Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway.

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.

Sialic Acid-Binding Lectin from Bullfrog Eggs Exhibits an Anti-Tumor Effect Against Breast Cancer Cells Including Triple-Negative Phenotype Cells.

Sialic acid-binding lectin from Rana catesbeiana eggs (cSBL) is a multifunctional protein that has lectin and ribonuclease activity. In this study, the anti-tumor activities of cSBL were assessed using a panel of breast cancer cell lines. cSBL suppressed the cell growth of all cancer cell lines tested here at a concentration that is less toxic, or not toxic at all, to normal cells. The growth suppressive effect was attributed to the cancer-selective induction of apoptosis. We assessed the expressions of several key molecules associated with the breast cancer phenotype after cSBL treatment by western blotting. cSBL decreased the expression level of estrogen receptor (ER) α, while it increased the phosphorylation level of p38 mitogen-activated protein kinase (MAPK). cSBL also suppressed the expression of the progesterone receptor (PgR) and human epidermal growth factor receptor type 2 (HER2). Furthermore, it was revealed that cSBL decreases the expression of the epidermal growth factor receptor (EGFR/HER1) in triple-negative breast cancer cells. These results indicate that cSBL induces apoptosis with decreasing ErbB family proteins and may have great potential for breast cancer chemotherapy, particularly in triple-negative phenotype cells.

Role of Spinal Cholecystokinin Octapeptide, Nociceptin/Orphanin FQ, and Hemokinin-1 in Diabetic Allodynia.

A complication of diabetes is neuropathic pain, which is difficult to control with medication. We have confirmed that neuropathic pain due to mechanical allodynia in diabetic mice is mediated by a characteristic neuropeptide in the spinal cord. We evaluated the strength of mechanical allodynia in mice using von Frey filaments. When mice were intravenously injected with streptozotocin, mechanical allodynia appeared 3 days later. Antibodies of representative neuropeptides were intrathecally (i.t.) administered to allodynia-induced mice 7 days after the intravenous administration of streptozotocin, and allodynia was reduced by anti-cholecystokinin octapeptide antibodies, anti-nociceptin/orphanin FQ antibodies, and anti-hemokinin-1 antibodies. In contrast, i.t.-administered anti-substance P antibodies, anti-somatostatin antibodies, and anti-angiotensin II antibodies did not affect streptozotocin-induced diabetic allodynia mice. Mechanical allodynia was attenuated by the i.t. administration of CCK-B receptor antagonists and ORL-1 receptor antagonists. The mRNA level of CCK-B receptors in streptozotocin-induced diabetic allodynia mice increased in the spinal cord, but not in the dorsal root ganglion. These results indicate that diabetic allodynia is caused by cholecystokinin octapeptide, nociceptin/orphanin FQ, and hemokinin-1 released from primary afferent neurons in the spinal cord that transmit pain to the brain via the spinal dorsal horn.

Everolimus prevents doxorubicin-induced apoptosis in H9c2 cardiomyocytes but not in MCF-7 cancer cells: Cardioprotective roles of autophagy, mitophagy, and AKT.

Cardiotoxicity is a severe side effect of the chemotherapeutic agent doxorubicin (DOX). We recently showed that DOX-induced cardiomyocyte apoptosis and death were attenuated through autophagy pre-induction. Herein, we assessed how the autophagy/mitophagy-inducing antitumor drug everolimus (EVL) affected DOX-induced cytotoxicity in the rat cardiomyocyte cell line H9c2 and human breast cancer cell line MCF-7. Apoptosis was assessed using annexin V assay. Autophagy and mitophagy were assessed using fluorescence assays. Cellular protein levels were determined using western blotting. Pretreatment with EVL (1 nM) before DOX exposure inhibited mammalian target of rapamycin (mTOR) activity, induced autophagy and mitophagy, and activated protein kinase B (AKT) in H9c2 cells. In mitochondria, DOX (1 µM) induced structural damage (decreased membrane potential and release of cytochrome c), increased superoxide levels, decreased apoptosis inhibitor Bcl-2, and increased apoptosis inducer Bax, leading to apoptosis and reduced viability in H9c2 cells. EVL pretreatment suppressed DOX-induced changes. EVL anti-apoptotic effects were inhibited by treatment with MK-2206, a selective AKT inhibitor. Furthermore, EVL suppressed DOX-induced cardiotoxicity through autophagy/mitophagy and AKT activation but did not attenuate DOX-induced apoptosis or reduction in viability in MCF-7 cells. Altogether, EVL can protect cardiomyocytes from DOX-induced apoptosis and toxicity without reducing DOX antitumor effects, allowing safer chemotherapy.

