Antifungal And Anticancer Activities Study Of Natural Products And Their Underlying Mechanisms

Natural plant has been an important source of natural medicines, but also a major source of treating and preventing human disease. The incidence of fungal infections and cancer are increasing every year. An increase number of reports of clinical resistance to antifungal agents and anticancer agents highlight the need for searching for new antifungal and anticancer drugs which may have a new target and treat better. So far the research on antifungal and anticancer drugs has been supported by many countries. Millions of plants, animals, microorganisms, and marine organisms are an important source of new drug discovery. Lichens are quite a special large group in the the plant kingdom as high degree of integration of the symbiotic complex between fungi and algae. Recently, a lot of new bioactive compounds have been isolated as reported in the literature. The vitro antifungal activity of retigeric acid B (RAB), a pentacyclic triterpenoid from the lichen species Lobaria kurokawae, was evaluated alone and in combination with fluconazole, ketoconazole, and itraconazole against Candida albicans using checkerboard microdilution, agar diffusion assay, and time-killing tests. The minimum inhibitory concentrations (MICs) for RAB against ten different C. albicans isolates ranged from 8 to 16μg/ml.A synergistic action of RAB and azole was observed in azole-resistant strains, whereas synergistic or indifferent effects observed in azole-sensitive strains, when interpreted by separate approach of FICI (fractional inhibitory concentration index) and AE model (difference between the predicted and measured fungal growth percentages). In agar diffusion assay, the combination yielded significantly clearer and larger zones than those of either drug alone on the plain agar plate. In time-killing tests, we used both colony counts and a colorimetric assay to evaluate the combinational antifungal effects of RAB and azoles. For the tested concentration of RAB and azole hardly have activity against C. albicans but the combination yielded a>2log10CFU/ml decrease compared with either drug used alone which further confirmed their synergistic interactions. These findings suggest natural products of RAB may play a certain role in increasing the susceptibilities of azole-resistant C. albicans strains. Next we studied the underlying synergistic antifungal mechanisms of RAB in combination with azoles against C. albicans. Increased accumulation of rhodamine 123 (Rh123) in C. albicans was measured by both spectrophotometric method and flow cytometry. The inhibitory properties to the drug efflux of C. albicans were determined spectrophotometrically. A special indicator organism, CASA1 which has a green fluorescent protein gene inserted in place of one allele of the CDR1 structural gene, was used to investigate regulation of CDR1. After treated by RAB, the distribution of green fluorescence was changed, the intensity of fluorescence was decreased compared with the control, and some of them lost the green fluorescence. The downregulation expression level of CDR1 was detected by real-time reverse transcription polymerase chain reaction. Above all, RAB synergize the antifungal effect of azoles against C. albicans by inhibiting efflux pump activity. Cellular ergosterol synthesis was measured using its unique spectrophotetric absorbance profile. Both ergosterol and 24(28)-DHE absorb at 281.5 nm, whereas only 24(28)-DHE absorbs at 230 nm. The decreased cellular ergosterol synthesis was measured using its unique spectrophotetric absorbance profile and we found that when cells were treated with RAB, the expression of ERG11 was downregulated and ergosterol level was decreased. ERG11 expression in C. albicans after treatment by 16μg/ml RAB was markedly lower than untreated isolates (0.42-fold for azole-sensitive strain and 0.23-fold for azole-resistant strain, respectively). Low ergosterol content will enhance the membrane fluidity. An increase in membrane fluidity directly results in increased passive diffusion of drugs and sensitization of Candida cells to antifungals. Transmission electron microscopy investigation found the wrinkled cell membrane and the impaired cell wall due to RAB treatment. RAB synergize the antifungal effect of azoles against C. albicans by inhibiting efflux pump activity, targeting the ergosterol biosynthesis pathway resulted ergosterol depletion, and impairing the function of fungal cell membranes and increasing penetration of an antifungal agent.About the anticancer activity, the paper studied the activity of steroidal alkaloid glycoside isolated from Chinese herb Solanum Solanum nigrum L. Solamargine (SM), solasonine,β2-solamargine, and solasodine have the same aglycon solanidine, but different saccharide chains. We discussed the structural activity relationships between the steroidal alkaloidal saponins and inhibition of the growth of cancer cells. SM with a two-rhamnosyl on the