Ribosome inactivating proteins (RIPs) from momordica charantia for anti viral therapy

This review describes the nature and applications of ribosome inactivating proteins (RIPs) from Momordica charantia (bitter melon). RIPs from the plant kingdom have received much attention in biomedical research because they target conserved host protein synthesis machinery and show specificity towards human and animal cell targets. Recent studies aimed at unravelling the enzymatic activities of the M charantia RIPs provide a structural basis for their activities. It has been reported that RIPs are member of the single chain ribosome inactivating protein (SCRIP) family which act irreversibly on ribosome by removing adenine residue from eukaryotic ribosomal RNA. Various activities of RIPs include anti-tumor, broad anti-viral, ribonuclease and deoxyribonuclease. MAP30 (Momordica Anti-HIV Protein), alpha- and beta-momorcharins inhibit HIV replication in acutely and chronically infected cells and thus are considered potential therapeutic agent in HIV infection and AIDS. Further, MAP30 improved the efficacy of anti-HIV therapy when used in combination with other anti-viral drugs. MAP30 holds therapeutic promise over other RIPs because not only it is active against infection and replication of both HSV and HIV but is non toxic to normal cells. Here we review the nature, action, structure function relationship and applications of RIPs from Momordica charantia and evaluate their potential for anti-cancer and anti-viral therapy.

A novel nanoplatform for oral delivery of anti-cancer biomacromolecules

Oral administration of bio–macromolecules is an uphill task and the challenges from varying pH and enzymatic activity are difficult to overcome. In this regard, nanotechnology promises the new hope and offers advantages such as controlled release, target specific delivery, combinatorial therapy and many more. In this study, we demonstrate the formulation of a novel alginate enclosed, chitosan coated ceramic, anti cancer nano carrier (ACSC NC). These NC were loaded with multi functional anti cancer bovine lactoferrin (Lf), a natural milk based protein, for improvement of intestinal absorption, in order to develop a novel platform to carry anti cancer protein and/or peptides for oral therapy. Here we demonstrate the size, morphology, internalisation and release profiles of the nanoparticles (NC) under varying pH as perceived in human digestive system. We further determine the uptake of these particles by colon cancer cell lines by measuring the endocytosis and transcytosis of the NC. These NC can be used for future targeted protein/peptide or nucleic acid based drug delivery to treat difficult diseases including cancer.

Ribosome inactivating proteins (RIPs) : a promising candidate for industrial biotechnology

The ribosome inactivating proteins (RIPs) from plants possess RNA N- glycosidase activity that depurinates the major rRNA, thus damaging ribosomes in an irreversible manner and arresting protein synthesis. RIPs are presently classified as rRNA N-glycosidase in the enzyme nomenclature (EC 3.2.2.22) and do exhibit other enzymatic activities such as ribonuclease and deoxyribonuclease activities. RIPs have been shown to manifest anti-tumor, anti-viral and anti-microbial activities. RIPs are detected in some medicinal plants but the yields are insufficient to warrant their availability to conduct clinical trials thus limiting its therapeutic potential. Here, an approach based on "bioprocess development" shall be discussed that may enhance the yield of RIPs. It is anticipated; with the involvement of “Industrial biotechnology” the eventual availability of RIPs in large quantities shall be accomplished.

Ribosome inactivating proteins purification from a medicinal plant

The ribosome inactivating proteins (RIPs) from plants possess RNA N- glycosidase activity that depurinates the major rRNA, thus damaging ribosomes in an irreversible manner and arresting protein synthesis. RIPs are presently classified as rRNA N-glycosidase in the enzyme nomenclature (EC 3.2.2.22) and do exhibit other enzymatic activities such as ribonuclease and deoxyribonuclease activities. RIPs have been shown to manifest abortifacient, anti-tumor, anti-viral and anti-microbial activities. RIPs are detected in some medicinal plants but the yields are insufficient to warrant their availability to conduct clinical trials for therapeutic application. Here, we describe an approach based on “bioprocess development” that may enhance the yield of RIPs and eventually their availability for exploiting their therapeutic potential.

