Caffeic Acid Phenethyl Ester-Incorporated Radio-Sensitive Nanoparticles of Phenylboronic Acid Pinacol Ester-Conjugated Hyaluronic Acid for Application in Radioprotection.

We synthesized phenylboronic acid pinacol ester (PBPE)-conjugated hyaluronic acid (HA) via thiobis(ethylamine) (TbEA) linkage (abbreviated as HAsPBPE conjugates) to fabricate the radiosensitive delivery of caffeic acid phenetyl ester (CAPE) and for application in radioprotection. PBPE was primarily conjugated with TbEA and then PBPE-TbEA conjugates were conjugated again with hyaluronic acid using carbodiimide chemistry. CAPE-incorporated nanoparticles of HAsPBPE were fabricated by the nanoprecipitation method and then the organic solvent was removed by dialysis. CAPE-incorporated HAsPBPE nanoparticles have a small particle size of about 80 or 100 nm and they have a spherical shape. When CAPE-incorporated HAsPBPE nanoparticles were irradiated, nanoparticles became swelled or disintegrated and their morphologies were changed. Furthermore, the CAPE release rate from HAsPBPE nanoparticles were increased according to the radiation dose, indicating that CAPE-incorporated HAsPBPE nanoparticles have radio-sensitivity. CAPE and CAPE-incorporated HAsPBPE nanoparticles appropriately prevented radiation-induced cell death and suppressed intracellular accumulation of reactive oxygen species (ROS). CAPE and CAPE-incorporated HAsPBPE nanoparticles efficiently improved survivability of mice from radiation-induced death and reduced apoptotic cell death. We suggest that HAsPBPE nanoparticles are promising candidates for the radio-sensitive delivery of CAPE.

P2Y2 receptor activation by nucleotides released from highly metastatic breast cancer cells increases tumor growth and invasion via crosstalk with endothelial cells.

INTRODUCTION: Extracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y2R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y2R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs). METHODS: ATP release and P2Y2R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y2R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y2R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y2R-shRNA-transfected MDA-MB-231 cells. RESULTS: We found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y2R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y2R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y2R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y2R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y2R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y2R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells. CONCLUSION: This study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.

P2Y2 R activation by nucleotides promotes skin wound-healing process.

P2Y2 R has been shown to be upregulated in a variety of tissues in response to stress or injury and to mediate tissue regeneration through its ability to activate multiple signalling pathways. This study aimed to investigate the role of P2Y2 R in the wound-healing process and the mechanisms by which P2Y2 R activation promotes wound healing in fibroblasts. The role of P2Y2 R in skin wound healing was examined using a full-thickness skin wound model in wildtype (WT) and P2Y2 R(-/-) mice and an in vitro scratch wound model in control or P2Y2 R siRNA-transfected fibroblasts. WT mice showed significantly decreased wound size compared with P2Y2 R(-/-) mice at day 14 post-wounding, and immunohistochemical analysis showed that a proliferation marker Ki67 and extracellular matrix (ECM)-related proteins VEGF, collagen I, fibronectin and α-SMA were overexpressed in WT mice, which were reduced in P2Y2 R(-/-) mice. Scratch-wounded fibroblasts increased ATP release, which peaked at 5 min. In addition, scratch wounding increased the level of P2Y2 R mRNA. Activation of P2Y2 R by ATP or UTP enhanced proliferation and migration of fibroblasts in in vitro scratch wound assays and were blocked by P2Y2 R siRNA. Finally, ATP or UTP also increased the levels of ECM-related proteins through the activation of P2Y2 R in fibroblasts. This study suggests that P2Y2 R may be a potential therapeutic target to promote wound healing in chronic wound diseases.

Higenamine reduces HMGB1 during hypoxia-induced brain injury by induction of heme oxygenase-1 through PI3K/Akt/Nrf-2 signal pathways.

