Ribonuclease A variants with potent cytotoxic activity

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI–RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.

Cytotoxic activity of Fagonia cretica against human breast cancer cells

In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC). Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response. Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry. However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

Characterization of volatile compounds of Albertisia papuana Becc root extracts and cytotoxic activity in breast cancer cell line T47D

Purpose: To evaluate the cytotoxic activity of chloroform and water root extracts of Albertisia papuana Becc. on T47D cell line and identify the volatile compounds of the extracts. Methods: The plant roots were extracted with chloroform and water using maceration and boiling methods, respectively. The cytotoxicity of the extracts on T47D were determined using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as reference drug in the cytotoxicity test while Probit analysis was used to calculate the Median Growth Inhibitory Concentration IC50 of the extracts. The volatile compounds in the chloroform and water root extracts were analyzed using Gas Chromatography-Mass Spectrophotometry GC-MS. Results: The IC50 of the chloroform and water extracts were 28.0 ± 6.0 and 88.0 ± 5.5 μg/mL, respectively whereas that of doxorubicin was 8.5 ± 0.1 μg/mL. GC-MS results showed that there were 46 compounds in the chloroform extract, out of which the five major components are ethyl linoleate (49.68 %), bicyclo (3.3.1) non-2-ene (29.29 %), ethyl palmitate (5.06 %), palmitic acid (3.67 %) and ethyl heptadecanoate (1.57 %).The water extract consisted of three compounds, butanoic acid (15.58 %); methyl cycloheptane (3.45 %), and methyl 2-O-methylpentofuranoside (80.96 %). Conclusion: The chloroform root extract of A. papuana Becc. had a fairly potent anticancer activity against breast cancer cells and may be further developed as an anticancer agent. Its major components were fatty acids and fatty acid esters.

Leishmanicidal and cytotoxic activity of extracts and saponins from Ilex laurina (Aquifoliaceae)

Purpose: To evaluate the leishmanicidal and cytotoxic activity of alcohol and non-alcohol extracts and saponins from Ilex laurina . Methods: Extracts were obtained by percolation with solvents of different polarities: hexane, dichloromethane, ethyl acetate and ethanol. The ethyl acetate extract was subjected to silica gel column chromatography eluting with a step gradient of dichloromethane-methanol. All products were evaluated in vitro for leishmanicidal activity against amastigotes of leishmania panamensis and cytotoxicity on U- 937 cells. Results: Two saponins were isolated from the ethyl acetate extract. The ethyl acetate extract showed high leishmanicidal activity against intracellular amastigotes of L. panamensis (EC50, 7.5 ± 1.5 μg/mL) and low activity against axenic amastigotes (EC50, 52.8 ±1.6 μg/mL); this extract showed also high cytotoxicity (LC50, 57.7 ± 12.1 μg/mL). Saponin 2 exhibited high activity against intracellular amastigotes (EC50, 5.9 ± 0.5 μg/mL) but also showed high cytotoxicity on U-937 cells (EC50, 25.7 ± 6.1 μg/mL). This compound showed similar leishmanicidal activity and cytotoxicity to meglumine antimoniate and amphotericin B, respectively, drugs currently used for the treatment of leishmaniasis. Conclusions: Based on these results, Ilex laurina is a potential source of compounds that can lead to the development of new therapeutic alternatives against leishmaniasis.

New antitumoral agents I: In vitro anticancer activity and in vivo acute toxicity of synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and derivatives

This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one 1 and its derivatives 2-5. This set of synthetic compounds exhibited high antitumoral activities regarding in vitro screening against several human tumor cell lines as lung carcinoma NCI-460, melanoma UACC-62, breast MCF-7, colon HT-29, renal 786-O, ovarian OVCAR-03 and ovarian expressing the resistance phenotype for adriamycin NCI-ADR/ RES, prostate PC-3, and leukemia K-562. Compounds were also tested against murine tumor cell line B16F10 melanoma and lymphocytic leukemia L1210 as well as to their effect toward normal macrophages. Specific activity against colon cancer cells HT-29 was observed for all tested compounds and suggests further studies with models of colon cancer. Compounds 1, 2, and 4 showed significant cytotoxic activity with IC(50) values <= 2.3 mu M for all human cancer cell lines. Intraperitoneal acute administration of compound 1 and 2 showed very low toxicity rate. (C) 2010 Elsevier Ltd. All rights reserved.

Myotoxic phospholipases A(2) isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A(2) (PLA(2)S) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M-r similar to 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2S from snake venoms, MTX-I belonging to Asp49 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)S, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA(2)S induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA(2) proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. (C) 2008 Elsevier Inc. All rights reserved.

