induce apoptosis via DNA damage-mediated activation of p53, the data presented here may provide a new therapeutic approach for the use of CDK inhibitors and MDM2 antagonists in combinatorial drug therapy.     

The dichotomous role of TGF-β in controlling liver cancer cell survival and proliferation

Hepatocellular carcinoma (HCC) is the major form of primary liver cancer and one of the most prevalent and life-threatening malignancies globally. One of the hallmarks in HCC is the sustained cell survival and proliferative signals, which are determined by the balance between oncogenes and tumor suppressors. Transforming growth factor beta (TGF-β) is an effective growth inhibitor of epithelial cells including hepatocytes, through induction of cell cycle arrest, apoptosis, cellular senescence, or autophagy. The antitumorigenic effects of TGF-β are bypassed during liver tumorigenesis via multiple mechanisms. Furthermore, along with malignant progression, TGF-β switches to promote cancer cell survival and proliferation. This dichotomous nature of TGF-β is one of the barriers to therapeutic targeting in liver cancer. Thereafter, understanding the underlying molecular mechanisms is a prerequisite for discovering novel antitumor drugs that may specifically disable the growth-promoting branch of TGF-β signaling or restore its tumor-suppressive arm. This review summarizes how TGF-β inhibits or promotes liver cancer cell survival and proliferation, highlighting the functional switch mechanisms during the process.     

Calcitonin gene-related peptide and hypertension

Capsaicin-sensitive sensory nerves participate in the regulation of cardiovascular functions both in the normal state and the pathophysiology of hypertension through the actions of potent vasodilator neuropeptides, including calcitonin gene-related peptide (CGRP). CGRP, a very potent vasodilator, is the predominant neurotransmitter in capsaicin-sensitive sensory nerves, and plays an important role in the initiation, progression and maintenance of hypertension via: (1) the alterations in its synthesis and release and/or in vascular sensitivity response to it; (2) interactions with pro-hypertensive systems, including renin-angiotensin-aldosterone system, sympathetic nervous system and endothelin system; and (3) anti-hypertrophy and anti-proliferation of vascular smooth muscle cells. The decrease in CGRP synthesis and release contributes to the elevated blood pressure, as shown in the spontaneously hypertensive rats, alpha-CGRP knockout mice, Dahl-salt or phenol-induced hypertensive rats. In contrast, the increase in CGRP levels or the enhancement of vascular sensitivity response to CGRP plays a beneficial compensatory depressor role in the development of hypertension, as shown in deoxycorticosterone-salt, sub-total nephrectomy-salt, N(omega)-nitro-L-arginine methyl ester or two-kidney, one-clip models of hypertension in rats. We found that rutaecarpine causes a sustained depressor action by stimulation of CGRP synthesis and release via activation of vanilloid receptor subtype 1 (VR1) in hypertensive rats, which reveals the therapeutic implications of VR1 agonists for treatment of hypertension.     

Molecular mechanism of 'mitocan'-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2 family proteins

Mitochondria have emerged recently as effective targets for novel anti-cancer drugs referred to as 'mitocans'. We propose that the molecular mechanism of induction of apoptosis by mitocans, as exemplified by the drug alpha-tocopheryl succinate, involves generation of reactive oxygen species (ROS). ROS then mediate the formation of disufide bridges between cytosolic Bax monomers, resulting in the formation of mitochondrial outer membrane channels. ROS also cause oxidation of cardiolipin, triggering the release of cytochrome c and its translocation via the activated Bax channels. This model may provide a general mechanism for the action of inducers of apoptosis and anticancer drugs, mitocans, targeting mitochondria via ROS production.     

Contribution of cytochrome P450 1B1 to hypertension and associated pathophysiology: a novel target for antihypertensive agents

The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone (DOCA)-salt-, and N(ω)-nitro-L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src.     

Perspective on hypertension in the elderly.

More than half of elderly men and women have hypertension, leading to a significant risk of increased morbidity and mortality. The cause of hypertension in this age group is unknown. Left ventricular hypertrophy is frequently present, often associated with diastolic dysfunction. Systolic hypertension in the elderly increases the risk of cardiovascular disease, but there are no good data to show that the treatment of isolated systolic hypertension reduces the morbidity or mortality. Good evidence indicates that antihypertensive treatment in this group decreases cardiovascular morbidity and mortality up to age 80, so most elderly hypertensive patients should be treated. An empiric trial of nonpharmacologic therapy can be initiated in those with mild hypertension and no cardiovascular disease, but most patients will require drug therapy. Most elderly hypertensive patients have accompanying illnesses for which they may or may not be taking medications. Some antihypertensive drugs exacerbate coexisting diseases while others augment treatment regimens. Similarly, drugs may interact in a beneficial or adverse way. Finally, drug metabolism is altered by age, leading to problems with toxicity or diminished efficacy. The choice of medication should be based on all such considerations, including the cost and convenience of the drugs available.     