Growing lines of evidence suggests that high mobility group box-1 (HMGB1) plays an important role for promoting inflammation and apoptosis in brain ischemia. Previously, we demonstrated that inducers of heme oxygenase-1 (HO-1) significantly reduce HMGB1 release in inflammatory conditions in vitro and in vivo. Thus, we tested our hypothesis that higenamine protects brain injury by inhibition of middle cerebral artery occlusion (MCAO)-mediated HMGB1 release in vivo, and glucose/glucose oxidase (GOX)-induced apoptosis in C6 cells in vitro due to HO-1 induction. Higenamine increased HO-1 expression in C6 cells in both hypoxia and normoxia, in which the former was much more significant than the latter. Higenamine increased Nrf-2 luciferase activity, translocated Nrf-2 to nucleus, and increased phosphorylation of Akt in C6 cells. Consistent with this, LY 294002, a PI3K inhibitor, inhibited HO-1 induction by higenamine and apoptosis induced by glucose/GOX in C6 cells was prevented by higenamine, which effect was reversed by LY 294002. Importantly, administration of higenamine (i.p) significantly reduced brain infarct size, mortality rate, MPO activity and tissue expression of HMGB1 in MCAO rats. In addition, recombinant high mobility group box 1 induced apoptosis in C6 cells by increasing ratio of Bax/bcl-2 and cleaved caspase c, which was inhibited by higenamine, and all of these effects were reversed by co-treatment with ZnPPIX. Therefore, we conclude that higenamine, at least in part, protects brain cells against hypoxic damages by up-regulation of HO-1. Thus, higenamine may be beneficial for the use of ischemic injuries such as stroke.

Ethanol extract of Prunella vulgaris var. lilacina inhibits HMGB1 release by induction of heme oxygenase-1 in LPS-activated RAW 264.7 cells and CLP-induced septic mice.

The ethanol extract of the flower of P. vulgaris var. lilacina (EEPV) has been used traditionally as an antiinflammatory agent in many countries. Inducers of heme oxygenase-1 (HO-1) reduce high mobility group box 1 (HMGB1), a late phase cytokine, in sepsis. Although EEPV has been used as an antiinflammatory agent, no report is available as to whether it modifies HMGB1 in sepsis due to HO-1 induction. It was found that EEPV increased HO-1 protein expression in RAW 264.7 cells, which was significantly inhibited by LY294002, but not PD98059, SB203580 or SP600125. In addition, EEPV activated NF-E2-related factor (Nrf2) to move from the cytosol to the nucleus, and EEPV-induced HO-1 and activation of ARE-luciferase activity were significantly reduced by siNrf2 transfection and LY294002 but not SB203508. EEPV also significantly inhibited NF-κB luciferase activity, and decreased both iNOS/NO and COX-2/PGE(2) production in lipopolysaccharide (LPS)-stimulated macrophages which was reversed by siHO-1 RNA transfection. Importantly, EEPV inhibited HMGB1 release in LPS-activated macrophages in a PI3K-sensitive manner and reduced serum HMGB1 level and lung HMGB1 expression in cecal ligation and puncture (CLP)-induced septic mice. It is concluded that EEPV induces HO-1 expression through PI3K/Nrf2 signal pathways, which may be beneficial for the treatment of sepsis due to a reduction of HMGB1 release.

Transcriptional up-regulation of antioxidant genes by PPARδ inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells.

This study evaluated peroxisome proliferator-activated receptor (PPAR) δ as a potential target for therapeutic intervention in Ang II-induced senescence in human vascular smooth muscle cells (hVSMCs). Activation of PPARδ by GW501516, a specific agonist of PPARδ, significantly inhibited the Ang II-induced premature senescence of hVSMCs. Agonist-activated PPARδ suppressed the generation of Ang II-triggered reactive oxygen species (ROS) with a concomitant reduction in DNA damage. Notably, GW501516 up-regulated the expression of antioxidant genes, such as glutathione peroxidase 1, thioredoxin 1, manganese superoxide dismutase and heme oxygenase 1. siRNA-mediated down-regulation of these antioxidant genes almost completely abolished the effects of GW501516 on ROS production and premature senescence in hVSMCs treated with Ang II. Taken together, the enhanced transcription of antioxidant genes is responsible for the PPARδ-mediated inhibition of premature senescence through sequestration of ROS in hVSMCs treated with Ang II.

Peroxisome proliferator-activated receptor {delta} regulates extracellular matrix and apoptosis of vascular smooth muscle cells through the activation of transforming growth factor-{beta}1/Smad3.

