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.

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.

Zingiberaceae plants are well known for their use in ethnomedicine. Curcuma mutabilis Škorničk., 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.

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.

No abstract