Anticancer potential of rhizome extract and a labdane diterpenoid from Curcuma mutabilis plant endemic to Western Ghats of India.

Zingiberaceae plants are well known for their use in ethnomedicine. Curcuma mutabilis Skornick., M. Sabu & Prasanthk., is an endemic Zingiberaceae species from Western Ghats of Kerala, India. Here, we report for the first time, the anticancer potential of petroleum ether extract from C. mutabilis rhizome (CMRP) and a novel labdane diterpenoid, (E)-14, 15-epoxylabda-8(17), 12-dien-16-al (Cm epoxide) isolated from it. CMRP was found to be a mixture of potent bioactive compounds including Cm epoxide. Both the extract and the compound displayed superior antiproliferative activity against several human cancer cell lines, without any display of cytotoxicity towards normal human cells such as peripheral blood derived lymphocytes and erythrocytes. CMRP treatment resulted in phosphatidylserine externalization, increase in the levels of intracellular ROS, Ca2+, loss of mitochondrial membrane potential as well as fragmentation of genomic DNA. Analyses of transcript profiling and immunostained western blots of extract-treated cancer cells confirmed induction of apoptosis by both intrinsic and extrinsic pathways. The purified compound, Cm epoxide, was also found to induce apoptosis in many human cancer cell types tested. Both CMRP and the Cm epoxide were found to be pharmacologically safe in terms of acute toxicity assessment using Swiss albino mice model. Further, molecular docking interactions of Cm epoxide with selected proteins involved in cell survival and death were also indicative of its druggability. Overall, our findings reveal that the endemic C. mutabilis rhizome extract and the compound Cm epoxide isolated from it are potential candidates for development of future cancer chemotherapeutics.

Spondias pinnata (L.f.) Kurz Leaf Extract Derived Zinc Oxide Nanoparticles Induce Dual Modes of Apoptotic-Necrotic Death in HCT 116 and K562 Cells.

This study evaluates the effects of phyto-derived zinc oxide nanoparticles (ZnONPs) on human cancer cells, colon carcinoma HCT 116, and chronic myelogenous leukemic K562, along with normal lymphocytes/erythrocytes. The commercial, chemically synthesized ZnONPs (cZnONPs) were also assessed in parallel. Using an eco-friendly approach devoid of harmful chemicals, biogenic nanoparticles were synthesized from aqueous leaf extract of Spondias pinnata (SpLZnONPs) by a sol-gel method. Optical, structural, and elemental characterization of both particle types were carried out deploying UV-Vis/photoluminescence spectroscopy, FTIR, XRD, FESEM, HRTEM, and EDX. Both SpLZnONPs and cZnONPs displayed hexagonal wurtzite structure with particle sizes averaging 30 and 48.5 nm, respectively. SpLZnONPs were found to be cytotoxic to both cancer cell types while cZnONPs exhibited toxicity only against HCT 116 cells. Interestingly, the cytomorphological changes and analysis of DNA laddering pattern observed in these treated cells were indicative of simultaneous induction of dual modes of death involving apoptosis and necrosis. Flow cytometric analysis of cell-cycle distribution, clonogenic, wound healing, and comet assays provided evidences of the antiproliferative potential of the tested nanoparticles. Apoptosis induction via oxidative stress-mediated Ca2+ release, ROS generation, loss of mitochondrial membrane potential, and externalization of phosphatidylserine was also determined biochemically. Relative expression of apoptotic genes was quantified using RT-qPCR and Western blot analysis. Mitotic index analysis, MTT, and hemolytic assays on lymphocytes and erythrocytes clearly revealed the absence of any deleterious effect(s) of SpLZnONPs in these cells compared with the toxicity of the chemically derived cZnONPs, thereby attesting to the biocompatibility and selective action of the biogenic nanoparticles.

Chemical composition, antioxidant and antiproliferative activities of essential oil from rhizome and leaves of Curcuma mutabilis Skornick., M. Sabu & Prasanthk., endemic to Western Ghats of India.

