Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) has recently emerged as a promising therapeutic target in cancer. In this study, we explored the biological function of MAP4K4 in radioresistant breast cancer cells using two MAP4K4 inhibitors, namely PF06260933 and GNE-495. Radioresistant SR and MR cells were established by exposing SK-BR-3 and MCF-7 breast cancer cells to 48-70 Gy of radiation delivered at 4-5 Gy twice a week over 10 months. Surprisingly, although radioresistant cells were derived from two different subtypes of breast cancer cell lines, MAP4K4 was significantly elevated regardless of subtype. Inhibition of MAP4K4 with PF06260933 or GNE-495 selectively targeted radioresistant cells and improved the response to irradiation. Furthermore, MAP4K4 inhibitors induced apoptosis through the accumulation of DNA damage by inhibiting DNA repair systems in radioresistant cells. Notably, Inhibition of MAP4K4 suppressed the expressions of ACSL4, suggesting that MAP4K4 functioned as an upstream effector of ACSL4. This study is the first to report that MAP4K4 plays a crucial role in mediating the radioresistance of breast cancer by acting upstream of ACSL4 to enhance DNA damage response and inhibit apoptosis. We hope that our findings provide a basis for the development of new drugs targeting MAP4K4 to overcome radioresistance.

Oxidative tryptamine dimers from Corynebacterium durum directly target survivin to induce AIF-mediated apoptosis in cancer cells.

Accumulating evidence indicates that microbial communities in the human body crucially affect health through the production of chemical messengers. However, the relationship between human microbiota and cancer has been underexplored. As a result of a biochemical investigation of the commensal oral microbe, Corynebacterium durum, we identified the non-enzymatic transformation of tryptamine into an anticancer compound, durumamide A (1). The structure of 1 was determined using LC-MS and NMR data analysis as bis(indolyl)glyoxylamide, which was confirmed using one-pot synthesis and X-ray crystallographic analysis, suggesting that 1 is an oxidative dimer of tryptamine. Compound 1 displayed cytotoxic activity against various cancer cell lines with IC50 values ranging from 25 to 35 µM. A drug affinity responsive target stability assay revealed that survivin is the direct target protein responsible for the anticancer effect of 1, which subsequently induces apoptosis-inducing factor (AIF)-mediated apoptosis. Inspired by the chemical structure and bioactivity of 1, a new derivative, durumamide B (2), was synthesized using another indole-based neurotransmitter, serotonin. The anticancer properties of 2 were similar to those of 1; however, it was less active. These findings reinforce the notion of human microbiota-host interplay by showing that 1 is naturally produced from the human microbial metabolite, tryptamine, which protects the host against cancer.

Daidzein Inhibits Human Platelet Activation by Downregulating Thromboxane A2 Production and Granule Release, Regardless of COX-1 Activity.

Platelets play crucial roles in cardiovascular diseases (CVDs) by regulating hemostasis and blood coagulation at sites of blood vessel damage. Accumulating evidence indicates daidzein inhibits platelet activation, but the mechanism involved has not been elucidated. Thus, in this study, we investigated the mechanism responsible for the inhibition of collagen-induced platelet aggregation by daidzein. We found that in collagen-induced platelets, daidzein suppressed the production of thromboxane A2 (TXA2), a molecule involved in platelet activation and aggregation, by inhibiting the cytosolic phospholipase A2 (cPLA2) signaling pathway. However, daidzein did not affect cyclooxygenase-1 (COX-1). Furthermore, daidzein attenuated the PI3K/PDK1/Akt/GSK3αß and MAPK (p38, ERK) signaling pathways, increased the phosphorylation of inositol trisphosphate receptor1 (IP3R1) and vasodilator-stimulated phosphoprotein (VASP), and increased the level of cyclic adenosine monophosphate (cAMP). These results suggest that daidzein inhibits granule release (ATP, serotonin, P-selectin), integrin αIIbß3 activation, and clot retraction. Taken together, our study demonstrates that daidzein inhibits collagen-induced platelet aggregation and suggests that daidzein has therapeutic potential for the treatment of platelet aggregation-related diseases such as atherosclerosis and thrombosis.