Thromboxane A2 and prostaglandin F2alpha mediate inflammatory tachycardia.

Systemic inflammation induces various adaptive responses including tachycardia. Although inflammation-associated tachycardia has been thought to result from increased sympathetic discharge caused by inflammatory signals of the immune system, definitive proof has been lacking. Prostanoids, including prostaglandin (PG) D(2), PGE(2), PGF(2alpha), PGI(2) and thromboxane (TX) A(2), exert their actions through specific receptors: DP, EP (EP(1), EP(2), EP(3), EP(4)), FP, IP and TP, respectively. Here we have examined the roles of prostanoids in inflammatory tachycardia using mice that lack each of these receptors individually. The TXA(2) analog I-BOP and PGF(2alpha) each increased the beating rate of the isolated atrium of wild-type mice in vitro through interaction with TP and FP receptors, respectively. The cytokine-induced increase in beating rate was markedly inhibited in atria from mice lacking either TP or FP receptors. The tachycardia induced in wild-type mice by injection of lipopolysaccharide (LPS) was greatly attenuated in TP-deficient or FP-deficient mice and was completely absent in mice lacking both TP and FP. The beta-blocker propranolol did not block the LPS-induced increase in heart rate in wild-type animals. Our results show that inflammatory tachycardia is caused by a direct action on the heart of TXA(2) and PGF(2alpha) formed under systemic inflammatory conditions.

Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes.

Doxorubicin (DOX) is a potent chemotherapeutic agent; however, it causes severe heart injury via apoptosis induction in many patients. DOX-induced cardiotoxicity is attenuated by activated autophagy in the heart. We previously found that programmed cell death 1 (Pdcd1), an immune checkpoint receptor, inhibits DOX-induced cardiomyocyte apoptosis. In this study, we investigated whether autophagy contributes to the protective role of Pdcd1 against DOX-induced cardiomyocyte apoptosis. We also examined the role of Pdcd1 in DOX-induced apoptosis in cancer cells. Rat cardiomyocyte cell line H9c2 and human cancer cell lines K562 and MCF-7 were transfected with Pdcd1-encoding plasmid DNA to establish Pdcd1-overexpressing cells. Apoptosis and autophagy were determined using a luciferase assay. In H9c2 cells, DOX-induced apoptosis and viability reduction occurred through caspase activation. In particular, Pdcd1 overexpression activated the autophagy pathway through the inhibition of the mammalian target of rapamycin, a major negative regulator of autophagy. Moreover, it prevented DOX-induced cardiomyocyte apoptosis; a similar cardioprotection was observed when normal H9c2 cells (without Pdcd1 overexpression) were treated with rapamycin, an autophagy inducer, before the DOX treatment. Conversely, in cancer cells, Pdcd1 overexpression increased both basal and DOX-induced apoptosis. The role of Pdcd1 in DOX-induced apoptosis in cardiomyocytes and cancer cells was opposing. Pdcd1 signaling prevented DOX-induced apoptosis in cardiomyocytes, through autophagy induction; it enhanced DOX-induced apoptosis in cancer cells. Therefore, Pdcd1 could be a critical molecule for more effective and safer DOX chemotherapy.

The mRNA expression of Il6 and Pdcd1 are predictive and protective factors for doxorubicin­induced cardiotoxicity.

