Antitumor effect of snake venoms

The search for biological antitumor agents has been pursued for over half a century. Snake venom has been shown to possess a wide spectrum of biological activities. The objectives of the present review are to evaluate the existing controversies on this subject published in a number of papers and to propose probable explanations for the phenomena observed. We reported our results obtained in a study, in which we evaluated the action of the venoms of Crotalus durissus terrificus and Bothrops jararaca on Ehrlich ascites tumor cells. We noticed an important antitumor effect, mainly with Bothrops jararaca venom, as well as an increase in the functional activity of macrophages. We also observed an increase in the number of mononuclear and polymorphonuclear cells with Bothrops jararaca venom. Considering these findings, we postulate that both Borhrops jararaca and Crotalus durissus terrificus venoms can act directly on tumor cells. In addition, we propose an indirect mechanism, based on the stimulation of the inflammatory response, to inhibit tumor growth and to promote its rejection.

New ruthenium(II)/phosphines/diimines complexes: Promising antitumor (human breast cancer) and Mycobacterium tuberculosis fighting agents

The synthesis and characterization of ruthenium compounds of the type [RuCl2(P)2(N-N)] [(P)2 = (PPh3) 2, dppb = 1,4-bis(diphenylphosphino)butano; dppp = 1,3-bis(diphenylphosphino)propane; N-N = 5,5′-dimethyl-2,2′dipyridyl (5,5′-mebipy) or 4,4′-dimethyl-2,2′dipyridyl (4,4′-mebipy)] are described. The complexes were characterized using elemental analysis, UV-Vis and infrared spectroscopies, cyclic voltammetry, and X-ray crystallography. In vitro evaluation of the complexes, using the MTT methodology, revealed their cytotoxic activities in a range of 5.4-15.7 μM against the MDA-MB-231 breast tumor cells and showed that, in this case, they are more active than the reference metallodrug cisplatin. The in vitro antimycobacterial activities of the complexes had their Minimum Inhibitory Concentration (MIC) for MTB cell growth measured, by the REMA method. The MICs for these complexes were found to be between 12.5 and 25.0 μg/mL. The results are comparable with the second line drug cycloserine (MIC = 12.5-50.0 μg/mL), commonly used in the treatment of TB. © 2013 Elsevier Ltd. All rights reserved.

Development and characterization of biocompatible isotropic and anisotropic oil-in-water colloidal dispersions as a new delivery system for methyl dihydrojasmonate antitumor drug

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Morphological and biochemical alterations activated by antitumor clerodane diterpenes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antitumor and Anti-Inflammatory Effects of Palladium(II) Complexes on Ehrlich Tumour

Palladium(II) complexes are an important class of cyclopalladated compounds that play a pivotal role in various pharmaceutical applications. Here, we investigated the antitumour, anti-infl ammatory, and mutagenic effects of two complexes: [Pd(dmba)(Cl)tu] (1) and [Pd(dmba)(N3)tu] (2) (dmba = N,N-dimethylbenzylamine and tu = thiourea), on Ehrlich ascites tumour (EAT) cells and peritoneal exudate cells (PECs) from mice bearing solid Ehrlich tumour. The cytotoxic effects of the complexes on EAT cells and PECs were assessed using the 3-(4,5-dimethylthiazol-3-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The effects of the complexes on the immune system were assessed based on the production of nitric oxide (NO) (Griess assay) and tumour necrosis factor-Į (TNF-Į), interleukin-12 (IL-12), and interleukin-10 (IL-10) (ELISA). Finally the mutagenic activity was assessed by the Ames test using the Salmonella typhimurium strain TA 98. Cisplatin was used as a standard. The IC50 ranges for the growth inhibition of EAT cells and PECs were found to be (72.8 ± 3.23) µM and (137.65 ± 0.22) µM for 1 and (39.7 ± 0.30) µM and (146.51 ± 2.67) µM for 2, respectively. The production of NO, IL-12, and TNF-Į, but not IL-10, was induced by both complexes and cisplatin. The complexes showed no mutagenicity in vitro, unlike cisplatin, which was mutagenic in the strain. These results indicate that the complexes are not mutagenic and have potential immunological and antitumour activities. These properties make them promising alternatives to cisplatin.