Hydro-distilled essential oils, from fresh rhizomes and leaves of Curcuma mutabilis Skornick., M.Sabu & Prasanthk., characterized by GC-MS revealed the presence of thirty three and twenty three compounds therein respectively. Whilst estrone methyl ether (3-Methoxyestra-1,3,5(10)-trien-17-one) was the major component in rhizome oil (47.35%), sesquiterpene hydrocarbons predominated as the major group (63.92%) in leaf oil with a higher preponderance of ß-caryophyllene (25.48%), ß-farnesene (19.47%) and α-humulene (11.01%). Weak antioxidant activities observed in these oils determined by DPPH and ABTS methods were apparently influenced both by the oil composition and the assay conditions. Rhizome oil showed higher antiproliferative activity than leaf oil against leukemic K562 (IC50-6.8µg/mL) and colorectal HCT116 (IC50-8.5µg/mL) cancer cell lines. This first report reveals composition and biological activities of essential oils from C. mutabilis.

Manilkara zapota (L.) P. Royen Leaf Extract Derived Silver Nanoparticles Induce Apoptosis in Human Colorectal Carcinoma Cells Without Affecting Human Lymphocytes or Erythrocytes.

Plant-derived synthesis of silver nanoparticles (AgNPs) has found wide biomedical applications including cancer cure. This report deals with biosynthesis of silver nanoparticles (MZLAgNPs) employing leaf extracts of Manilkara zapota (L.) under optimized conditions. Characterization of MZLAgNPs using UV-Vis spectroscopy, FTIR, XRD, and FESEM analyses revealed that the particles were predominantly spherical averaging 24 nm in size. Their cellular effects were assessed by MTT assay, fluorescence, and scanning electron microscopy of cells stained with propidium iodide, acridine orange/ethidium bromide, and annexin V-FITC to visualize signs of apoptosis. Evaluation of cell proliferation by clonogenic assay, wound healing ability by scratch assay and cell cycle distribution by flow-cytometry was also carried out. Apoptosis-related gene expressions were analyzed by RTq-PCR and western blot analysis. MZLAgNPs selectively inhibited growth of colorectal carcinoma HCT116, HeLa, and non-small lung carcinoma A549 cells, dose-dependently with IC50 concentrations of 8, 16, and 29 µg/mL respectively, following 72-h treatment, without affecting growth of normal human lymphocytes and erythrocytes. Apoptosis induction was observed by fluorescence and scanning electron microscopy. Overproduction of reactive oxygen species (ROS), reduction of mitochondrial membrane potential, upregulation of apoptotic-related genes - PUMA, cas-3, cas-8, cas-9, and BAX, expression of caspase 3, and occurrence of PARP cleavage were observed in MZLAgNPs/cisplatin treated cells. Taken together, our results clearly demonstrate the therapeutic potential of biogenic MZLAgNPs as an effective agent for killing colorectal carcinoma cells by apoptosis induction.

Mutational analyses of regulatory genes, mexR, nalC, nalD and mexZ of mexAB-oprM and mexXY operons, in efflux pump hyperexpressing multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

The present study deals with membrane-bound efflux pumps, MexAB-OprM and MexXY and their respective regulatory genes mexR, nalC, nalD and mexZ in multidrug resistant (MDR) Pseudomonas aeruginosa. Following antibiotic sensitivity testing and detection of various beta-lactamases, hyperexpression of efflux pump genes, mexB and mexY in the isolates was investigated using semi-quantitative and real-time reverse transcription-PCR. Amplicons from regulatory genes were sequenced and subjected to mutational and phylogenetic analysis. Twenty-nine clinical isolates of P. aeruginosa were obtained from a total of 144 MDR gram-negative bacteria collected from Kerala State, South India. All strains were found to be resistant to ampicillin and nalidixic acid with 13.8, 44.8 and 31% testing positive for extended-spectrum beta-lactamases, metallo-beta-lactamases and AmpC producers respectively. Increased mexB and mexY transcription was detected respectively in 10.3 and 20.7% of the isolates in comparison with P. aeruginosa reference strain, PAO (MTCC). Co-expression of MexY was also observed in MexB overproducers. Various synonymous/and non-synonymous mutations in regulatory gene sequences of efflux pump operons were detected. In the strain designate Pa16, mexR was found to harbour four novel point mutations with one transversion and three transitions which included a substitution of an ochre codon with that for serine. The gene also displayed a novel mutation involving insertion of a cysteine at the 444th base position, followed by an opal codon. The genetic divergence and homogeneity of the concatenated (mexR, nalC and nalD) regulatory gene sequences of mexAB-oprM operon was apparent in the phylogram generated with similar sequences retrieved from public database.