Anthracyclines, such as doxorubicin (DOX), have been widely used in the treatment of a number of different solid and hematological malignancies. However, these drugs can inflict cumulative dose­dependent and irreversible damage to the heart, and can occasionally lead to heart failure. The cardiotoxic susceptibility varies among patients treated with anthracycline, and delays in the recognition of cardiotoxicity can result in poor prognoses. Accordingly, if the risk of cardiotoxicity could be predicted prior to drug administration, it would aid in safer and more effective chemotherapy treatment. The present study was carried out to identify genes that can predict DOX­induced cardiotoxicity (DICT). In an in vivo study, mice cumulatively treated with DOX demonstrated increases in serum levels of cardiac enzymes (aspartate aminotransferase, lactate dehydrogenase, creatine kinase MB isoenzyme and troponin T), in addition to decreases in body and heart weights. These changes were indicative of DICT, but the severity of these effects varied among individual mice. In the current study, the correlation in these mice between the extent of DICT and circulating blood concentrations of relevant transcripts before DOX administration was analyzed. Among various candidate genes, the plasma mRNA levels of the genes encoding interleukin 6 (Il6) and programmed cell death 1 (Pdcd1) in blood exhibited significant and positive correlations with the severity of DICT. In an in vitro study using cardiomyocyte H9c2 cells, knockdown of Il6 or Pdcd1 by small interfering RNA was revealed to enhance DOX­induced apoptosis, as determined by luminescent assays. These results suggested that the levels of transcription of Il6 and Pdcd1 in cardiomyocytes serve a protective role against DICT, and that the accumulation of these gene transcripts in blood is a predictive marker for DICT. To the best of our knowledge, this is the first report to demonstrate a role for Il6 and Pdcd1 mRNA expression in DICT.

Sialic acid-binding lectin from bullfrog eggs inhibits human malignant mesothelioma cell growth in vitro and in vivo.

Malignant mesothelioma is an aggressive cancer that results from exposure to asbestos. The therapeutic options for this type of cancer are limited; therefore, the development of novel therapeutic agents is urgently required. Sialic acid-binding lectin isolated from Rana catesbeiana oocytes (cSBL) is a novel therapeutic candidate for cancer, which exhibits antitumor activity mediated through RNA degradation. In the present study, we evaluated the effect of cSBL in vitro and in vivo. Xenograft-competent H2452 and MSTO human mesothelioma cell lines were treated with cSBL, and the pathway by which cSBL induces apoptosis was analyzed. In vivo studies were performed using nude mice inoculated with one of the two cell lines, and the effects of cSBL and pemetrexed were monitored simultaneously. Furthermore, the pharmacological interactions between the three agents (pemetrexed, cisplatin and cSBL) were statistically assessed. It was demonstrated that cSBL treatments caused morphological and biochemical apoptotic changes in both cell lines. Caspase cascade analysis revealed that an intrinsic pathway mediated cSBL-induced apoptosis. The administration of cSBL significantly inhibited tumor growth in two xenograft models, without any adverse effects. Furthermore, the combination index and dose reduction index values indicated that the cSBL + pemetrexed combination showed the highest synergism, and thus potential for reducing dosage of each drug, compared with the other combinations, including the existing pemetrexed + cisplatin regimen. cSBL exerted prominent antitumor effects on malignant mesothelioma cells in vitro and in vivo, and showed favorable effects when combined with pemetrexed. These results suggest that cSBL has potential as a novel drug for the treatment of malignant mesothelioma.

Decreased susceptibility to renovascular hypertension in mice lacking the prostaglandin I2 receptor IP.

Persistent reduction of renal perfusion pressure induces renovascular hypertension by activating the renin-angiotensin-aldosterone system; however, the sensing mechanism remains elusive. Here we investigated the role of PGI2 in renovascular hypertension in vivo, employing mice lacking the PGI2 receptor (IP-/- mice). In WT mice with a two-kidney, one-clip model of renovascular hypertension, the BP was significantly elevated. The increase in BP in IP-/- mice, however, was significantly lower than that in WT mice. Similarly, the increases in plasma renin activity, renal renin mRNA, and plasma aldosterone in response to renal artery stenosis were all significantly lower in IP-/- mice than in WT mice. All these parameters were measured in mice lacking the four PGE2 receptor subtypes individually, and we found that these mice had similar responses to WT mice. PGI2 is produced by COX-2 and a selective inhibitor of this enzyme, SC-58125, also significantly reduced the increases in plasma renin activity and renin mRNA expression in WT mice with renal artery stenosis, but these effects were absent in IP-/- mice. When the renin-angiotensin-aldosterone system was activated by salt depletion, SC-58125 blunted the response in WT mice but not in IP-/- mice. These results indicate that PGI2 derived from COX-2 plays a critical role in regulating the release of renin and consequently renovascular hypertension in vivo.