A new function for MAP4K4 inhibitors during platelet aggregation and platelet-mediated clot retraction.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is implicated in type 2 diabetes mellitus, insulin tolerance, inflammation, cancer, and atherosclerosis. We found that GNE 495 and PF 06260933 (both potent and selective MAP4K4 inhibitors) regulated human platelet activation. Immunoblotting revealed human platelets express MAP4K4, and that GNE 495 and PF 06260933 inhibited collagen-, ADP-, and thrombin-induced platelet aggregation and eventually suppressed granule release, TXA2 generation, integrin αIIbß3 activation, and clot retraction. In addition, both inhibitors elevated intracellular levels of cAMP, and coincubation with GNE 495 and aspirin or dipyridamole (a phosphodiesterase inhibitor) synergistically inhibited collagen-induced platelet aggregation and TXA2 generation. Moreover, both inhibitors phosphorylated VASP (ser157), IP3 receptor, and PKA and attenuated MAPK and PI3K/Akt/GSK3ß signaling pathways. This study is the first to demonstrate that MAP4K4 inhibitors reduce thrombus formation by inhibiting platelet activation. These findings also suggest MAP4K4 be considered an emerging target protein for the treatment of thrombosis.

The antithrombotic effect of caffeic acid is associated with a cAMP-dependent pathway and clot retraction in human platelets.

Caffeic acid (CA) is a polyphenol widely distributed in the plant kingdom. Studies have shown CA possesses antithrombotic activity in mouse cerebral arterioles in vivo and inhibits platelet aggregation in vitro. However, little is known regarding the effects of CA on platelet-mediated clot retraction. We investigated the effects of CA on platelet activation and clot retraction in response to thrombin. CA inhibited thrombin-induced platelet aggregation, calcium mobilization, adenosine 1,4,5-tri-phosphate (ATP) release, P-selectin expression and fibrinogen binding to integrin αIIbß3 activation without inducing any cytotoxic effect, and inhibited the phosphorylations of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) in thrombin-stimulated platelets. In addition, CA enhanced cyclic adenosine monophosphate (cAMP) generation, which led to the phosphorylations of vasodilator-stimulated phosphoprotein (VASP) and inositol trisphosphate (IP3) receptor, and reduced clot retraction without any anticoagulation effect. Dipyridamole, a phosphodiesterase 3 (PDE3) inhibitor, reduced clot retraction, which suggested CA-mediated cAMP generation is the main signaling pathway responsible for its inhibition of clot retraction. Taken together, the findings of the present study suggest that CA may have potential as a therapeutic for the prevention of thrombotic disorders.

Arctigenin attenuates platelet activation and clot retraction by regulation of thromboxane A2 synthesis and cAMP pathway.

Pathophysiological reaction of platelets in the blood vessel is an indispensable part of thrombosis and cardiovascular disease, which is the most common cause of death in the world. In this study, we performed in vitro assays to evaluate antiplatelet activity of arctigenin in human platelets and attempted to identify the mechanism responsible for thromboxane A2 (TXA2) generation, integrin αIIbß3 activation and cAMP pathway. Arctigenin exhibited obvious inhibitory effects on collagen-, thrombin-, and ADP-induced human platelet aggregation, granule secretion, TXA2 generation, integrin αIIbß3 activation, and clot retraction. Additionally, we found that arctigenin attenuated PI3K/Akt/mTOR/GSK-3ß and MAPK signaling pathways, and increased cAMP level. Accordingly, the findings support that arctigenin attenuates thrombotic events through the inhibition of platelet activation and platelet plug formation. Therefore, we suggest that arctigenin may have therapeutic potential as an antiplatelet and antithrombotic agent.

Anti­platelet activity of mineral­balanced deep sea water is mediated via the regulation of Akt and ERK pathway crosstalk.