trisaccharide chain achieved higher intracellular concentrations and was potently toxic. As compared with the tumor cell lines, SM had a lower cytotoxicity on the normal cell retinal pigment epithelial cell line RPE-1 and mesangial cells MC. The cytotoxicity of SM did not correlate with the expression level of the multidrug resistance MDR1 and had the strong cytotoxicity on K562/A02 and KB/VCR. Solasonine, which has a trisacharide chain, has also exhibited moderate cytotoxicity, while other two compounds, without the trisacharide chain, had no anticancer activity. These studies revealed the trisaccharide chain was critical for activity in these steroidal alkaloid saponins. It is likely that apoptosis can be induced by low doses of SM, and oncosis by high doses, both types of cell death are induced by intermediate concentration of SM. Apoptosis was evaluated by DAPI staining and annexin V/PI double staining. Further investigation with human K562 leukemia cells found that SM could induce an early lysosomal rupture within 2h as assessed by acridine-orange relocation and alkalinization of lysosomes. Intracellular lysosomal rupture is also confirmed with the release of cathepsin B to cytosol detected by western blot. Subsequent mitochondrial damage including mitochondrial membrane permeabilization detected by decrease membrane potential as well as the release of cytochrome c from mitochondria was also observed. The down-expression of Bcl-2, up-regulation of Bax, caspase-3 and caspase-9 activities followed by above changes revealed that the cytoxicity of SM was involved in a lysosomal-mitochondrial death pathway induced by SM.Cell membrane integrity was assessed by detecting the leakage of cytoplasmic content, the release of lactate dehydrogenase (LDH), and the uptake of propidium iodide (PI). The rapid ability of SM (IC80) to make PI permeate into tumor cells, LDH release, and leakage of cytoplasmic content at the same rate (within minutes), suggests a killing mechanism that involves plasma membrane perturbation. We use whole-cell recording to measure the time courses of membrane blebbing and disruption in human K562 leukemia and squamous cell carcinoma KB cells. SM induced membrane blebbing within 5 min of sustained application. Both chelating extracellular calcium with EGTA and clamping intracellular calcium concentrations with a high concentration of the cell-permeable chelator BAPTA-AM did not prevent blebbing. Polyethylene glycols having molecular weights≥3350 blocked membrane blebbing. We also evaluated the effects of PEGs of various molecular weights on antitumor effect of SM. PEG 1000 had no effect on antitumor effect of SM. Chelating extracellular calcium with EGTA did not prevent blebbing after a delay, showing that the formation of blebs does not depend on the influx of calcium. Blebbing was also not affected when the intracellular calcium concentration was clamped by the cell-permeable chelator BAPTA-AM. Blebbing is independent of calcium. SM reduced intracellular ATP levels, disrupted the cytoskeletal systems, and had no effect on cell cycle.In addition, the quantity drugs associated with intracellular compartments was studied. We used a magnetic capture technique that allows for isolation of lysosomes and sucrose density gradient centrifugation for isolation of mitochondrial. After incubation with SM, cells were separated into fractions containing cytosol, lysosomes, and mitochondria and quantitation of compounds by LC/MS/MS. It is reported that receptor-mediated endocytosis cause lysosomal alkalinization and swollen. The amounts of SM that accumulated in the lysosomes and mitochondrial were determined. SM was found to preferentially accumulate within lysosomes in drug-resistant cells. These results suggest the internalization of SM in cancer cells may be related to rhamnose receptor-mediated endocytosis through endosomes, leading to localization in the lysosomes. In addition, the lumenal PH of lysosomes is known to be considerably lower in MDR cells, resulting in an enhanced lysosome-to-cytosol pH differential. SM, as a weakly basic compound, may become ionized and unable to diffuse back through the lipid monolayer of lysosomal membrane.In this thesis, we described the antifungal activity of RAB and its capacity to reverse azole resistance. It can be used clinically as an antifungal agent to potentiate azole antifungal agents. About the anticancer activity, it's the first report SM (IC80) can induce oncosis on cancer cell. Apoptosis is the process of programmed cell death and has little effect upon surrounding tissue. However, oncosis resulting from cell membrane damage and the leakage of contents can cause inflammation of surrounding tissue. Rational use of anticancer agents will reduce the dose toxicity and achieve a better anticancer effect. The intracellular distribution of SM was also studied in this paper. SM was found to preferentially accumulate within lysosomes in drug-resistant cells, which is important to drug delivery and its target research...