BRCA1/TP53 tumor proteins inhibited by novel analogues of curcumin - Insight from computational modelling, dynamic simulation and experimental validation.

The current study aimed to design novel curcumin analogue inhibitors with antiproliferative and antitumor activity towards BRCA1 and TP53 tumor proteins and to study their therapeutic potential by computer-aided molecular designing and experimental investigations. Four curcumin analogues were computationally designed and their drug-likeness and pharmacokinetic properties were predicted. The binding of these analogues against six protein targets belonging to BRCA1 and TP53 tumor proteins were modelled by molecular docking and their binding energies were compared with that of curcumin and the standard drug cyclophosphamide and its validated target. The stabilities of selected docked complexes were confirmed by molecular dynamic simulation (MDS) and MMGBSA calculations. The best-docked analogue was chemically synthesized, characterized, and used for in vitro cytotoxic screening using DLA, EAC, and C127I cell lines. In vivo antitumor studies were carried out in Swiss Albino Mice. The study revealed that the designed analogues satisfied drug-likeness and pharmacokinetic properties and demonstrated better binding affinity to the selected targets than curcumin. Among the analogues, NLH demonstrated significant interaction with the BRCA1-BRCT-c domain (TG3; binding energy -8.3 kcal/mol) when compared to the interaction of curcumin (binding energy -6.19 kcal) and cyclophosphamide (binding energy -3.8 kcal/mol) and its usual substrate (TG7). The MDS and MM/GBSA studies revealed that the binding free energy of the NLH-TG3 complex (-61.24 kcal/mol) was better when compared to that of the cyclophosphamide-TG7 complex (-21.67 kcal/mol). In vitro, cytotoxic studies showed that NLH demonstrated significant antiproliferative activities against tumor cell lines. The in vivo study depicted NLH possesses the potential for tumor inhibition. Thus, the newly synthesized curcumin analogue is probably used to develop novel therapeutic agents against breast cancer.

Structural and functional validation of a cloned parasporin from Bacillus thuringiensis isolate KAU 41 native to Western Ghats of India.

Parasporins of Bacillus thuringiensis (Bt) exhibit specific toxicity to cancer cells. PCR based mining has identified apoptosis inducing parasporin in KAU41 Bt isolate from the Western Ghats of India. The study aimed to clone and overexpress the parasporin of native KAU41 Bt isolate for determining structural and functional characteristics of the protein. Parasporin gene was cloned in pGEM-T, sequenced, sub-cloned in pET30+ and overexpressed in Escherichia coli. The expressed protein was characterized by SDS-PAGE and in silico methods. Cytotoxicity of cleaved peptide was assessed by MTT assay. SDS-PAGE displayed a 31 kDa protein (rp-KAU41) overexpressed. Upon proteinase K digestion, the protein was cleaved into 29 kDa peptide which was found to be cytotoxic to HeLa cells. The protein has a deduced sequence of 267 amino acids with ß-strands folding pattern of crystal protein. Even though rp-KAU41 shared a 99.15% identity to chain-A of non-toxic crystal protein, it only showed a less similarity to the existing parasporins like PS4 (38%) and PS5 (24%) in UPGMA analysis, emphasizing the novelty of rp-KAU41. The protein is predicted to have more similarity to the pore forming toxins of Aerolysin superfamily and an additional loop in rp-KAU41 may be contributing towards its cytotoxicity. The molecular docking with caspase 3 resulted in higher Z dock and Z rank score substantiating its role in the activation of intrinsic pathway of apoptosis. The recombinant parasporin protein, rp-KAU41 is presumed to belong to the Aerolysin superfamily. An interaction with caspase 3 substantiates its role in activating the intrinsic pathway of apoptosis in cancer cells.

Desmodium gyrans dc modulates lipid trafficking in cultured macrophages and improves functional high-density lipoprotein in male wistar rats.