Mineral­balanced deep sea water (MBDSW), an unlimited natural sea source, has been demonstrated to minimize the risk of developing cardiovascular diseases, such as obesity, hypertension, inflammation and hyperlipidemia. This study investigated the effects of MBDSW [magnesium (Mg):calcium (Ca) ratio, 3:1] on platelet activation. MBDSW significantly inhibited the collagen­ and thrombin­induced platelet aggregation of human platelets. In collagen­induced platelets, MBDSW inhibited intracellular calcium mobilization, granule secretion [serotonin, adenosine triphosphate (ATP) and P­selectin expression] and thromboxane A2 (TXA2) production. Moreover, MBDSW markedly inhibited Akt and extracellular signal­regulated kinase (ERK) phosphorylation, but not that of c­Jun N­terminal kinase (JNK) and p38. Moreover, MBDSW phosphorylated inositol 1,4,5­triphosphate receptor (IP3R) and vasodilator­stimulated phosphoprotein (VASP), and it increased the cyclic adenosine monophosphate (cAMP) level in collagen­induced human platelets. Dipyridamole, a phosphodiesterase (PDE) inhibitor, significantly increased the cAMP level and regulated the Akt, ERK and VASP (Ser157) levels in a manner similar to that of MBDSW. In addition, LY294002, an Akt inhibitor, inhibited the phosphorylation of ERK, and U0126, an ERK inhibitor, inhibited the phosphorylation of Akt. Taken together, the results of the present investigation suggest that the inhibitory effects of MBDSW on platelet aggregation may be associated with the cross­inhibition of Akt and ERK phosphorylation. These results strongly indicate that MBDSW may have preventive or therapeutic potential for platelet aggregation­mediated diseases, such as thrombosis, atherosclerosis and myocardial infarction.

Inhibition of TPA­induced metastatic potential by morin hydrate in MCF­7 human breast cancer cells via the Akt/GSK­3ß/c­Fos signaling pathway.

Plant flavonoid 2',3,4',5,7­pentahydroxyflavone (morin hydrate), isolated from the family Moraceae (Morus alba L.), is known to have anti­inflammatory and anticancer effects. However, its pharmaceutical effects on metastasis have not been fully elucidated to date. Therefore, the current study investigated the effects of morin hydrate on cancer metastasis in MCF­7 human breast cancer cells. The results showed that morin hydrate suppressed 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced cell migration and invasion via the inhibition of matrix metalloproteinase (MMP)­9 activity. Furthermore, gene expression level of MMP­9, MMP­7, urokinase plasminogen activator (uPA), uPA receptor (uPAR) and fibronectin were significantly decreased by morin hydrate treatment. Morin hydrate inhibited the phosphorylation of Akt and glycogen synthase kinase (GSK)­3ß, and downregulated the expression of an activator protein­1 subunit c­Fos. In addition, the GSK­3ß phosphorylation and c­Fos expression were suppressed by PI3K/Akt pathway inhibitors, LY294002 and wortmannin. Taken together, these results demonstrated that morin hydrate reduced the metastatic potential in TPA­treated MCF­7 human breast cancer cells via the inhibition of MMPs, uPA and uPAR, and the underlying Akt/GSK­3ß/c­Fos pathway. Therefore, the present investigation suggested that morin hydrate may be a natural substance with a preventive potential for metastasis in breast cancer cells.

Orostachys japonicus exerts antipancreatic cancer activity through induction of apoptosis and cell cycle arrest in PANC-1 cells.