Homeostasis of the extracellular matrix and apoptosis of vascular smooth muscle cells (VSMCs) are key components in the regulation of the stability of atherosclerotic plaques. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR)delta regulates extracellular matrix synthesis and degradation through transforming growth factor-beta1 and its effector, Smad3. Activation of PPARdelta strongly amplified the expression of types I and III collagen, fibronectin, elastin, and TIMP-3 (tissue inhibitor of metalloproteinases 3), but not of TIMP-1, matrix metalloproteinase-2 or -9. The effect of PPARdelta on the expression of type III collagen was dually regulated by the direct binding of PPARdelta and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, in the type III collagen gene promoter. The activation of PPARdelta attenuated apoptotic cell death in VSMCs induced by oxidized low-density lipoprotein, and similar antiapoptotic effects were observed on treatment of cells with exogenous type I and/or III collagen. Administration of a PPARdelta ligand GW501516 to mice also suppressed elastase-induced cell death of aortic VSMCs. These results suggest that PPARdelta-induced upregulation of extracellular matrix proteins exerts an antiapoptotic effect, thereby maintaining the stability of atherosclerotic plaques. Specific ligands of PPARdelta may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.

PPARdelta promotes wound healing by up-regulating TGF-beta1-dependent or -independent expression of extracellular matrix proteins.

Although the peroxisome proliferator-activated receptor (PPAR) delta has been implicated in the wound healing process, its exact role and mechanism of action have not been fully elucidated. Our previous findings showed that PPARdelta induces the expression of the transforming growth factor (TGF)-beta1, which has been implicated in the deposit of extracellular matrix proteins. Here, we demonstrate that administration of GW501516, a specific PPARdelta ligand, significantly promoted wound closure in the experimental mouse and had a profound effect on the expression of collagen types I and III, alpha-smooth muscle actin, pSmad3 and TGF-beta1, which play a pivotal role in wound healing processes. Activation of PPARdelta increased migration of human epidermal keratinocytes and dermal fibroblasts in in vitro scrape-wounding assays. Addition of a specific ALK5 receptor inhibitor SB431542 significantly suppressed GW501516-induced migration of human keratinocytes and fibroblasts. In these cells, activated PPARdelta also induced the expression of collagen types I and III and fibronectin in a TGF-beta1-dependent or -independent manner. The effect of PPARdelta on the expression of type III collagen was dually regulated by the direct binding of PPARdelta and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, of the type III gene promoter. Taken together, these results demonstrated that PPARdelta plays an important role in skin wound healing in vivo and that it functions by accelerating extracellular matrix-mediated cellular interactions in a process mediated by the TGF-beta1/Smad3 signaling-dependent or - independent pathway.

Heme-oxygenase-1 induction and carbon monoxide-releasing molecule inhibit lipopolysaccharide (LPS)-induced high-mobility group box 1 release in vitro and improve survival of mice in LPS- and cecal ligation and puncture-induced sepsis model in vivo.

We examined our hypothesis that heme-oxygenase-1 (HO-1)-derived carbon monoxide (CO) inhibits the release of high-mobility group box 1 (HMGB1) in RAW264.7 cells activated with lipopolysaccharide (LPS) in vitro and in LPS- or cecal ligation and puncture (CLP)-induced septic mice in vivo, so that HO-1 induction or CO improves survival of sepsis in rodents. We found that pretreatment with HO-1 inducers (hemin, cobalt protoporphyrin IX) or transfection of HO-1 significantly inhibited HMGB1 release, which was blocked by HO-1 small interfering RNA, in cells activated by LPS. Carbon monoxide-releasing molecule 2 (CORM-2) but not bilirubin or deferoxamine inhibited HMGB1 release in LPS-activated macrophages. Oxyhemoglobin reversed the effect of HO-1 inducers on HMGB1 release. Translocation of HMGB1 from nucleus to cytosol was significantly inhibited by HO-1 inducers, CORM-2, or HO-1 transfection. Neutralizing antibodies to tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, interferon-beta, and N(omega)-nitro-L-arginine methyl ester hydrochloride but not N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) significantly inhibited HMGB1 release in LPS-activated cells. Production of TNF-alpha, IL-1beta, and IFN-beta was significantly reduced by pretreatment of HO-1 inducers, CORM-2, or HO-1 transfection in LPS-activated cells. Plasma levels of HMGB1 in mice challenged with LPS or CLP were significantly reduced by the administration of HO-1 inducers or CORM-2, which was accompanied by either reduction (pretreatment) or no change (delayed administration) of serum TNF-alpha and IL-1beta levels. Regardless of pretreatment or delayed administration, CORM-2 and hemin rescued mice from lethal endotoxemia and sepsis induced by LPS or CLP. Taken together, we concluded that HO-1-derived CO reduces HMGB1 release in LPS-activated cells and LPS- or CLP-induced animal model of sepsis.

EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells.