Glutathione peroxidase 3 is a protective factor against acetaminophen­induced hepatotoxicity in vivo and in vitro.

Acetaminophen (APAP) is a widely available antipyretic and analgesic; however, overdose of the drug inflicts severe damage to the liver. It is well established that the hepatotoxicity of APAP is initiated by formation of a reactive metabolite, N­acetyl­p­benzoquinone imine (NAPQI), which can be detoxified by conjugation with reduced glutathione (GSH), a typical antioxidant. We recently found that the blood mRNA expression level of glutathione peroxidase 3 (Gpx3), which catalyzes the oxidation of GSH, is associated with the extent of APAP­induced hepatotoxicity in mice. The present study was carried out to determine the in vivo and in vitro role of GPx3 in APAP­induced hepatotoxicity. In in vivo experiments, oral administration of APAP to mice induced liver injury. Such liver injury was greater in males than in females, although no gender difference in the plasma concentration of APAP was found. Female mice had a 2­fold higher expression of Gpx3 mRNA and higher plasma GPx activity than male mice. 17ß­estradiol, a major female hormone, decreased APAP­induced hepatotoxicity and increased both the expression of blood Gpx3 mRNA and plasma GPx activity, suggesting that the cytoprotective action of this hormone is mediated by the increase in GPx3. To further clarify the role of GPx3 in APAP­induced hepatotoxicity, we evaluated the effect of a change in cellular GPx3 expression resulting from transfection of either siRNA­GPx3 or a GPx3 expression vector on NAPQI­induced cellular injury (as assessed by a tetrazolium assay) in in vitro experiments using heterogeneous cultured human cell lines (Huh­7 or K562). NAPQI­induced cell death was reduced by increased GPx3 and was enhanced by decreased GPx3. These results suggest that GPx3 is an important factor for inhibition of APAP­induced hepatotoxicity both in vivo and in vitro. To our knowledge, this is the first report to show a hepatoprotective role of cellular GPx3 against APAP­induced liver damage.

Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant mesothelioma.

Malignant mesothelioma is an aggressive cancer with limited therapeutic options. Sialic acid-binding lectin isolated from Rana catesbeiana oocytes (cSBL) is a multifunctional protein with anti-cancer activity. The effects of pemetrexed, cisplatin, and cSBL were evaluated in mesothelioma and normal mesothelial cell lines. We evaluated cytotoxicity, apoptosis, caspase-3 cleavage and activation, cell proliferation, cell cycle arrest, and levels of cell cycle proteins in H28 cells treated with pemetrexed, cisplatin, and cSBL alone or in combination. Treatment with cSBL alone was cytotoxic to mesothelioma cells. The anti-cancer effect of cSBL was observed in a broader range of cell lines and exhibited greater cancer cell selectivity than pemetrexed or cisplatin. Combination treatment with pemetrexed + cSBL resulted in greater dose-dependent cytotoxicity than pemetrexed + cisplatin, the standard of care in mesothelioma. The synergistic effect of pemetrexed + cSBL was mediated by the cytostatic effect of pemetrexed and the cytotoxic effect of cSBL. It thus appears that cSBL has therapeutic potential for the treatment of mesothelioma.

Augmented cardiac hypertrophy in response to pressure overload in mice lacking the prostaglandin I2 receptor.