Anti-Cancer Effect of HIV-1 Viral Protein R on Doxorubicin Resistant Neuroblastoma

Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX). To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.     

Induction of hemeoxygenase-1 reduces glomerular injury and apoptosis in diabetic spontaneously hypertensive rats

Induction of hemeoxygenase-1 (HO-1) lowers blood pressure and reduces organ damage in hypertensive animal models; however, a potential protective role for HO-1 induction against diabetic-induced glomerular injury remains unclear. We hypothesize that HO-1 induction will protect against diabetes-induced glomerular injury by maintaining glomerular integrity and inhibiting renal apoptosis, inflammation, and oxidative stress. Diabetes was induced with streptozotocin in spontaneously hypertensive rats (SHR) as a model where the coexistence of hypertension and diabetes aggravates the progression of diabetic renal injury. Control and diabetic SHR were randomized to receive vehicle or the HO-1 inducer cobalt protoporphyrin (CoPP). Glomerular albumin permeability was significantly greater in diabetic SHR compared with control, consistent with an increase in apoptosis and decreased glomerular nephrin and α(3)β(1)-integrin protein expression in diabetic SHR. CoPP significantly reduced albumin permeability and apoptosis and restored nephrin and α(3)β(1)-integrin protein expression levels in diabetic SHR. Glomerular injury in diabetic SHR was also associated with increases in NF-κB-induced inflammation and oxidative stress relative to vehicle-treated SHR, and CoPP significantly blunted diabetes-induced increases in glomerular inflammation and oxidative stress in diabetic SHR. These effects were specific to exogenous stimulation of HO-1, since incubation with the HO inhibitor stannous mesoporphyrin alone did not alter glomerular inflammatory markers or oxidative stress yet was able to prevent CoPP-mediated decreases in these parameters. These data suggest that induction of HO-1 reduces diabetic induced-glomerular injury and apoptosis and these effects are associated with decreased NF-κB-induced inflammation and oxidative stress.     

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation

In vivo imaging of apoptosis in a preclinical setting in anticancer drug development could provide remarkable advantages in terms of translational medicine. So far, several imaging technologies with different probes have been used to achieve this goal. Here we describe a bioluminescence imaging approach that uses a new formulation of Z-DEVD-aminoluciferin, a caspase 3/7 substrate, to monitor in vivo apoptosis in tumor cells engineered to express luciferase. Upon apoptosis induction, Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase generating measurable light. Thus, the activation of caspase 3/7 can be measured by quantifying the bioluminescent signal. Using this approach, we have been able to monitor caspase-3 activation and subsequent apoptosis induction after camptothecin and temozolomide treatment on xenograft mouse models of colon cancer and glioblastoma, respectively. Treated mice showed more than 2-fold induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D-luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, we confirmed that it is possible to follow non-invasively tumor growth inhibition and induction of apoptosis after treatment in the same animal over time. Moreover, here we have proved that following early apoptosis induction by caspase 3 activation is a good biomarker that accurately predicts tumor growth inhibition by anti-cancer drugs in engineered colon cancer and glioblastoma cell lines and in their respective mouse xenograft models.     

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.     

Reappraisal of the Therapeutic Role of Celecoxib in Cholangiocarcinoma

Cholangiocarcinoma (CCA), a lethal disease, affects many thousands worldwide yearly. Surgical resection provides the best chance for a cure; however, only one-third of CCA patients present with a resectable tumour at the time of diagnosis. Currently, no effective chemotherapy is available for advanced CCA. Cyclooxygenase-2 (COX-2) is a potential oncogene expressing in human CCA tissues and represents a candidate target for treatment; however, COX-2 inhibitors increase the risk of negative cardiovascular events as application for chemoprevention aim. Here, we re-evaluated the effectiveness and safety of celecoxib, one widely used COX-2 inhibitor, in treating CCA. We demonstrated that celecoxib exhibited an anti-proliferative effect on CGCCA cells via cell cycle arrest at G2 phase and apoptosis induction. Treatment for 5 weeks high dose celecoxib (160 mg/kg) significantly repressed thioacetamide-induced CCA tumour growth in rats as monitored by animal positron emission tomography through apoptosis induction. No obviously observable side effects were noted during the therapeutic period. As retrospectively reviewing 78 intrahepatic mass-forming CCA patients, their survival was strongly and negatively associated with a positive resection margin and high COX-2 expression. Based on our result, we concluded that short-term high dose celecoxib may be a promising therapeutic regimen for CCA. Yet its clinical application still needs more studies to prove its safety.     

Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.