Targeted therapy at the molecular level is important for pancreatic cancer treatment. This study looked over the anticancer activity of Orostachys japonicus in a human pancreatic cancer cell line, PANC-1. An ethyl acetate fraction containing quercetin, kaempferol, and flavonol glycosides from O. japonicus (OJE) exhibited significant anticancer activity against the PANC-1. OJE activated caspase-3, caspase-8, and caspase-9, leading to the induction of both intrinsic and extrinsic apoptosis pathways. It also inhibited cyclin D1, cyclin B1, and cyclin-dependent kinase 4, representing cell cycle arrest at both G1/S and G2/M phases. In addition, OJE phosphorylated MAPKs such as p38, JNK, and ERK, which are important upstream signaling factors in apoptosis and arrest of cell cycle inducing system. In conclusion, OJE effectively exerted antipancreatic cancer activity via induction of apoptosis directed by both intrinsic and extrinsic pathways and arrest of cell cycle regulated at both G1/S and G2/M stages, which is activated by MAPKs, p38, JNK, and ERK.

Morin hydrate inhibits platelet activation and clot retraction by regulating integrin αIIbß3, TXA2, and cAMP levels.

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbß3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule. In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbß3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25-100 µM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbß3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbß3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.

Antiplatelet and antithrombotic effects of cordycepin-enriched WIB-801CE from Cordyceps militaris ex vivo, in vivo, and in vitro.

BACKGROUND: A species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, we prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo, in vivo, and in vitro antiplatelet and antithrombotic effects. METHODS: Ex vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation, serotonin release, thromboxane A2 (TXA2) production and its associated activities of enzymes [cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS)], arachidonic acid (AA) release and its associated phosphorylation of phospholipase Cß3, phospholipase Cγ2 or cytosolic phospholipase A2, mitogen-activated protein kinase (MAPK) [p38 MAPK, extracellular signal-regulated kinase (ERK)], and blood coagulation time in rats were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. In vitro effects of WIB-801CE on cytotoxicity, and fibrin clot retraction in human platelets, and nitric oxide (NO) production in RAW264.7 cells or free radical scavenging activity were studied. RESULTS: Cordycepin-enriched WIB-801CE inhibited ex vivo platelet aggregation, TXA2 production, AA release, TXAS activity, serotonin release, and p38 MAPK and ERK2 phosphorylation in collagen- and ADP-activated rat platelets without affecting blood coagulation. Furthermore, WIB-801CE manifested in vivo inhibitory effect on collagen plus epinephrine-induced pulmonary thromboembolism mice model. WIB-801CE inhibited in vitro NO production and fibrin clot retraction, but elevated free radical scavenging activity without affecting cytotoxicity against human platelets. CONCLUSION: WIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. These were resulted from down-regulation of TXA2 production and its related AA release and TXAS activity, and p38MAPK and ERK2 activation. These results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.

Cordycepin-enriched WIB801C from Cordyceps militaris inhibits ADP-induced [Ca(2+)] i mobilization and fibrinogen binding via phosphorylation of IP 3R and VASP.

In this study, we investigated the effect of cordycepin-enriched (CE)-WIB801C from Cordyceps militaris on ADP (20 µM)-stimulated platelet aggregation. CE-WIB801C dose-dependently inhibited ADP-induced platelet aggregation, and its IC50 value was 18.5 µg/mL. CE-WIB801C decreased TXA2 production, but did not inhibit the activities of COX-1 and thromboxane synthase (TXAS) in ADP-activated platelets, which suggests that the inhibition of TXA2 production by CE-WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. CE-WIB801C inhibited ATP release and [Ca(2+)]i mobilization, and increased cAMP level and IP3RI (Ser(1756)) phosphorylation in ADP-activated platelets. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased CE-WIB801C-inhibited [Ca(2+)]i mobilization, and strongly inhibited CE-WIB801C-increased IP3RI (Ser(1756)) phosphorylation. CE-WIB801C elevated the phosphorylation of VASP (Ser(157)), an A-kinase substrate, but inhibited fibrinogen binding to αIIb/ß3. These results suggest that CE-WIB801C-elevated cAMP involved in IP3RI (Ser(1756)) phosphorylation to inhibit [Ca(2+)]i mobilization and, VASP (Ser(157)) phosphorylation to inhibit αIIb/ß3 activation. Therefore, in this study, we demonstrate that CE-WIB801C may have a preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.