OBJECTIVE: High-density lipoprotein (HDL) cholesterol-mediated atherosclerotic plaque regression has gained wide therapeutic attention. The whole plant methanolic extract of the medicinal plant Desmodium gyrans Methanolic Extract (DGM) has shown to mitigate hyperlipidemia in high fat- and-cholesterol fed rats and rabbits with significant HDL enhancing property. The study aimed to assess the functionality and mechanistic basis of HDL promoting effect of DGM. MATERIALS AND METHODS: Macrophage cholesterol efflux and foam cell formation assays were performed in THP-1 macrophages. Male Wistar rats were given DGM extract over 1 month and assessed the serum HDL, Apolipoprotein A1 (Apo-A1), and paraoxonase activity. Quantitative Polymerase chain reaction was carried out to assess the expression level of Apo-A1, SR-B1 (Scavenger receptor B1), and Cholesteryl ester transfer protein (CETP) on cDNA of HepG2 cells exposed to DGM. RESULTS: Pretreatment of DGM inhibited uptake of oxidized lipids and enhanced the lipid efflux by THP-1-derived macrophages. Oral administration of DGM (100 and 250 mg/kg) progressively enhanced the serum HDL, Apo-A1 level, and associated paraoxonase activity in normal male Wistar rats. In support to this, DGM exposed HepG2 cells documented dose-dependent increase in the expression of SR-B1 and Apo-A1 mRNA, while reduced the CETP expression. CONCLUSION: Overall the results indicated that DGM modulates lipid trafficking and possesses functional HDL enhancing potential through increased Apo-A1 levels and paraoxonase activity. Further, reduced CETP expression and increased expression of SR-B1 suggest the reverse cholesterol transport promoting role of DGM.

Variations in natural polyphenols determine the anti-inflammatory potential of virgin coconut oils.

Virgin coconut oil (VCO), an edible oil prepared from fresh coconut kernel by natural or mechanical means without undergoing chemical refining, has been in the limelight of research as functional food oil. The phenolic components in VCO have been accredited with these pharmacological benefits. The present study compared the phenolic constituents of freshly prepared fermentation processed (FVCO) and hot-pressed VCO (HVCO) and their anti-inflammatory efficacies. The biochemical analysis documented quantitative variation in the phenolic content, being higher in HVCO than FVCO (40.03 ± 5.8 µg and 25.55 ± 5.8 µg/mL of oil, respectively). In vitro studies observed nitric oxide radical scavenging efficacy (IC50 value of 14.84 ± 0.81 µg/mL) for HVCO polyphenols, which shows higher inhibition efficacy than FVCO (29.41 ± 1.7 µg/mL). In dextran and formalin mediated acute and chronic inflammation in mice, HVCO displayed more protective efficacy (40.5 and 46.4% inhibition) than FVCO (33.3 and 43.8% inhibition), which is similar to the standard diclofenac (55.6 and 59.8% inhibition). The study, thus, concludes that compared to FVCO, HVCO is a more active anti-inflammatory agent. PRACTICAL APPLICATION: Virgin coconut oil, a widely used edible oil in South Asian countries, has been shown to have health benefits possibly exerted by the natural phenolics it contains. However, different modes of preparations of VCO determine the phenolic combinations and efficacy as well. Our study compared two different VCO preparations and suggests that the VCO prepared by the traditional way (HVCO) is pharmacologically potent than that prepared by simple fermentation process (FVCO) in reducing inflammation. The efficacy is attributed to the variations in phenolic profile revealed by LC-MS analysis. Hence, the current study suggests HVCO as a potential food supplement that can reduce the incidence of degenerative diseases.

A parasporin from Bacillus thuringiensis native to Peninsular India induces apoptosis in cancer cells through intrinsic pathway.