The Ruthenium Complex cis-(Dichloro) Tetraammineruthenium(III) Chloride Presents Immune Stimulatory Activity on Human Peripheral Blood Mononuclear Cells

Ruthenium compounds in general are well suited for medicinal applications. They have been investigated as immunosuppressants, nitric oxide scavengers, antimicrobial agents, and antimalarials. The aim of this study is to evaluate the immunomodulatory activity of cis-(dichloro) tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) on human peripheral blood mononuclear cells (PBMC). The cytotoxic studies performed here revealed that the ruthenium( III) complex presents a cytotoxic activity towards normal human PBMC, only at very high concentration. Results also showed that cis-[ RuCl(2)(NH(3))(4)] Cl presents a dual role on PBMC stimulating proliferation and interleukin-2 (IL-2) production at low concentration and inducing cytotoxicity, inability to proliferate, and inhibiting IL-2 production at high concentration. The noncytotoxic activity of cis-[RuCl(2)(NH(3))(4)] Cl at low concentration towards PBMC, which correlates with the small number of annexin V positive cells and also the absence of DNA fragmentation, suggest that this compound does not induce apoptosis on PBMC. For the first time, we show that, at low concentration (10-100 mu g L(-1)), the cis-[ RuCl(2)(NH(3))(4)] Cl compound induces peripheral blood lymphocytes proliferation and also stimulates them to IL-2 production. These results open a new potential applicability of ruthenium(III) complexes as a possible immune regulatory compound acting as immune suppressor at high concentration and as immune stimulator at low concentration.

In vitro Antitumour Activity of Orsellinates

Lichen phenolic compounds exhibit antioxidant, antimicrobial, antiproliferative. and cytotoxic activities. The purpose of this study was to evaluate the anticancer activity of lecanoric acid, a secondary metabolite of the lichen Parmotrema tinctorum, and its derivatives, orsellinates, obtained by structural modification. A cytotoxicity assay was carried out hi vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma, MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity index of the compounds. n-Butyl orsellinate was the most active compound, with IC(50) Values (the concentration that inhibits 50% of growth) ranging from 7.2 to 14.0 mu g/ml, against all the cell lines tested. The compound was more active (IC(50), = 11.4 mu g/mL) against B16-F10 cells than was cisplatin (12.5 mu g/mL). Conversely, lecanoric acid and methyl orsellinate were less active against all cell lines, having an IC(50) value higher than 50 mu g/mL. Ethyl orsellinate was more active against HEp-2 than against MCF7, 786-0, or B16-F10 cells. The same pattern was observed for n-propyl and n-butyl orsellinates. n-Pentyl orsellinate was less active than n-propyl or n-butyl orsellinates against HEp-2 cells. The orsellinate activity increased with chain elongation (from methyl to n-butyl), a likely consequence of an increase in lipophilicity. The results revealed that the structural modification of lecanoric acid increases the cytotoxic activity of the derivatives tested.

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)(3) selectively potentiates its photodynamic activity

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)(3) into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)(3) and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

Bariatric Surgery Reverses Natural Killer (NK) Cell Activity and NK-Related Cytokine Synthesis Impairment Induced by Morbid Obesity

Background Obesity is related to a higher rate of infections and some types of cancer. Here we analyzed the impact of obesity and weight loss induced by Roux-en-Y gastric bypass (RYGB) on immunological parameters, i.e., cytokine productions and natural killer cell function. Methods We analyzed 28 morbidly obese patients before and 6 months after RYGB. Biochemical parameters were analyzed in plasma. The percent of natural killer (NK) cells, their cytotoxicity, and the production of cytokines by peripheral blood mononuclear cells were analyzed. The percent of NK cells was determined by flow cytometry and cytokine production determined by enzyme-linked immunosorbent assay. NK cytotoxicity was determined by the lactate dehydrogenase release assay. Results The weight loss 6 months following surgery was 35.3 +/- 4.5 kg. RYGB also improves biochemical parameters. No significant difference was found in the percent of NK cells after surgery. We found an increase in the production of interferon-gamma, interleukin (IL)-12 and IL-18, but not in IL-2, 6 months after RYGB. Cytotoxic activity of NK cells was significantly enhanced 6 months after RYGB [17.1 +/- 14.7% before RYGB vs 51.8 +/- 11.3% at 6 months after, at 40: 1 effector to target cell ratio; p<0.001]. We observed significant post-surgical improvement in the cytotoxic activity curve in 22 out of 28 patients (78.6%), irrespective of the target to effector cell ratio. Conclusions The weight loss induced by RYGB modifies the production of cytokines related with NK cell function and improves its activity.