Characterization of Type 1 ribosome inactivating proteins in edible plants

The ribosome inactivating proteins (RIPs) from plants possess RNA N-glycosidase activity that depurinates the major rRNA, thus damaging ribosome in an irreversible manner and arresting protein synthesis. RIPs occur in fungi, bacteria and plants and are abundant in angiosperms, where they appear to have defensive role. RIPs are presently classified as rRNA N-glycosidase in the enzyme nomenclature (EC 3.2.2.22) and do exhibit other enzymatic activities such as ribonuclease and deoxyribonuclease activities. RIPs are classified into two groups based on their difference in their primary structure. Type I RIPs consist of a single polypeptide chain of approximately 26–35 kDa that possess an RNA N-glycosidase activity. These proteins have attracted a great deal of attention because of their anti-viral, anti-tumor, and anti-microbial activities, which is useful in medical research and development. Here, we describe isolation of a novel protein from Momordica sp, a highclimbing vine from family Cucurbitaceae which is native to the tropical regions of Africa, Asia, Arabia and Caribbean. The purified protein has been verified by SDS-PAGE and mass spectrometry to contain only single chain Type-1 ribosome inactivating proteins (RIPs). With present experiments, we determined the presence of RIPs in edible plant materials, including some that are eaten raw by human beings. The novel protein is further characterized to validate its therapeutic potential.

Synthesis and biological evaluation of novel folic acid receptor-targeted, β-cyclodextrin-based drug complexes for cancer treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5-2.5 nm. The host-guest association constant Ka was 1,639 M-1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Studies on the interaction between malanin and metal ions by fluorescence spectra methods

A novel protein with anti-tumor activities named malanin was isolated and purified from an endemic plant in Yunnan and Guangxi provinces. Effects of copper ion, silver ion and calcium ion on malanin and apo-malanin fluorescence spectra were studied. The results showed that copper ion leads to obvious statistic quenching of malanin and apo-malanin fluorescence. The dissociation constant of them from malanin and apo-malanin were about 2.37×10-4 and 2.66×10-4 mol·L-1, respectively. The silver ion did not have quenching action on malanin fluorescence, but it had statistic quenching effect on apo-malanin fluorescence, and its dissociation constant was 2.37×10-4 mol·L-1. Calcium ion did not have quenching action on malanin and apo-malanin fluorescence. It plays an important role in keeping malanin natural conformation.

EpCAM Aptamer-siRNA chimera targets and regress epithelial cancer

Epithelial cell adhesion molecule (EpCAM), a cancer stem cell (CSC) marker is over expressed in epithelial cancers and in retinoblastoma (RB). We fabricated an EpCAM targeting aptamer-siRNA chimera and investigated its anti-tumor property and EpCAM intracellular domain (EpICD) mediated signaling in epithelial cancer. The anti-tumor efficacy of EpCAM aptamer-siEpCAM chimera (EpApt-siEp) was evaluated by qPCR, northern and Western blotting in WERI-Rb1- RB cell line, primary RB tumor cells and in MCF7- breast cancer cell line. Anti-tumor activity of EpApt-siEp was studied in vivo using epithelial cancer (MCF7) mice xenograft model. The mechanism and pathways involved in the anti-tumor activity was further studied using protein arrays and qPCR. EpApt-siEp chimera was processed in vitro by dicer enzyme. Treatment of the WERI-Rb1 and MCF7 cells with EpApt-siEp revealed statistically significant down regulation of EpCAM expression (P<0.005) and concomitant reduction in cellular proliferation. In primary RB cells cultured from RB tumors, EpApt-siEp silenced EpCAM, significantly inhibited (P<0.01) cell proliferation and induced cytotoxicity. Knockdown of EpICD expressed in RB primary tumors led to repression of pluripotency markers, SOX2, OCT4, NANOG, and CD133. In vivo studies showed complete tumor growth regression without any toxicity in animals (P<0.001) and tumor tissues showed significant downregulation (P<0.05) of EpCAM, MRP1, ABCG2, stathmin, survivin and upregulation of ATM (P<0.05) leading to apoptosis by intrinsic pathway with minor alteration in cytokines. Our results revealed that EpApt-siEp potentially eradicated EpCAM positive cancer cells through CSC marker suppression and apoptosis, while sparing normal EpCAM negative adjacent cells.

Probing the biophysical interaction between Neocarzinostatin toxin and EpCAM RNA aptamer

Neocarzinostatin (NCS) a potent DNA-damaging, anti-tumor toxin extracted from Streptomyces carzinostaticus that recognizes double-stranded DNA bulge and induces DNA damage. 2 Fluoro (2F) Modified EpCAM RNA aptamer is a 23-mer that targets EpCAM protein, expressed on the surface of epithelial tumor cells. Understanding the interaction between NCS and the ligand is important for carrying out the targeted tumor therapy. In this study, we have investigated the biophysical interactions between NCS and 2-fluro Modified EpCAM RNA aptamer using Circular Dichroism (CD) and Infra-Red (IR) spectroscopy. The aromatic amino acid residues spanning the β sheets of NCS are found to participate in intermolecular interactions with 2 F Modified EpCAM RNA aptamer. In-silico modeling and simulation studies corroborate with CD spectra data. Furthermore, it reinforces the involvement of C and D1 strand of NCS in intermolecular interactions with EpCAM RNA aptamer. This the first report on interactions involved in the stabilization of NCS-EpCAM aptamer complex and will aid in the development of therapeutic modalities towards targeted cancer therapy.