Parasporins, a class of non-insecticidal crystal proteins of Bacillus thuringiensis (Bt) are being explored as promising anticancer agents due to their specific toxicity to cancer cells. The present study has identified 25 Bt isolates harbouring parasporin genes from Western Ghats region, the hotspot of biodiversity in India. Among these, the isolate, KAU 41 (Kerala Agricultural University isolate 41) contained non-hemolytic homogenous crystals showing specific cytotoxicity towards cancer cells. SDS-PAGE analysis of this crystal, isolated by aqueous biphasic separation, revealed a 31 kDa sized peptide. The N-terminal sequence deciphered in BLAST analysis showed homology to a hypothetical Bt protein. Upon proteolysis, a 29 kDa active peptide was generated which exhibited heterogenic cytotoxic spectrum on various cancer cells. HeLa cells were highly susceptible to this peptide with IC 50 1 lg/mL and showed characteristics of apoptosis. RT-qPCR analysis revealed the overexpression of APAF1, caspase 3 and 9 by 14.9, 8 and 7.4 fold, respectively which indicates the activation of intrinsic pathway of apoptosis. However, at higher concentrations of peptide (greater than 3 lg/mL), necrotic death was prominent. The results suggest that the 31 kDa protein from Bt isolate, KAU 41 is a parasporin that may have high therapeutic potential.

Kingiodendron pinnatum, a pharmacologically effective alternative for Saraca asoca in an Ayurvedic preparation, Asokarishta.

Saraca asoca (Fabaceae) is a prime ingredient in Asokarishta, a well-known Ayurvedic preparation for gynecological ailments. Due to scarcity, adulteration or substitution of related raw drugs is a common practice in its preparation. The bark of Kingiodendron pinnatum (Roxb. ex DC.) Harms, morphologically similar to S. asoca (Asoka) is a widely used substitute. The present study aimed to evaluate the pharmacological effectiveness of K. pinnatum as an alternative for S. asoca in Asokarishta by determining the inhibitory effect of estrogen induced uterus endometrial thickening in immature female rats. Arishta was prepared using S. asoca and with the substitute, K. pinnatum as per Ayurvedic Pharmacopeia. Uterus endometrial thickening was induced by the administration of estradiol (20 µg/kg b. wt, i.p) to 8-day-old rats for 5 alternate days. On day 16, following estradiol administration, the serum estrogen level was found elevated to 156.5 ± 8 pg/ml from the normal value 32.4 ± 5 pg/ml and consequently increased the thickness of uterus endometrium from 16.7 ± 1.4 to 75.2 ± 15.3 µm. Upon oral administration of 400 µl/kg b. wt Asokarishta (ASA) and Arishta made with K. pinnatum (AKP), the thickening was reduced to 42.5 ± 12.7 and 47.1 ± 10.5 µm and the estrogen level diminished to 102.6 ± 10 and 97.3 ± 8 pg/ml, respectively. Arishta also reduced the chronic/acute inflammations in mice and improved the antioxidant status of rats. No toxic symptom was observed in the animals by the treatment of Arishta. The study supports the use of K. pinnatum as an alternative to S. asoca in Asokarishta and gives a scientific validation for Asokarishta in gynecological ailments.

Ameliorative effect of Apodytes dimidiata on cisplatin-induced nephrotoxicity in Wistar rats.

Context Nutraceuticals possessing antioxidant potential have been used to alleviate side effects exerted by many chemotherapeutics, including cisplatin. Since Apodytes dimidiata E. Mey. Ex Arn. (Icacinaceae) shows antioxidant potential, it may possess significant chemoprotective effects. Objectives The study investigated whether A. dimidiata could attenuate cisplatin-induced renal damage. Materials and methods Nephrotoxicity was induced by cisplatin (single i.p., 16 mg/kg b wt.) in Wistar rats. Methanolic leaf extract of A. dimidiata (AMF) was administered at a dose of 250 mg/kg b. wt. orally for 5 consecutive days before/after cisplatin administration. Blood and renal parameters were analysed. Total phenolic and flavonoid content in AMF and its NO scavenging effect was determined. Results Significant protective effect of AMF on cisplatin-induced nephrotoxicity was observed in pre-treated animals. The reduction of urea, creatinine and lipid peroxidation was 58.31%, 42.19% and 60%, respectively, and the increase in haemoglobin and leucocyte count was 28.25% and 42.91%, respectively. The increase calculated for GSH, GPx, SOD and catalase was 35.64%, 18.14%, 74.42% and 35.46%, respectively. Tissue architecture of kidney was almost normal in AMF treated animals. The results were comparable to the standard drug, silymarin. AMF contained high level of polyphenols and flavonoids and was found to scavenge NO radicals (IC50 121.8 µg/mL). Discussion and conclusion AMF can effectively counteract cisplatin mediated renal acute toxicity possibly by scavenging reactive oxygen and nitrogen species. Accordingly, the study suggests that AMF can ameliorate free radical-induced damage associated with chemotherapeutic drugs.