The relationship of natural killer cell counts, perforin mRNA and CD2 expression to post-exercise natural killer cell activity in humans

The aim of this study was to further investigate the mechanism of suppression of natural killer (NK) cell cytotoxic activity In peripheral blood following strenuous exercise. Blood was collected for analysis of NK cell concentration, cytotoxic activity, CD2 surface expression and perforin gene expression from runners (RUN, n = 6) and resting controls (CONTROL, n = 4) pre-exercise, 0, 1.5, 5, and 24 h following a 60-min treadmill run at 80% of VO2 peak. Natural killer cytotoxic activity, measured using a whole blood chromium release assay, fluctuated minimally in the CONTROL group and increased by 63% and decreased by 43% 0 and 1.5 h post-exercise, respectively, in the RUN group (group x time, P < 0.001). Lytic index (cytotoxic activity per cell) did not change. Perforin mRNA, measured using quantitative real-time polymerase chain reaction (ORT-PCR) decreased from pre- to post-exercise and remained decreased through 24 h, The decrease from pre- to 0 In post-exercise was seen predominately in the RUN group and was inversely correlated r = - 0.95) to pre-exercise perform mRNA. The NK cell surface expression of CD2 (lymphocyte function-associated antigen-2) was determined using fluorescent antibodies and flow cytometry, There was no change in the proportion of NK cells expressing CD2 or CD2 density, We conclude that (1) numerical redistribution accounted for most of the change in NK cytotoxic activity following a strenuous run, (2) decrease in perforin gene expression during the run was inversely related to pre-exercise levels but did not parallel changes in cytotoxic activity, and (3) CD2 surface expression was not affected by exercise.

Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcus

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

Redox-active cytotoxic diorganotin(IV) cycloalkylhydroxamate complexes with different ring sizes: Reduction behaviour and theoretical interpretation

Two series of new diorganotin(IV) cycloalkylhydroxamate complexes with different ring sizes (cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), formulated as the mononuclear [R2Sn(HL)(2)] (1:2) (a, R=Bu-n and Ph) and the polymeric [R2SnL](n) (1:1) (b, R=Bu-n) compounds, were prepared and fully characterized. Single crystal X-ray diffraction for [(Bu2Sn)-Bu-n{C5H9C(O)NHO}(2)] (3a) discloses the cis geometry and strong intermolecular NH center dot center dot center dot O interactions. The in vitro cytotoxic activities of the complexes were evaluated against HL-60, Bel-7402, BGC-823 and KB human tumour cell lines, the greater activity concerning [(Bu2Sn)-Bu-n(HL)(2)] [HL=C3H5C(O)NHO (1a), C6H11C(O)NHO (4a)] towards BGC-823. The complexes undergo, by cyclic voltammetry and controlled-potential electrolysis, one irreversible overall two-electron cathodic process at a reduction potential that does not appear to correlate with the antitumour activity. The electrochemical behaviour of [R2Sn(C5H9C(O)NHO)(2)] [R=Bu-n (3a), Ph (7a)] was also investigated using density functional theory (DFT) methods, showing that the ultimate complex structure and the mechanism of its formation are R dependent: for the aromatic (R = Ph) complex, the initial reduction step is centred on the phenyl ligands and at the metal, being followed by a second reduction with Sn-O and Sn-C ruptures, whereas for the alkyl (R=Bu-n) complex the first reduction step is centred on one of the hydroxamate ligands and is followed by a second reduction with Sn-O bond cleavages and preservation of the alkyl ligands. In both cases, the final complexes are highly coordinative unsaturated Sn-II species with the cis geometry, features that can be of biological significance.

Natural killer cell activity in experimental paracoccidioidomycosis of the Syrian hamster

The study evaluated the activity of NK cells during the course of experimental infection of hamsters with Paracoccidioides brasiliensis. Eigthy hamsters were infected with P. brasiliensis by intratesticular route and sacrificed at 24h, 48h, 96h, 1, 2, 4, 8 and 11 weeks of infection and compared to 40 noninfected hamsters employed as controls. These animals were submitted to the study of NK cytotoxic activity by a single-cell assay and humoral immune response by immunodiffusion and ELISA tests. The production of macrophage migration inhibitory factor in the presence of Phyto-hemagglutinin and P. brasiliensis antigen and histopathology of the lesions were evaluated at 1, 4, 8 and 11 weeks of infection. The infected animals displayed significantly high levels of NK activity during the four weeks of infection that decreased from the 8th week on when compared to controls. This impairment of NK activity was associated with depression of cell-mediated immune response and with increase in the extension of the histopathologic lesions. There was an inverse correlation between NK cell activity and specific antibody levels. The results suggest that after initial activation, NK cells were unable to control the fungus dissemination. The impairment of NK activity in the late stages of the infection might be related to immunoregulatory disturbances associated with paracoccidioidomycosis.