BACKGROUND: In the heart, the expressions of several types of prostanoid receptors have been reported. However, their roles in cardiac hypertrophy in vivo remain unknown. We intended to clarify the roles of these receptors in pressure overload-induced cardiac hypertrophy using mice lacking each of their receptors. METHODS AND RESULTS: We used a model of pressure overload-induced cardiac hypertrophy produced by banding of the transverse aorta in female mice. In wild-type mice subjected to the banding, cardiac hypertrophy developed during the observation period of 8 weeks. In mice lacking the prostaglandin (PG) I2 receptor (IP(-/-)), however, cardiac hypertrophy and cardiomyocyte hypertrophy were significantly greater than in wild-type mice at 2 and 4 weeks but not at 8 weeks, whereas there was no such augmentation in mice lacking the prostanoid receptors other than IP. In addition, cardiac fibrosis observed in wild-type hearts was augmented in IP(-/-) hearts, which persisted for up to 8 weeks. In IP(-/-) hearts, the expression level of mRNA for atrial natriuretic peptide, a representative marker of cardiac hypertrophy, was significantly higher than in wild-type hearts. In vitro, cicaprost, an IP agonist, reduced platelet-derived growth factor-induced proliferation of wild-type noncardiomyocytes, although it could not inhibit cardiotrophin-1-induced hypertrophy of cardiomyocytes. Accordingly, cicaprost increased cAMP concentration efficiently in noncardiomyocytes. CONCLUSIONS: IP plays a suppressive role in the development of pressure overload-induced cardiac hypertrophy via the inhibition of both cardiomyocyte hypertrophy and cardiac fibrosis. Both effects have been suggested as originating from the action on noncardiomyocytes rather than cardiomyocytes.

Prostaglandin E2 protects the heart from ischemia-reperfusion injury via its receptor subtype EP4.

BACKGROUND: In the heart with acute myocardial infarction, production of prostaglandin (PG) E2 increases significantly. In addition, several subtypes of PGE2 receptors (EPs) have been reported to be expressed in the heart. The role of PGE2 in cardiac ischemia-reperfusion (I/R) injury, however, remains unknown. We intended to clarify the role of PGE2 via EP4, an EP subtype, in I/R injury using mice lacking EP4 (EP4-/- mice). METHODS AND RESULTS: In murine cardiac ventricle, competitive reverse transcription-polymerase chain reaction revealed the highest expression level of EP4 mRNA among EP mRNAs. EP4-/- mice had larger infarct size than wild-type mice in a model of I/R; the left anterior descending coronary artery was occluded for 1 hour, followed by 24 hours of reperfusion. In addition, isolated EP4-/- hearts perfused according to the Langendorff technique had greater functional and biochemical derangements in response to I/R than wild-type hearts. In vitro, AE1-329, an EP4 agonist, raised cAMP concentration remarkably in noncardiomyocytes, whereas the action was weak in cardiomyocytes. When 4819-CD, another EP4 agonist, was administered 1 hour before coronary occlusion, it reduced infarct size significantly in wild-type mice. Notably, a similar cardioprotective effect was observed even when it was administered 50 minutes after coronary occlusion. CONCLUSIONS: Both endogenous PGE2 and an exogenous EP4 agonist protect the heart from I/R injury via EP4. The potent cardioprotective effects of 4819-CD suggest that the compound would be useful for treatment of acute myocardial infarction.

Thromboxane A2 regulates vascular tone via its inhibitory effect on the expression of inducible nitric oxide synthase.

BACKGROUND: Circulatory failure in sepsis arises from vascular hyporesponsiveness, in which nitric oxide (NO) derived from inducible NO synthase (iNOS) plays a major role. Details of the cross talk between thromboxane (TX) A2 and the iNOS-NO system, however, remain unknown. We intended to clarify the role of TXA2, via the cross talk, in vascular hyporesponsiveness. METHODS AND RESULTS: We examined cytokine-induced iNOS expression and NO production in cultured vascular smooth muscle cells (VSMCs) and cytokine-induced hyporesponsiveness of the aorta from mice lacking the TXA2 receptor (TP-/- mice). The cytokine-induced iNOS expression and NO production observed in wild-type VSMCs were significantly augmented in TP-/- VSMCs, indicating an inhibitory effect of endogenous TXA2 on iNOS expression. Furthermore, in indomethacin-treated wild-type VSMCs, U-46619, a TP agonist, inhibited cytokine-induced iNOS expression and NO production in a concentration-dependent manner, effects absent from TP-/- VSMCs. In an ex vivo system, the cytokine-induced hyporesponsiveness of aortas to phenylephrine was significantly augmented in TP-/- aorta but was almost completely canceled by aminoguanidine, an iNOS inhibitor. Accordingly, cytokine-induced NO production was significantly higher in TP-/- aorta than in wild-type aorta. Moreover, U-46619 significantly suppressed lipopolysaccharide-induced NO production in vivo only in wild-type mice. CONCLUSIONS: These results suggest that TXA2 has a protective role against the development of vascular hyporesponsiveness via its inhibitory action on the iNOS-NO system under pathological conditions such as sepsis.