Selective induction of DNA damage, G2 abrogation, and mitochondrial apoptosis by leaf extract of traditional medicinal plant Wrightia arborea in K562 cells.

Plants have proved to be an important source of anti-cancer drugs. Wrightia arborea, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. This study evaluates the antiproliferative/apoptotic potential of Wrightia arborea leaf extracts, prepared in different organic solvents, on cancer cell lines. MTT assay, light and fluorescence microscopy, flow cytometry, DNA laddering, alkaline comet assay, and western blotting were some of the techniques used for evaluation. Combinations of camptothecin, either with CHK1 inhibitor-PD407824 or with W. arborea leaf extract, were deployed to determine the G2 abrogating potential of the extract. The chloroform extract (WAC) selectively killed K562 cells, without affecting cancerous MCF-7, Hep G2 cells, and normal human peripheral blood lymphocytes. Cell death was characterized by observation of apoptotic bodies, increased Ca2+ and ROS, phosphatidyl serine externalization, mitochondrial membrane depolarization, DNA laddering, increased sub-G1 population, and altered expression of caspase 3, -9, and PARP. WAC also induced DNA damage, alterations in key G2/M phase protein expression, cell cycle perturbation, and potent G2 abrogation. The present study showed that W. arborea leaf extract, WAC, is capable of selectively killing leukemic cancer cells leaving normal lymphocytes unaffected. Our results indicate that this is effectuated through DNA damage and G2 abrogation leading to mitochondrial apoptosis. Taken together, this report contributes toward a better understanding of the anticancer properties of this traditional medicinal plant extract possessing valuable bioactive constituents which can serve as a bioresource for promising complimentary/alternative/chemopreventive therapeutics.

Analysis of beta-lactamases, blaNDM-1phylogeny & plasmid replicons in multidrug-resistant Klebsiella spp. from a tertiary care centre in south India.

BACKGROUND & OBJECTIVES: ß-lactamases play a predominant role in drug-resistance amongst Enterobacteriaceae. Presence of genes on transferable plasmids encoding these enzymes favours their dissemination across species and genera within and outside geographical boundaries. This study was aimed to understand the presence of ß-lactamases and transferable plasmids in clinical isolates of Klebsiella spp. which can contribute to the spread of resistance determinants. METHODS: A total of 41 clinical isolates of Klebsiella spp., collected from a tertiary care centre in Kerala, India, were checked for antibiotic sensitivity and the presence of plasmids. The ability to produce extended-spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs) was screened for and confirmed in 29 plasmid-harbouring isolates. blaNDM-1-specific primers were used for polymerase chain reaction amplification with plasmid DNA as template to determine episomal prevalence of this gene and its sequence-based phylogeny employing similar sequences from GenBank. Plasmid replicon typing was also carried out to determine the presence of transferable plasmids. RESULTS: Our results showed a high degree of multidrug-resistant (MDR) pathogens with ESBL production confirmed in 52 per cent, MBL in 31 per cent and co-production of both enzymes in seven per cent of the plasmid-bearing isolates. Plasmid DNA from 14 per cent of the isolates produced blaNDM-1-specific amplicons which showed sequence homology with those from bacteria of different genera and geographical areas. The predominant replicon type was found to be that of conjugative plasmids belonging to the incompatibility group - IncFIIK. INTERPRETATION & CONCLUSIONS: This study provides insight into the predominance of various ß-lactamases and potent gene-disseminating agents in Klebsiella spp. and emphasizes the need for constant surveillance of these pathogens to determine appropriate treatment strategies.

Zerumbone induces mitochondria-mediated apoptosis via increased calcium, generation of reactive oxygen species and upregulation of soluble histone H2AX in K562 chronic myelogenous leukemia cells.

