Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

BACKGROUND: Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. INTRODUCTION: Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. METHODS: Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. RESULT: A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. CONCLUSION: KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Antioxidant and Antithrombotic Activities of Kenaf Seed (Hibiscus cannabinus) Coat Ethanol Extract in Sprague Dawley Rats.

Oxidative stress has been implicated in deadly lifestyle diseases, and antioxidants from plant sources are the primary option in the treatment regime. Kenaf seeds are the storehouse of potential natural antioxidant phytoconstituents. Perhaps, none of the studies documented the phytoconstituents and their antioxidant potential from Kenaf seed coat so far. Thus, the current study focuses on exploring the protective effect of Kenaf Seed Coat Ethanol Extract (KSCEE) against sodium nitrite and diclofenac-induced oxidative stress in vitro (red blood cell and platelets model) and in vivo (female Sprague Dawely rat's model) along with the antithrombotic activity. The infrared spectrophotometry data showed the heterogeneous functional groups (CH, OH, C = C, C = C-C) and aromatic rings. Reverse phase high-performance liquid chromatography and gas chromatography-mass spectrometry chromatogram of KSCEE also evidenced the presence of several phytochemicals. KSCEE displayed about 76% of DPPH scavenging activity with an IC50 value of 34.94 µg/ml. KSCEE significantly (***p < 0.001) normalized the stress markers such as lipid peroxidation, protein carbonyl content, superoxide dismutase, and catalase in sodium nitrite and diclofenac-induced oxidative stress in RBC, platelets, liver, kidney, and small intestine, respectively. Furthermore, KSCEE was found to protect the diclofenac-induced tissue destruction of the liver, kidney, and small intestine obtained from seven groups of female Sprague Dawely rats. KSCEE delayed the clotting time of platelet-rich plasma and platelet-poor plasma and activated partial thromboplastin time, suggesting its anticoagulant property. In addition, KSCEE also exhibited antiplatelet activity by inhibiting both adenosine diphosphate and epinephrine-induced platelet aggregation. In conclusion, KSCEE ameliorates the sodium nitrite and diclofenac-induced oxidative stress in red blood cells, platelets, and experimental animals along with antithrombotic properties.

Loss of miR-936 leads to acquisition of androgen-independent metastatic phenotype in prostate cancer.

Prostate cancer (PCa) progresses from a hormone-sensitive, androgen-dependent to a hormone-refractory, androgen-independent metastatic phenotype. Among the many genes implicated, ANXA2, a calcium-dependent phospholipid binding protein, has been found to have a critical role in the progression of PCa into more invasive metastatic phenotype. However, the molecular mechanisms underlying the absence of ANXA2 in early PCa and its recurrence in advanced stage are yet unknown. Moreover, recent studies have observed the deregulation of microRNAs (miRNAs) are involved in the development and progression of PCa. In this study, we found the down-regulation of miR-936 in metastatic PCa wherein its target ANXA2 was overexpressed. Subsequently, it has been shown that the downregulation of miRNA biogenesis by siRNA treatment in ANXA2-null LNCaP cells could induce the expression of ANXA2, indicating the miRNA mediated regulation of ANXA2 expression. Additionally, we demonstrate that miR-936 regulates ANXA2 expression by direct interaction at coding as well as 3'UTR region of ANXA2 mRNA by luciferase reporter assay. Furthermore, the overexpression of miR-936 suppresses the cell proliferation, cell cycle progression, cell migration, and invasion abilities of metastatic PCa PC-3 cells in vitro and tumor forming ability in vivo. These results indicate that miR-936 have tumor suppressor properties by regulating the over expression of ANXA2 in hormone-independent metastatic PCa. Moreover, our results suggest that this tumor suppressor miR-936 could be developed as a targeted therapeutic molecule for metastatic PCa control and to improve the prognosis in PCa patients.

Carbidopa is an activator of aryl hydrocarbon receptor with potential for cancer therapy.

Carbidopa is used with l-DOPA (l-3,4-dihydroxyphenylalanine) to treat Parkinson's disease (PD). PD patients exhibit lower incidence of most cancers including pancreatic cancer, but with the notable exception of melanoma. The decreased cancer incidence is not due to l-DOPA; however, the relevance of Carbidopa to this phenomenon has not been investigated. Here, we tested the hypothesis that Carbidopa, independent of l-DOPA, might elicit an anticancer effect. Carbidopa inhibited pancreatic cancer cell proliferation both in vitro and in vivo Based on structural similarity with phenylhydrazine, an inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1), we predicted that Carbidopa might also inhibit IDO1, thus providing a molecular basis for its anticancer effect. The inhibitory effect was confirmed using human recombinant IDO1. To demonstrate the inhibition in intact cells, AhR (aryl hydrocarbon receptor) activity was monitored as readout for IDO1-mediated generation of the endogenous AhR agonist kynurenine in pancreatic and liver cancer cells. Surprisingly, Carbidopa did not inhibit but instead potentiated AhR signaling, evident from increased CYP1A1 (cytochrome P450 family 1 subfamily A member 1), CYP1A2, and CYP1B1 expression. In pancreatic and liver cancer cells, Carbidopa promoted AhR nuclear localization. AhR antagonists blocked Carbidopa-dependent activation of AhR signaling. The inhibitory effect on pancreatic cancer cells in vitro and in vivo and the activation of AhR occurred at therapeutic concentrations of Carbidopa. Chromatin immunoprecipitation assay further confirmed that Carbidopa promoted AhR binding to its target gene CYP1A1 leading to its induction. We conclude that Carbidopa is an AhR agonist and suppresses pancreatic cancer. Hence, Carbidopa could potentially be re-purposed to treat pancreatic cancer and possibly other cancers as well.

Structural basis for the carbohydrate recognition of the Sclerotium rolfsii lectin.

The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and in complex with N-acetyl-d-galactosamine (GalNAc) and N-acetyl- d -glucosamine (GlcNAc) has been determined at 1.1 A, 2.0 A, and 1.7 A resolution, respectively. The protein structure is composed of two beta-sheets, which consist of four and six beta-strands, connected by two alpha-helices. Sequence and structural comparisons reveal that SRL is the third member of a newly identified family of fungal lectins, which includes lectins from Agaricus bisporus and Xerocomus chrysenteron that share a high degree of structural similarity and carbohydrate specificity. The data for the free SRL are the highest resolution data for any protein of this family. The crystal structures of the SRL in complex with two carbohydrates, GalNAc and GlcNAc, which differ only in the configuration of a single epimeric hydroxyl group, provide the structural basis for its carbohydrate specificity. SRL has two distinct carbohydrate-binding sites, a primary and a secondary. GalNAc binds at the primary site, whereas GlcNAc binds only at the secondary site. Thus, SRL has the ability to recognize and probably bind at the same time two different carbohydrate structures. Structural comparison to Agaricus bisporus lectin-carbohydrate complexes reveals that the primary site is also able to bind the Thomsen-Friedenreich antigen (Galbeta1-->3GalNAc-alpha- glycan structures) whereas the secondary site cannot. The features of the molecular recognition at the two sites are described in detail.