Sinomenine and magnoflorine, major constituents of Sinomeni caulis et rhizoma, show potent protective effects against membrane damage induced by lysophosphatidylcholine in rat erythrocytes.

The effects of the water extract of Sinomeni Caulis et Rhizoma (SCR-WE) and its major constituents, sinomenine (SIN) and magnoflorine (MAG), on moderate hemolysis induced by lysophosphatidylcholine (LPC) were investigated in rat erythrocytes and compared with the anti-hemolytic effects of lidocaine (LID) and propranolol (PRO) as reference drugs. LPC caused hemolysis at concentrations above the critical micelle concentration (CMC), and the concentration of LPC producing moderate hemolysis (60 %) was approximately 10 µM. SCR-WE at 1 ng/mL-100 µg/mL significantly inhibited the hemolysis induced by LPC. SIN and MAG attenuated LPC-induced hemolysis in a concentration-dependent manner from very low to high concentrations (1 nM-100 µM and 10 nM-100 µM, respectively). In contrast, the inhibiting effects of LID and PRO on LPC-induced hemolysis were observed at higher concentrations (1-100 µM) but not at lower concentrations (1-100 nM). Neither SIN nor MAG affected micelle formation of LPC, nor, at concentrations of 1 nM-1 µM, did they attenuate the hemolysis induced by osmotic imbalance (hypotonic hemolysis). Similarly, SCR-WE also did not modify micelle formation or hypotonic hemolysis, except at the highest concentration. These results suggest that SIN and MAG potently protect the erythrocyte membrane from LPC-induced damage and contribute to the beneficial action of SCR-WE. The protective effects of SIN and MAG are mediated by some mechanism other than prevention of micelle formation or protection of the erythrocyte membrane against osmotic imbalance.

Effects of the prostanoids on the proliferation or hypertrophy of cultured murine aortic smooth muscle cells.

Effects of the prostanoids on the growth of cultured aortic vascular smooth muscle cells (VSMCs) were examined using mice lacking prostanoid receptors. Proliferation of VSMCs was assessed by measuring [(3)H]-thymidine incorporation and the cell number, and their hypertrophy by [(14)C]-leucine incorporation and protein content. In VSMCs from wild-type mice, expressions of mRNAs for the EP(4) and TP were most abundant, followed by those for the IP, EP(3) and FP, when examined by competitive reverse transcriptase-PCR. Those for the EP(1), EP(2) and DP, however, could not be detected. AE1-329, an EP(4) agonist, and cicaprost, an IP agonist, inhibited platelet derived growth factor (PDGF)-induced proliferation of VSMCs from wild-type mice; these inhibitory effects disappeared completely in VSMCs from EP(4)(-/-) and IP(-/-) mice, respectively. In accordance with these effects, AE1-329 and cicaprost stimulated cAMP production in VSMCs from wild-type mice, which were absent in VSMCs from EP(4)(-/-) and IP(-/-) mice, respectively. Effects of PGE(2) on cell proliferation and adenylate cyclase were almost similar with those of AE1-329 in VSMCs from wild-type mice, which disappeared in VSMCs from EP(4)(-/-) mice. PGD(2) inhibited PDGF-induced proliferation of VSMCs from both wild-type and DP(-/-) mice to a similar extent. This action of PGD(2) was also observed in VSMCs from EP4(-/-) and IP(-/-) mice. In VSMCs from wild-type mice, I-BOP, a TP agonist, showed potentiation of PDGF-induced hypertrophy. I-BOP failed to show this action in VSMCs from TP(-/-) mice. The specific agonists for the EP(1), EP(2) or EP(3), and PGF(2)alpha showed little effect on the growth of VSMCs. These results show that PGE(2), PGI(2) and TXA(2) modulate PDGF-induced proliferation or hypertrophy of VSMCs via the EP(4), IP and TP, respectively, and that the inhibitory effect of PGD(2) on PDGF-induced proliferation is not mediated by the DP, EP(4) or IP.

Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation.

The effect of selective activation of platelet prostaglandin (PG) E2 receptor subtype EP2 or EP4 on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE2 inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE2 on platelets lacking EP2 (EP2-/- platelets) and EP4 (EP4-/- platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1-259 and AE1-329, agonists for EP2 and EP4, respectively, potently inhibited U-46619 -induced aggregation with respective IC50 values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP2-/- and EP4-/- platelets. In human platelets, AE1-259 and AE1-329 inhibited U-46619-induced aggregation with respective IC50 values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1-329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1-259. AE1-329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3-208, an EP4 antagonist. In addition, AE1-329 increased intracellular cAMP concentrations in a concentration- and EP4-dependent manner in human platelets. These results indicate that selective activation of EP2 or EP4 can inhibit platelet aggregation and that EP4 agonists are particularly promising as novel anti-platelet agents.

[Pathophysiological roles of the prostanoids in the cardiovascular system: studies using mice deficient in prostanoid receptors].

Prostanoids, consisting of the prostaglandins (PGs) and thromboxanes (TXs), exert various actions through activation of their specific receptors. They include the DP, EP, FP, IP, and TP receptors for PGD2, PGE2, PGF2alpha, PGI2, and TXA2, respectively. Moreover, EP receptors are classified into four subtypes, the EP1, EP2, EP3 and EP4 receptors. Using mice lacking prostanoid receptors, we intended to clarify in vivo roles of prostanoids under pathophysiological conditions of the cardiovascular system, which include ischemia-induced cardiac injury, pressure overload-induced cardiac hypertrophy, renovascular hypertension, tachycardia during systemic inflammation and thromboembolism. The results demonstrated that 1) PGI2 plays an important role in attenuating the ischemic injury and the pressure overload-induced hypertrophy of the hearts, and also contributes to the development of renovascular hypertension; 2) PGE2 plays a cardioprotective role against the ischemic injury via both the EP3 and EP4, and also participates in acute thromboembolism via the EP3; and 3) both PGF2alpha and TXA2, which have been produced during systemic inflammation, are responsible for tachycardia.

Protective effects of quinaprilat and trandolaprilat, active metabolites of quinapril and trandolapril, on hemolysis induced by lysophosphatidylcholine in human erythrocytes.

We examined the effects of the angiotensin converting enzyme (ACE) inhibitors captopril, enalaprilat, quinapril, and trandolapril, and their active metabolites quinaprilat and trandolaprilat, on hemolysis induced by lysophosphatidylcholine (LPC) in human erythrocytes. LPC induced hemolysis at the concentrations above the critical micelle concentration (4 microM). Propranolol, used as a reference drug, attenuated the 50% hemolysis induced by 6 microM LPC at concentrations ranging from 100 nM to 100 microM. Similarly, quinaprilat (10 microM) and trandolaprilat (10, 100 microM) significantly attenuated the LPC-induced hemolysis, but other ACE inhibitors did not. Since propranolol possesses a membrane stabilizing action correlated with high lipophilicity, it appears that the high lipophilicity of quinaprilat or trandolaprilat is responsible for the protection from the damage induced by LPC. However, quinapril and trandolapril were not effective, although both drugs have higher lipophilicity than quinaprilat and trandolaprilat. Hence, it is suggested that the high lipophilicity alone may not contribute to the protective effects of ACE inhibitors against LPC-induced hemolysis. None of ACE inhibitors attenuated the hypotonic hemolysis (60 mM NaCl), although propranolol did. Furthermore, neither propranolol (100 microM) nor quinaprilat (50 microM) and trandolaprilat (50 microM) affected LPC micelle formation, suggesting that these drugs do not directly bind to LPC. We therefore believe that the protective effects of quinaprilat and trandolaprilat on the LPC-induced hemolysis may be related physicochemically to their highly lipophilic and ACE inhibitory structures, which probably maintain erythrocyte membrane integrity by a mechanism other than ACE inhibition, prevention of LPC micelle formation or protection against osmotic imbalance.