ABSTRACT
Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.
Subject(s)
Arthropod Proteins/pharmacology , Carrier Proteins/pharmacology , Drug Design , Invertebrate Hormones/pharmacology , Models, Molecular , Penaeidae/drug effects , Reproductive Control Agents/pharmacology , Vitellogenesis/drug effects , Amino Acid Sequence , Animals , Antibodies, Neutralizing/pharmacology , Aquaculture , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Arthropod Proteins/metabolism , Biological Assay , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Conserved Sequence , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/pharmacology , Eye , Female , Invertebrate Hormones/chemistry , Invertebrate Hormones/genetics , Invertebrate Hormones/metabolism , Neurosecretory Systems/cytology , Neurosecretory Systems/drug effects , Neurosecretory Systems/physiology , Penaeidae/cytology , Penaeidae/physiology , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Reproductive Control Agents/antagonists & inhibitors , Reproductive Control Agents/chemistry , Reproductive Control Agents/metabolism , Sequence Alignment , Structural Homology, Protein , Thioredoxins/chemistry , Thioredoxins/genetics , Thioredoxins/metabolism , Thioredoxins/pharmacology , Vitellins/antagonists & inhibitors , Vitellins/genetics , Vitellins/metabolism , Vitellogenins/antagonists & inhibitors , Vitellogenins/genetics , Vitellogenins/metabolismABSTRACT
It has been shown earlier that plumbagin, a naturally occurring naphthaquinone has specific anticancer activity in BRCA1 blocked ovarian cancer cells. Plumbagin can induce estrogen dependent cell signaling and apoptosis in BRCA1 blocked ovarian cancer cells. Being a reactive oxygen species (ROS) generator and apoptosis inducing agent, plumbagin has immense potential as a promising anticancer agent. In this study we analyzed whether there would be increased anticancer activity if the positions of the functional groups on plumbagin were altered and further to analyze the detailed molecular mechanism of action of the lead molecule. Methods like MTT assay, apoptosis analysis by flow cytometry, assessment of mitochondrial membrane potential-Δψm , suppression subtractive hybridization, microarray, molecular docking and estrogen receptor-DNA binding activity by electrophoresis mobility shift assay (EMSA) were adopted for assessing the anticancer activity. Consequently we found that, plumbagin was the most potent anticancer agent when compared to structurally related compounds. The anti-cancer activities were in the order plumbagin > 1,4-naphthaquinone > juglone > lawsone > menadione. Molecular docking studies showed that plumbagin could be well docked in the receptor ligand complex of TRAIL-DR5 complexes to activate the extrinsic pathway of apoptosis. Since the antiproliferative activity of plumbagin could be reduced by inhibiting ERα, we speculated that plumbagin interferes with the binding of ERα to ERE and we confirmed this by EMSA. This study clearly indicates that plumbagin can induce multiple pathways of apoptosis and cell cycle arrest in BRCA1 blocked cells compared to unblocked cells.
