In vitro anticancer activity of Hirudinaria manillensis methanolic extract and its validation using in silico molecular docking approach.

Cancer has emerged as a potentially lethal illness, which recently upsurged in the mortality rate. Animal-derived compounds could be promising targets with higher efficacy and low toxicity in anticancer therapy. The present study aimed to explore the presence of anticancer potential compounds in Hirudinaria manillensis methanolic extract and their anticancer potential against various cancer cell types and target identification by Auto dock method. Initially, the identification of bioactive compounds was achieved by GC-MS analysis followed by the anticancer activity by MTT assay against A549, HeLa, MDA-MB-231, MG-63, and MOLT-4. Further, the effect of a lead compound on the cancer cell target was analyzed by the Auto dock method. GC-MS analysis results revealed the presence of 25 different bioactive compounds including anticancer potential compounds, such as Lupeol, Carvacrol, and Demecolcine. Interestingly, MTT assay results demonstrated the anticancer potential of Hirudinaria manillensis extract (LE) against various cancer cell lines, such as A549 (54.60 µg/ml), HeLa (19.93 µg/ml), MDA-MB-231 (20.23 µg/ml), MG-63 (20.04 µg/ml), and MOLT-4 (171.8 µg/ml), respectively. Among these cell types, the maximum inhibition was observed against HeLa with the IC50 concentration of 19.93 µg/ml. Furthermore, Demecolcine compound was docked with the EGFR tyrosine kinase showed the binding affinity of the docked complex was predicted to be - 6.2 kcal/mol. Thus, we conclude that H. manillensis has a significant anticancer effect on human cancer cell lines and could be used as a natural target which paves the way for further studies on biomedical applications in cancer therapeutics.

Antimicrobial activity of nanoemulsion on drug-resistant bacterial pathogens.

The appearance of drug-resistant (DR) bacteria in the community is a crucial development, and is associated with increased morbidity, mortality, healthcare costs, and antibiotic use. Natural oil nanoemulsions (NEs) have potential for antimicrobial applications. In the present study, we determined the antimicrobial activity of an NE against DR bacterial pathogens in vitro. The NE comprised Cleome viscosa essential oil, Tween 80 nonionic surfactant, and water. We found that an NE with a droplet size of 7 nm and an oil:surfactant (v/v) ratio of 1:3 was effective against methicillin-resistant Staphylococcus aureus (MRSA), DR Streptococcus pyogenes, and DR extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Fourier-transform infrared (FTIR) spectroscopy revealed that NE treatment modified the functional groups of lipids, proteins, and nucleic acids in DR bacterial cells. Scanning electron microscopy (SEM) showed damage to the cell membranes and walls of NE-treated DR bacteria. These alterations were caused by bioactive compounds with wide-spectrum enzyme-inhibiting activity in the NE, such as ß-sitosterol, demecolcine, campesterol, and heneicosyl formate. The results suggest that the nanoemulsion is effective against DR bacteria, and acts by inhibiting the drug efflux mechanism of DR strains.

Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding.

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a "substrate-induced fit" mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol or trans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds.

Effects of polyphenolic anthrone derivatives, resistomycin and hypercin, on apoptosis in human megakaryoblastic leukemia CMK-7 cell line.