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E(2), one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE(2) increased HO-1 expression in C6 cells in vitro. The effects of PGE(2) were mimicked by PGE(2) receptor EP(2) agonists, 11-deoxy PGE(2), and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE(2) was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP(2)-specific antagonist, AH8006 also inhibited PGE(2)-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE(2) inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE(2) also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE(2) induces HO-1 protein expression through PKA and PI3K signaling pathways via EP(2) receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE(2) is responsible for anti-apoptosis against oxidant stress.

(S)-1-(alpha-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) reduces rat myocardial apoptosis against ischemia and reperfusion injury by activation of phosphatidylinositol 3-kinase/Akt signaling and anti-inflammatory action in vivo.

We examined our hypothesis that (S)-1-(alpha-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) inhibits apoptosis in myocardial ischemia and reperfusion (I/R) injury in vivo via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and by reducing inflammation during I/R. To do this, we induced a 30-min period of ischemia by occlusion of the left anterior descending coronary artery of the rat followed by a 2-h (for phosphorylation of Akt), 6-h (for biochemical analysis), or 24-h (for functional analysis) period of reperfusion to determine the effect of CKD712 treatment. Pretreatment with CKD712 significantly improved myocardial function as evidenced by an increase in the +/-dP/dt and a decrease in the infarct size, which were antagonized by a PI3K inhibitor, wortmannin (WT). Interestingly, CKD712 increased the phosphorylation of Akt and cAMP-response element-binding protein and increased the expression of the Bcl-2 gene, but it reduced the expression of the Bax gene. CKD712 decreased not only the expression but also the activity of the caspase-3 protein in the myocardium after reperfusion. Thus, all of the antiapoptotic effects of CKD712 were significantly inhibited by WT. Furthermore, the antiapoptotic effects of CKD712 and its inhibition by WT in myocardium after reperfusion were confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining. Finally, CKD712 was found to reduce the serum levels of the high-mobility group box 1 protein, tumor necrosis factor-alpha, and the cardiac troponin I protein in addition to tissue levels of malondialdehyde and myeloperoxidase activity in I/R hearts. Taken together, both the activation of PI3K/Akt and its anti-inflammatory action prevent apoptosis in myocardial I/R injury by CKD712.

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride.

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.

Daidzein administration in vivo reduces myocardial injury in a rat ischemia/reperfusion model by inhibiting NF-kappaB activation.

AIMS: We tested the hypothesis that daidzein may reduce myocardial damage by both inhibiting the release of cytokines and limiting the nuclear translocation of NF-kappaB. MAIN METHODS: Male Sprague-Dawley rats were anesthetized, and the left anterior descending coronary artery (LAD) was ligated for 25 min. Twenty-four hours after reperfusion was established, the hemodynamics and infarct size were examined. KEY FINDINGS: Treatment with daidzein (10 mg/kg, i.p.) 1 h prior to the ischemia/reperfusion procedure (I/R) reduced the infarct size by 52.8% (P<0.05). Daidzein also significantly improved I/R-induced myocardial contractile dysfunction by improving the left ventricular diastolic pressure and the positive and negative maximal values of the first derivative of the left ventricular pressure. In addition, daidzein reduced the plasma levels of TNF-alpha and IL-6 in I/R rats and decreased malondialdehyde levels, myeloperoxidase activity, catalase activity and neutrophil infiltration in I/R rat myocardium. Interestingly, daidzein inhibited I/R-induced myocardial apoptosis by decreasing DNA strand breaks and cleaved caspase-3 activity. Furthermore, daidzein inhibited both the nuclear translocation of NF-kappaB in I/R rat hearts and the H(2)O(2)-induced activation of NF-kappaB-luciferase activity in human umbilical vein endothelial cells. SIGNIFICANCE: This study reveals that the administration of daidzein in vivo attenuates I/R-induced myocardial damage via inhibition of NF-kappaB activation, which in turn may suppress inflammatory cytokine expression.

NS398 protects cells from sodium nitroprusside-mediated cytotoxicity through enhancing HO-1 induction independent of COX-2 inhibition.