Cross-strand disulfides in the non-hydrogen bonding site of antiparallel β-sheet (aCSDns): poised for biological switching

Forbidden disulfides are stressed disulfides found in recognisable protein contexts previously defined as structurally forbidden. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. The meta-stability of forbidden disulfides makes them likely candidates as redox switches. Here we mined the Protein Data Bank for examples of the most common forbidden disulfide, the aCSDn. This is a canonical motif in which disulfide-bonded cysteine residues are positioned directly opposite each other on adjacent anti-parallel β-strands such that the backbone hydrogen bonded moieties are directed away from each other. We grouped these aCSDns into homologous clusters and performed an extensive physicochemical and informatic analysis of the examples found. We estimated their torsional energies using quantum chemical calculations and studied differences between the preferred conformations of the computational model and disulfides found in solved protein structures to understand the interaction between the forces imposed by the disulfide linkage and typical constraints of the surrounding β-sheet. In particular, we assessed the twisting, shearing and buckling of aCSDn-containing β-sheets, as well as the structural and energetic relaxation when hydrogen bonds in the motif are broken. We show the strong preference of aCSDns for the right-handed staple conformation likely arises from its compatibility with the twist, shear and Cα separation of canonical β-sheet. The disulfide can be accommodated with minimal distortion of the sheet, with almost all the strain present as torsional strain within the disulfide itself. For each aCSDn cluster, we summarise the structural and strain data, taxonomic conservation and any evidence of redox activity. aCSDns are known substrates of thioredoxin-like enzymes. This, together with their meta-stability, means they are ideally suited to biological switching roles and are likely to play important roles in the molecular pathways of oxidative stress.

Targeting HSP90/survivin using a cell permeable structure based peptido-mimetic shepherdin in retinoblastoma

BACKGROUND: Retinoblastoma (RB) is a childhood retinal malignancy. Effective therapeutic strategies are still being investigated in RB disease management. Here, the anti-cancer effect of shepherdin, a peptido-mimetic inhibiting heat shock protein (HSP90)-Survivin interaction has been analyzed. METHODS: We analyzed HSP (HSP70/90) and Survivin protein expressions by immunohistochemistry (29 archival tumors), qRT-PCR, FACS and Western analysis (10 un-fixed RB tumors). We also analyzed cellular cytotoxicity and anti-proliferative effect in peptide treated RB cells (Y79, Weri Rb1) and MIO-M1 cells. RESULTS: Heterogeneous expressions of HSP70/90 and Survivin with a significant association between HSP70 and HSP90 (r(2) = 0.59, p = 0.001) was observed. In RB cells, anti-tumor effects were detected with 0.42 μg/ml of shepherdin at 4 h s of serum starvation. Decreased Survivin, Bcl2, MMP-2 activity with increased Bax, Bim, and Caspase-9 protein expressions were noticed. No significant changes were observed in shepherdin treated non-neoplastic MIO-M1, nor in scramble-peptide treated RB cells. CONCLUSION: The presence of HSPs (HSP70/90) and Survivin reveals multiple cellular mechanisms adopted by RB cells during cancer progression. Serum starvation induced HSP90 whose interactions with Survivin were specifically inhibited by shepherdin. The associated molecular shuffling has been reported. These findings strongly implicate the potential of targeting HSP90-Survivin interaction as an adjuvant therapy in RB management.

Annetocin and TCTP expressions in the earthworm Eisenia fetida exposed to PAHs in artificial soil

The toxicity of sublethal polycyclic aromatic hydrocarbons (PAHs) levels in soils was assessed by testing their impact on expression of annetocin, a reproduction regulating gene, and translationally controlled tumor protein (TCTP), a tumorigenic response gene, in the earthworm Eisenia fetida cultured in artificial soil spiked with, phenanthrene (Phe), pyrene (Pyr), fluoranthene (Flu), or benzo(a)pyrene (Bap). Annetocin and TCTP were both up-regulated by 0.1 and 1.0 mg kg−1 benzo(a)pyrene and TCTP was down-regulated by 10.0 mg kg−1 phenanthrene. Weight loss and cocoon production of the worms were also analyzed. Only 10.0 mg kg−1 phenanthrene impacted earthworm weight loss significantly and no significant differences on cocoon production were observed. Our study indicated that the potential ecotoxicity of sublethal PAHs in soil should not be neglected and mRNA transcription level in earthworms was a more sensitive indicator of PAHs exposure than traditional indexes using cocoon production as endpoints and/or using the whole-organism as the test materials.