A tetrahydroxyanthrone derivative, resistomycin, was isolated from the culture broth of Streptomyces sulphureus and a similar polyphenolic dianthraquinone, hypericin, was isolated from an extract of Hypericum perforatum L. as modulators for apoptosis. Resistomycin inhibited apoptosis induced by actinomycin D (AD) with or without acceleration by colcemid (CL) in human megakaryoblastic leukemia CMK-7 cells, IC50 for inhibition against AD-induced apoptosis was about 0.5 microM and IC50 for inhibition against AD plus CL-induced apoptosis was about 1 microM. CL alone induced weak apoptosis in cells, which was enhanced by resistomycin. Hypericin did not inhibit AD-induced apoptosis and slightly enhanced CL-induced apoptosis. Emodin, corresponding to 1 of 2 anthraquinone units in hypericin, did not show any effect on this apoptotic system. AD-induced apoptosis was inhibited by the antioxidative flavonoid, luteolin (IC50 45 microM), and a protein kinase C (PKC) inhibitor, staurosporine (IC50 1.5 microM), but these compounds did not affect the CL-induced apoptosis. Hypericin and resistomycin scavenged superoxide anion radicals at the same rate as luteolin. PKC in CMK-7 cells was inhibited by hypericin and luteolin, but not significantly inhibited by resistomycin. This result suggests that the inhibition of AD-induced apoptosis by resistomycin is at least partly correlated with its antioxidative activity, and that the enhancement of CL-induced apoptosis by this compound depends upon the lack of PKC inhibitory activity. Though the mechanism is not clear, the enhancement of the CL-induced apoptosis might be hindered by PKC inhibition in the case of hypericin and luteolin.

Improvement of accuracy of chromosome aberration analysis for biological radiation dosimetry.

The frequency of chromosome aberrations in circulating lymphocytes is accepted as being the most reliable indicator of the absorbed dose of radiation. Researches done to improve the accuracy of cytogenetic analysis are described in this review. These include investigations of in vitro factors that affect the yield of radiation-induced aberrations and of in vivo factors that affect the chromosomal radiosensitivity of individuals. Improved chromosome-painting methods for accurate judgment of dicentrics and translocations are introduced. The practicality of these advanced cytogenetic techniques is shown by examinations of individuals exposed in the radiation accident at Tokaimura in 1999.

[A brief introduction of achievements in the study of the art of history of Shanghan (Cold Injury)].

The achievement is described in five subheadings, viz. relevant people, relevant works, relevant events, academic relationship, and characteristic of stages. It is held that, the early development is not enough to demonstrate its significance and historical position, even less to show its development direction. It is essential to make further investigation.

Induction of polyploidy and apoptosis after exposure to high concentrations of the spindle poison nocodazole.

The proportions of aneuploid/polyploid versus euploid cells formed after treatment with spindle poisons like nocodazole are of course dependent on the relative survival of cells with numerical chromosome aberrations. This work aimed at studying the survival of polyploid cells formed after treatment with a nocodazole concentration sufficient to significantly decrease tubulin polymerization (0.1 microg/ml). First, normal primary lymphocytes were analysed and the following complementary chromosomal parameters were quantified: mitotic index, frequency of abnormal mitoses, polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and the induction of polyploidy. They therefore led to a single cell approach in which both apoptosis and polyploidy induction could be scored in the same cell. For this purpose, actively proliferating cells are required and two human leukaemic cell lines were used, KS (p53-positive) and K562 (p53-negative), which have a near-triploid karyotype. Cells were separated into an apoptotic and a viable fraction by means of annexin-V staining and flow cytometry. In KS, treatment with nocodazole induced a similar fraction of hexaploid cells in both the viable and apoptotic fraction, but no dodecaploid cells were ever observed. In contrast, a population of dodecaploid cells (essentially viable) was clearly observed in the K562 cell line. The results in KS, as compared with K562, confirm that wild-type p53 can prevent further cycling of polyploid cells by blocking rereplication. The most probable explanation for these data is that not only the mitotic spindle but also interphase microtubules are sensitive to nocodazole treatment. Our data thus strongly suggest that besides the G(1)/S checkpoint under the control of p53, the G(2)/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho.

Microsome-mediated transformation of O-methylandrocymbine to demecolcine and colchicine.

Microsomal preparations from immature seeds of Colchicum autumnale L. catalyse the ring expansion reaction of O-methylandrocymbine to demecolcine in the presence of NADPH and O2. In addition evidence is given for further transformation of demecolcine to colchicine in the presence of acetyl-CoA and NADPH.

Changes in colchicine and demecolcine content during vegetation period of colchicum autumnale L.