Heme oxygenase-1 (HO-1) plays a preventive role in oxidative stress. In contrast, COX-2 is involved in the pathogenesis of many inflammatory diseases, thus COX-2 inhibitor is believed to exert anti-inflammatory properties by blocking COX-2. Recently, however, salicylate the active metabolite of aspirin showed COX-independent anti-inflammatory effects through induction of HO-1. Thus, we hypothesized that HO-1 are induced as an adaptive response to sodium nitroprusside (SNP) and play a protective role against cytotoxicity. Moreover, we investigated the protective effect of NS398 known as a selective COX-2 inhibitor on SNP-mediated cytotoxicity, and whether the protective effect of NS398 is due to COX-2 inhibition or not. SNP induced cytotoxicity in a dose-dependent manner, which was enhanced by inhibition of HO-1, suggesting that HO-1 acts in an adaptive response to SNP. Interestingly, NS398 decreased SNP-mediated cytotoxicity whereas COX-2 siRNA did not. Furthermore, NS398 enhanced SNP-induced HO-1 induction even though NS398 alone failed to induce HO-1 protein expression. In addition, NS398 enhanced SNP-induced COX-2, even though NS398 effectively inhibited SNP-mediated PGE(2) production. These results demonstrate that NS398 exerts cytoprotective effects by inducing HO-1 independent of COX-2 inhibition.

Tanshinone I suppresses growth and invasion of human breast cancer cells, MDA-MB-231, through regulation of adhesion molecules.

The role of cell adhesion molecules has been studied extensively in the process of inflammation, and these molecules are critical components of carcinogenesis and cancer metastasis. This study investigated the effect of tanshinone I derived from the traditional herbal medicine, Salvia miltiorrhiza Bunge, on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-alpha (TNF-alpha)-stimulated endothelial cells. Furthermore, this study investigated the effect of tanshinone I on cancer growth, invasion and angiogenesis on human breast cancer cells MDA-MB-231, both in vitro and in vivo. Tanshinone I dose dependently inhibited ICAM-1 and VCAM-1 expressions in human umbilical vein endothelial cells (HUVECs) that were stimulated with TNF-alpha for 6 h. Pretreatment with tanshinone I significantly reduced adhesion of either monocyte U937 or MDA-MB-231 cells to HUVECs. Interestingly, the inhibitory effect of tanshinone I on monocyte and cancer cell adhesion to HUVECs was mimicked by transfection with ICAM-1 and VCAM-1 small interfering RNA. In addition, tanshinone I effectively inhibited TNF-alpha-induced production of vascular endothelial growth factor (VEGF) and VEGF-mediated tube formation in HUVECs. Tanshinone I also inhibited TNF-alpha-induced VEGF production in MDA-MB-231 cells and migration of MDA-MB-231 cells through extracellular matrix. Additionally, reduction of tumor mass volume and decrease of metastasis incidents by tanshinone I were observed in vivo. In conclusion, this study provides a potential mechanism for the anticancer effect of tanshinone I on breast cancer cells, suggesting that tanshinone I may serve as an effective drug for the treatment of breast cancer.

Ran suppresses paclitaxel-induced apoptosis in human glioblastoma cells.

Yeast-based functional screening of a human glioblastoma cDNA library identified ras-related nuclear protein (Ran) as a novel suppressor of Bcl-2-associated X protein (Bax), a pro-apoptotic member of the Bcl-2 family of proteins. Yeast cells that expressed human Ran were resistant to Bax-induced cell death. In U373MG glioblastoma cells, stable overexpression of Ran significantly attenuated apoptotic cell death induced by the chemotherapeutic agent paclitaxel. FACS analysis demonstrated that Ran is involved in paclitaxel-induced cell cycle arrest. Stable overexpression of Ran also markedly inhibited the phosphorylation of Bcl-2 by paclitaxel, and inhibited the translocation of Bax, the release of cytochrome c and activation of caspase-3. Paclitaxel-induced phosphorylation of c-JUN N-terminal kinase (JNK), but not p38, extracellular signal-regulated kinase and Akt, was markedly suppressed in U373MG cells that stably expressed Ran. These results suggest that Ran suppresses paclitaxel-induced cell death through the downregulation of JNK-mediated signal pathways.

The obligatory role of COX-2 expression for induction of HO-1 in ischemic preconditioned rat brain.

The molecular mechanisms of preconditioning-induced ischemic tolerance (PCIT) have yet to be elucidated. We investigated whether minimal expression levels of COX-2 induced by preconditioning trigger HO-1, thereby inducing the synthesis of cytoprotective proteins. We show that both COX-2 and HO-1 are induced in rat brains subjected to preconditioning by middle cerebral artery (MCA) occlusion for 10 min followed by different amounts of reperfusion time (1-24 h). Although preconditioning significantly reduced the brain infarct size against severe ischemia (24 h MCA occlusion), pretreatment with the COX-2-selective inhibitor rofecoxib increased infarct size and abolished PCIT-induced COX-2 and HO-1 expression in vivo. We also found that PGE(2) increased the phosphorylation of Akt, which was significantly inhibited by the PI3 kinase inhibitor LY294002. Taken together, we conclude that the kinetic changes in COX-2 induction during the reperfusion period following preconditioning may be important for ischemic tolerance.