Zerumbone, a natural cyclic sesquiterpene, is known to exhibit selective toxicity toward various cancer cells. Sustained efforts to explore the potential of new agents for effective therapy are critical in the context of development of drug resistance especially in cancers like chronic myelogenous leukemia (CML). The present study evaluated the effect of zerumbone on CML-K562 cells. The cell viability of zerumbone-treated K562 cells was detected by MTT assay, and morphological changes were observed by light microscopy and scanning electron microscopy (SEM). Staining with Hoechst 33258, acridine orange/ethidium bromide, and AnnexinV-FITC were used to detect apoptosis. Intracellular reactive oxygen species (ROS), Ca(2+), and changes in mitochondrial membrane potential were measured using Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Fluo-3AM, and Rhodamine-123, respectively. Western blot analysis was carried out to detect key proteins involved in apoptosis. Zerumbone inhibited K562 cell proliferation with an IC50 value of 3.5 µg/mL and colony formation capability (P < 0.001). Interestingly, zerumbone did not affect the growth of normal human peripheral blood lymphocytes (hPBLs). Distinct morphological changes observed by light microscopy and fluorescent staining with Hoechst-33258, AO/EtBr, annexin V-FITC, and cytotoxicity evaluation by comet assay indicated induction of DNA damage and apoptosis. This was further confirmed by demonstration of pro-caspase-3, -9 activation and Poly(ADP-ribose) polymerase (PARP) cleavage on western blots. Apoptosis induction was found to be mitochondria mediated, involving increased free intracellular Ca(2+), ROS, and upregulation of soluble histone H2AX. Our results suggest that zerumbone holds promise as a potential candidate drug for CML.

Antioxidant potential and oxidative DNA damage preventive activity of unexplored endemic species of Curcuma.

Free radical scavenging activity, ferrous ion chelating capacity, reducing power and genoprotective effect of the aqueous leaf extracts of four unexplored endemic Curcuma spp. (C. vamana, C. neilgherrensis, C. mutabilis, C. haritha) were found to be dose-dependent and were highest in C. vamana. DNA protection property of the extracts was evaluated against H202/UV-induced oxidative damage. DNA-methyl green displacement assay showed that these extracts were free of DNA intercalating compounds. Further, hemolysis assay also showed that the extracts were non-toxic to human erythrocytes. The results highlight C. vamana as a promising source for herbal preparations possessing high antioxidant potential and genoprotective activity.

Assessment of cell cycle phase-specific effects of zerumbone on mitotically synchronous surface cultures of Physarum polycephalum.

Zerumbone, a natural cyclic sesquiterpene, has been the focus of recent research as it has been found to exhibit selective toxicity towards cancer cells compared to normal cells. Studies on the cell cycle phase-specific effects of this interesting compound, however, remain sparse. Hence, concentration and time-dependent effects of zerumbone were evaluated employing a suitable model system, the naturally synchronous surface cultures of Physarum polycephalum. Zerumbone treatment in S, early, and late G2 phases resulted in G2 arrest. Early G2 phase exhibited the highest sensitivity (P < 0.001) to the compound. Protein profiles showed a complete inhibition of cyclin B1 expression following zerumbone treatment. Furthermore, FACS and comet analysis revealed that zerumbone inhibited DNA synthesis (P < 0.001) without being genotoxic at the concentrations tested. Differential display of mRNA showed distinct zerumbone-induced variations in transcript profiles, an analysis of which suggested a likely link between cellular networks involving stress-related gene expression and G2 arrest in P. polycephalum.

Zingerone protects against stannous chloride-induced and hydrogen peroxide-induced oxidative DNA damage in vitro.

In this paper, we report the dose-dependent antioxidant activity and DNA protective effects of zingerone. At 500 µg/mL, the DPPH radical scavenging activity of zingerone and ascorbic acid as a standard was found to be 86.7 and 94.2 % respectively. At the same concentration, zingerone also showed significant reducing power (absorbance 0.471) compared to that of ascorbic acid (absorbance 0.394). The in vitro toxicity of stannous chloride (SnCl2) was evaluated using genomic and plasmid DNA. SnCl2-induced degradation of genomic DNA was found to occur at a concentration of 0.8 mM onwards with complete degradation at 1.02 mM and above. In the case of plasmid DNA, conversion of supercoiled DNA into the open circular form indicative of DNA nicking activity was observed at a concentration of 0.2 mM onwards; complete conversion was observed at a concentration of 1.02 mM and above. Zingerone was found to confer protection against SnCl2-induced oxidative damage to genomic and plasmid DNA at concentrations of 500 and 750 µg/mL onwards, respectively. This protective effect was further confirmed in the presence of UV/H2O2-a known reactive oxygen species (ROS) generating system-wherein protection by zingerone against ROS-mediated DNA damage was observed at a concentration of 250 µg/mL onwards in a dose-dependent manner. This study clearly indicated the in vitro DNA protective property of zingerone against SnCl2-induced, ROS-mediated DNA damage.