Colchicine and demecolcine were determined in raw and dried leaves, stems, mother and daughter corms of Colchicum autumnale L. in four stages of its ontogenesis. The colchicine content in raw material varies during plant growth. The content of both alkaloids decreases with drying. The HPLC method used is suitable both for the phytochemical analysis of Colchicum and for the toxicological evaluation in cases of intoxication by these plants.

Cell cycle effects of trimetrexate (CI-898).

The cell cycle phase specificity of trimetrexate (CI-898) was examined. CHO cells synchronized by mitotic selection were exposed to 50 microM trimetrexate for 2 h at various time points after release from Colcemid block. Only S phase cells were sensitive to trimetrexate when survival was measured by a cloning assay. Comparison of plateau phase and log phase cultures indicated that plateau phase CHO cells were relatively insensitive to 5 microM trimetrexate. Exponentially growing L1210 cells were continuously exposed to either 30 nM or 3 nM trimetrexate and analyzed by DNA flow cytometry. Incubation with 30 nM trimetrexate produced cell cycle arrest in late G1 or early S phase, while exposure to 3 nM trimetrexate caused only a delay in progression through S phase. In an in vivo schedule dependence study with mice bearing approximately 3 X 10(6) P388 leukemia cells, trimetrexate was most effective with frequent administration. Mice treated on the optimal schedule, every 3 h X 8 on days 1, 5, and 9 after tumor implant, had life-span increases in excess of 100%.

Planarians as a model system for in vitro teratogenesis studies.

Free-living flatworms such as planarians are inexpensive to culture, maintain, and use for toxicologic testing in the laboratory. A considerable number of basic studies by ourselves and others indicate that, in simplified miniature, they possess many features of biochemical and physiologic organization similar to higher animals such as mammals. These include a well-developed brain with a varied behavioral repertoire including complex maneuvers of prey capture and learning, with a number of the same neurotransmitters used in mammalian brain. They are sensitive to a variety of the same toxicants. Undifferentiated totipotent stem cells, i.e., "neoblasts," which are capable of mitosis and differentiation into any of the various specialized cell types, permit regeneration of complete planarians from fragments. They also provide new cells to replace those lost in the normal cellular turnover of nonregenerating planarians. Both regeneration of surgical fragments and aberrant remodeling of whole planarians model important features of embyrogenesis and are potentially useful for assaying teratogens. Results are described from studies in which various representative teratogenic toxicants were tested in these two different planarian paradigms. The potential of planarian cephalic regeneration for behavioral teratogenesis investigations is also indicated.

Comparison of direct harvest and cultures for karyotyping EDTA anticoagulated marrow.

Studies were undertaken to determine whether EDTA was a satisfactory anticoagulant for tissue specimens for cytogenetic analysis and to investigate a modification of a currently used culture technique for obtaining metaphases. The latter involved to prolonged exposure to very low-dose colcemid and was successful in qualitative or quantitative enhancement, or both, of the temperature yield over that obtained from direct harvest in 53% of the patients studied. EDTA is a suitable anticoagulant for cytogenetic studies of specimens from either direct harvest or short-term culture if the specimen is either processed within 24 hr after collection or diluted 1:1 with Eagles minimal essential media, supplemented with fetal bovine serum and refrigerated until processed. Success has been obtained with specimens stored up to 144 hr.

Mononuclear leucocyte chemotaxis in Boyden chambers: inhibition by subantimitotic concentrations of antitubulins.

The chemotaxis of Lymphoprep -isolated human mononuclear leucocytes (L-MNs) from peripheral blood was inhibited by subantimitotic concentrations of the antitubulins demecolcine, podophyllic acid ethylhydrazide, vinblastine and griseofulvin. It is suggest that L-MN chemotaxis, like polymorphonuclear leucocyte (PMN) chemotaxis, is composed of a direct antitubulin-insensitive chemotaxis and a leukocyte-induced antitubulin-sensitive chemotaxis.