Identification of cytochrome c oxidase subunit 6A1 as a suppressor of Bax-induced cell death by yeast-based functional screening.

Human cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1) was identified as a novel suppressor of Bcl-2-associated X protein (Bax)-mediated cell death using yeast-based functional screening of a mammalian cDNA library. The overexpression of COX6A1 significantly suppressed Bax- and N-(4-hydroxyphenyl)retinamide (4-HPR)-induced apoptosis in yeast and human glioblastoma-derived U373MG cells, respectively. The generation of reactive oxygen species (ROS) in response to Bax or 4-HPR was inhibited in yeast and U373MG cells that expressed COX6A1, indicating that COX6A1 exerts a protective effect against ROS-induced cell damage. 4-HPR-induced mitochondrial translocation of Bax, release of mitochondrial cytochrome c, and activation of caspase-3 were markedly attenuated in U373MG cells that stably expressed COX6A1. Our results demonstrate that yeast-based functional screening of human genes for inhibitors of Bax-sensitivity in yeast identified a protein that not only suppresses the toxicity of Bax in yeast, but also has a potential role in protecting mammalian cells from 4-HPR-induced apoptosis.

Tanshinone I effectively induces apoptosis in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells.

Danshen (Salvia miltiorrhiza Bunge) is a herb that has been widely and successfully used for treating inflammatory diseases in clinics in Asia. The relatively abundant tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone, have been isolated from Danshen. These tanshinones are the major diterpenes isolated from Danshen, and show cytotoxic effects on cell lines derived from human carcinomas of the colon, ovary, lung, mouth, and breast. Recently, anti-cancer activities of tanshinone IIA have been reported, which suggest that the structurally similar tanshinone I may possess similar cytotoxic effects on tumor cells. We investigated the effect of tanshinone I on the induction of apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231) in vitro. Tanshinone I inhibited cell proliferation of MCF-7 and MDA-MB-231 cells in a dose- and time-dependent manner, as assayed by MTT. In addition, TUNEL assay and flow cytometry showed that tanshinone I significantly induced apoptosis in MCF-7 and MDA-MB-231 cells. The induction of apoptotic cell death was mediated by the activation of caspase 3, the downregulation of the level of the anti-apoptotic protein, Bcl-2, and the upregulation of the level of the pro-apoptotic protein, Bax. Taken together, these results reveal a potential mechanism for the anti-cancer effect of tanshinone I on human breast cancer cells, and suggest that tanshinone I may serve as an effective adjunctive reagent in the treatment of human breast cancer.

Anti-apoptotic effect of magnolol in myocardial ischemia and reperfusion injury requires extracellular signal-regulated kinase1/2 pathways in rat in vivo.

Magnolol, an active component extracted from Magnolia officinalis, has been reported to have protective effect on ischemia and reperfusion (I/R)-induced injury in experimental animals. The aim of the present investigation was to further evaluate the mechanism(s) by which magnolol reduces I/R-induced myocardial injury in rats in vivo. Under anesthesia, left anterior descending (LAD) coronary artery was occluded for 30 min followed by reperfusion for 24 h (for infarct size and cardiac function analysis). In some experiments, reperfusion was limited to 1 h or 6 h for analysis of biochemical and molecular events. Magnolol and DMSO solution (vehicle) were injected intra-peritoneally 1 h prior to I/R insult. The infarct size was measured by TTC technique and heart function was monitored by Millar Catheter. Apoptosis related events such as p-ERK, p-Bad, Bcl-xl and cytochrome c expression were evaluated by Western blot analysis and myocardial caspase-3 activity was also measured. Magnolol (10 mg/kg) reduced infarct size by 50% (P < 0.01 versus vehicle), and also improved I/R-induced myocardial dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in magnolol-treated rats. Magnolol increased the expression of phosphor ERK and Bad which resulted in inhibition of myocardial apoptosis as evidenced by TUNEL analysis and DNA laddering experiments. Application of PD 98059, a selective MEK1/2 inhibitor, strongly antagonized the effect of magnolol. Taken together, we concluded that magnolol inhibits apoptosis through enhancing the activation of ERK1/2 and modulation of the Bcl-xl proteins which brings about reduction of infarct size and improvement of cardiac function in I/R-induced injury.