Cell cycle inhibitory effects of leaf extract from Curcuma vamana M. Sabu & Mangaly on mitotically synchronous cultures of Physarum polycephalum Schw.

Leaf extracts of C. vamana, endemic to Kerala state in India, were found to inhibit cell cycle progression in synchronous cultures of P. polycephalum in a concentration and phase-specific manner. Crude alkaloid extract (CAE) elicited maximum cell cycle delays in comparison to soxhletted chloroform, acetone and aqueous extracts. Total alkaloid content of CAE was found to be 64.9 mg/g. CAE showed lowest DPPH radical scavenging activity. Other extracts with higher free radical scavenging activity exhibited lesser cell cycle inhibiting potential. Upto 21% decrease in nuclear DNA was observed in CAE treated samples. However, genotoxicity as evidenced by comet assay was not observed. The extracts were also found to be non-toxic to human RBCs at the highest concentration tested (750 microg/mL). CAE treatment completely suppressed a 63 kDa polypeptide with a concomitant, but weak induction of a 60 kDa polypeptide suggesting that these may be cell cycle related. CAE was found to possess potent antiproliferative activity against PBLs. The study clearly demonstrates the cell cycle inhibitory activity of C. vamana leaf extracts, with CAE being the most potent of them.

In vitro antibacterial activity of Psidium guajava Linn. leaf extract on clinical isolates of multidrug resistant Staphylococcus aureus.

In the present study, antibacterial activity of aqueous and organic extracts of Psidium guajava leaves was evaluated against multidrug resistant (MDR) clinical isolates of Staphylococcus aureus strains collected from hospitals in northern (Malabar region) Kerala. The strains which exhibited resistance against all the antibiotics tested was selected for antibacterial assays. Minimum inhibitory concentration (MIC) for methanolic and aqueous extracts was found to be 625 ug/ml and 7.5 mg/ml, respectively. Minimum bactericidal concentration (MBC) recorded for methanolic and aqueous extracts was 1.25 and 12.5 mg/ml, respectively. Methanolic extract at minimum bactericidal concentration inhibited the growth of MDR strain by 80%. Time-kill assay revealed that methanolic extract (4 mg/ml) killed MDR bacteria within 10 hr. Total polypeptide profiling of bacterial cultures by SDS-PAGE indicated a high degree of protein degradative activity of the extract. Finally, a human RBC based haemolytic assay showed absence of haemolysis even at concentrations higher than that of MBC, advocating thereby its safety in therapeutic use.

Reduction of UV-induced mitotic delay by caffeine in BUdR-substituted plasmodia of Physarum polycephalum.

Chromosomal DNA of the synchronously mitotic plasmodia of P. polycephalum was substituted with 5-bromo-2'-deoxyuridine, by growing the plasmodia during S phase, on a medium containing this nucleoside analog. A strong synergism was observed between bromodeoxyuridine and UV-irradiation, in late G2-irradiated plasmodia in that, the mitotic delay obtained in them was much more than a simple sum of the delays induced by these two agents individually. It was also observed that the mitotic delay in this system is reduced significantly by different concentrations of caffeine applied immediately after irradiation and there was a stage specificity in this effect. The reduction in mitotic delay was maximum (80%) in those plasmodia irradiated 20-30 min before control metaphase, when mitogenic factors also reach their maximum activity in this system. It is proposed that the mitotic delay reducing effect of caffeine is due to its ability to promote the activity of the mitogenic factors, largely independent of the system which is responsible for monitoring the state of the chromosomal DNA.