Low-dose metronomic paclitaxel chemotherapy suppresses breast tumors and metastases in mice

This study investigated the use of low-dose metronomic (LDM) chemotherapy with paclitaxel in a highly metastatic mouse model of 4T1 breast cancers, and compared it with the maximum tolerable dose (MTD) therapy. LDM therapy displayed a stronger anti-tumor activity in suppressing primary and metastatic breast tumors with less degree of side effects, and stronger anti-angiogenic and anti-lymphangiogenic activities than MTD therapy. But MTD therapy showed stronger pro-apoptotic and anti-proliferative activities in situ. Paclitaxel therapy downregulated expression of vascular endothelial growth factor receptor-2 (VEGFR2) and up-regulated expression of thrombospondin-1. The results support the application of paclitaxel LDM therapy to treat advanced breast cancer.

Reperfusion-induced myocardial dysfunction is prevented by endogenous annexin-A1 and its N-terminal-derived peptide Ac-ANX-A12-26

BACKGROUND AND PURPOSE : Annexin-A1 (ANX-A1) is an endogenous, glucocorticoid-regulated anti-inflammatory protein. The N-terminal-derived peptide Ac-ANX-A12–26 preserves cardiomyocyte viability, but the impact of ANX-A1-peptides on cardiac contractility is unknown. We now test the hypothesis that ANX-A1 preserves post-ischaemic recovery of left ventricular (LV) function.

EXPERIMENTAL APPROACH : Ac-ANX-A12–26 was administered on reperfusion, to adult rat cardiomyocytes as well as hearts isolated from rats, wild-type mice and mice deficient in endogenous ANX-A1 (ANX-A1–/–). Myocardial viability and recovery of LV function were determined.

KEY RESULTS: Ischaemia–reperfusion markedly impaired both cardiomyocyte viability and recovery of LV function by 60%. Treatment with exogenous Ac-ANX-A12–26 at the onset of reperfusion prevented cardiomyocyte injury and significantly improved recovery of LV function, in both intact rat and wild-type mouse hearts. Ac-ANX-A12–26 cardioprotection was abolished by either formyl peptide receptor (FPR)-nonselective or FPR1-selective antagonists, Boc2 and cyclosporin H, but was relatively insensitive to the FPR2-selective antagonist QuinC7. ANX-A1-induced cardioprotection was associated with increased phosphorylation of the cell survival kinase Akt. ANX-A1−/− exaggerated impairment of post-ischaemic recovery of LV function, in addition to selective LV FPR1 down-regulation.

CONCLUSIONS AND IMPLICATIONS : These data represent the first evidence that ANX-A1 affects myocardial function. Our findings suggest ANX-A1 is an endogenous regulator of post-ischaemic recovery of LV function. Furthermore, the ANX-A1-derived peptide Ac-ANX-A12–26 on reperfusion rescues LV function, probably via activation of FPR1. ANX-A1-based therapies may thus represent a novel clinical approach for the prevention and treatment of myocardial reperfusion injury.

The inhibitory effect of stevioside on bacillus cereus growth in milk: validation and its response surface optimization

Stevioside, a glycoside present in the leaves of Stevia rebaudiana Bertoni, offers therapeutic benefits such as anti-hyperglycemic, anti-hypertensive, antiinflammatory, anti-tumor, diuretic and immune influencing properties. In this work antimicrobial activity of stevioside against Bacillus cereus, a major source of milk contamination was investigated. The isolate was confirmed by various biochemical and 16S rRNA gene sequencing. The effect of temperature, incubation time and concentration of stevioside was optimized from a central composite response surface design. The standard plate count (SPC) of pasteurized milk was drastically reduced in comparison to toned and fresh milk. The optimal temperature, incubation time and stevioside concentration were observed to be 60.23°C, 21 h, and 275 μg/ mL respectively. The synergism of stevioside with the external factors (temperature and time) against B. cereus was observed. Our studies showed that addition of stevioside in fresh as well as pasteurised milk would control growth of B. cereus in milk.