Evaluation of Cytotoxic and Antitumour Properties of Apodytes dimidiata and Characterisation of the Bioactive Component.

Apodytes dimidiata, belonging to the family Icacinaceae, is used for treating inflammation and various gastrointestinal ailments in Zulu traditional medicine. In the present study, significant cytotoxicity was exhibited by the methanolic extract of the A. dimidiata leaf against various cancer cell lines. The extract was purified partially through silica gel column by successive elution using various solvents of increasing polarity. Among these, the active methanolic fraction was found to be the most cytotoxic with IC50 values ranging from 0.92 to 3.95 µg/mL for Ehrlich's ascites carcinoma (a carcinoma cell line), Jurkat (human T lymphocyte cell line), and SK-BR-3 (mammary tumour cell line). The treated cells showed morphological alterations characteristic of apoptosis. Upon oral administration of active methanolic fraction at a dose of 250 mg/kg body weight, the solid tumour volume in mice was significantly reduced to 55.14% and the life span of the ascites tumour-bearing mice increased to 44.65% compared to untreated control. The active fraction with Rf value 0.56 was purified from the methanolic fraction by preparative thin-layer chromatography and was subjected to high-performance thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance analysis. The iridoid glycoside genipin was identified as the active component.

Effect of Saraca asoca (Asoka) on estradiol-induced keratinizing metaplasia in rat uterus.

BACKGROUND: Estrogen-mediated uterus endometrium instability is considered as one of the etiological factors in dysfunctional uterine bleeding (DUB) and uterine cancer. Saraca asoca (Family: Fabaceae) and its fermented preparation, Asokarishta, are extensively used as uterine tonic to treat gynecological disorders in Ayurveda. The present study evaluated the effect of S. asoca (Asoka) on estrogen-induced endometrial thickening of rat uterus. METHODS: Endometrial thickening was induced by intraperitoneal injection of estradiol (20 µg/kg b.wt) to 8-day-old immature rats for alternate 5 days. Methanolic extract (200 mg/kg b. wt) from S. asoca bark was given orally along with estradiol. Uterus endometrial thickening was analyzed histopathologically and serum estrogen level by radioimmunoassay (RIA). Cyclooxygenase (COX-2) expression in rat uterus was also estimated by Western blot. Anti-inflammatory activity of the extract was analyzed by formalin- and carrageenan-elicited paw edema models in mouse. RESULTS: Uterus endometrium proliferation and keratinized metaplasia with seven to eight stratified epithelial layers on day 16 was observed in rats administered with estradiol. Treatment with S. asoca reduced the thickening to two to four layers and the serum estrogen level diminished significantly to 82.9±12.87 pg/mL compared to rats administered with estrogen alone (111.2±10.68 pg/mL). A reduction of formalin- and carrageenan-induced paw edema in mouse by S. asoca extract was observed. Lower level of lipopolysaccharides (LPS)-induced COX-2 enzyme in rat uterus by the extract further confirms its anti-inflammatory activity. CONCLUSIONS: Present study reveals the antiproliferative and antikeratinizing effects of S. asoca in uterus endometrium possibly through its anti-estrogenic and anti-inflammatory properties.