Evaluating the microbicidal, antiparasitic and antitumor effects of CR-LAAO from Calloselasma rhodostoma venom

CR-LAAO is an l-amino acid oxidase from Calloselasma rhodostoma snake venom that has been broadly studied regarding its structural and biochemical characteristics, however, few studies have investigated its pharmacological effects. The present study aimed at the evaluation of the biotechnological potential of CR-LAAO by determining its bactericidal, antifungal, leishmanicidal and trypanocidal activity, as well as its cytotoxicity on human tumor and non-tumor cell lines. After 24h of preincubation, CR-LAAO showed bactericidal effects against both Staphylococcus aureus (MIC 0.78μg/mL) and Escherichia coli (MIC 31.25μg/mL) strains, inducing dismantle of bacterial cell walls. After 6h of preincubation with Candida albicans, CR-LAAO was able to inhibit 80% of the yeast growth, and it also showed cytotoxic activity on Leishmania species and Trypanosoma cruzi. Additionally, CR-LAAO showed high cytotoxicity on HepG2 and HL-60 tumor cells (IC50 10.78 and 1.7μg/mL), with lower effects on human mononuclear cells (PBMC). The cytotoxic effects of CR-LAAO were significantly inhibited in the presence of catalase, which suggests the involvement of hydrogen peroxide in its mechanisms of toxicity. Therefore, CR-LAAO showed promising pharmacological effects, and these results provide important information for the development of therapeutic strategies with directed action, such as more effective antimicrobial agents.

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I–truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA″ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA″, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.

Antitumor effect of SIRT1 inhibition in human HCC tumor models in vitro and in vivo

Sirtuins (SIRT1-7) are a highly conserved family of NAD(+)-dependent enzymes that control the activity of histone and nonhistone regulatory proteins. SIRT1 is purposed to promote longevity and to suppress the initiation of some cancers. Nevertheless, SIRT1 is reported to function as a tumor suppressor as well as an oncogenic protein. Our data show that compared with normal liver or surrounding tumor tissue, SIRT1 is strongly overexpressed in human hepatocellular carcinoma (HCC). In addition, human HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for the expression of the sirtuin family members and only SIRT1 was consistently overexpressed compared with normal hepatocytes. To determine its effect on HCC growth, SIRT1 activity was inhibited either with lentiviruses expressing short hairpin RNAs or with the small molecule inhibitor, cambinol. Knockdown or inhibition of SIRT1 activity had a cytostatic effect, characterized by an altered morphology, impaired proliferation, an increased expression of differentiation markers, and cellular senescence. In an orthotopic xenograft model, knockdown of SIRT1 resulted in 50% fewer animals developing tumors and cambinol treatment resulted in an overall lower tumor burden. Taken together, our data show that inhibition of SIRT1 in HCC cells impairs their proliferation in vitro and tumor formation in vivo. These data suggest that SIRT1 expression positively influences the growth of HCC and support further studies aimed to block its activity alone or in combination as a novel treatment strategy.

UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^

Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10 μg/mL VALE and 1 μg/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo.

Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma

We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte–macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

Erythropoietin induces tumor regression and antitumor immune responses in murine myeloma models

Recombinant human erythropoietin (rHuEpo) has been used successfully in the treatment of cancer-related anemia. Clinical observations with several patients with multiple-myeloma treated with rHuEpo has shown, in addition to the improved quality of life, a longer survival than expected, considering the poor prognostic features of these patients. Based on these observations, we evaluated the potential biological effects of rHuEpo on the course of tumor progression by using murine myeloma models (MOPC-315-IgAλ2 and 5T33 MM-IgG2b). Here we report that daily treatment of MOPC-315 tumor-bearing mice with rHuEpo for several weeks induced complete tumor regression in 30–60% of mice. All regressors that were rechallenged with tumor cells rejected tumor growth, and this resistance was tumor specific. The Epo-triggered therapeutic effect was shown to be attributed to a T cell-mediated mechanism. Serum Ig analysis indicated a reduction in MOPC-315 λ light chain in regressor mice. Intradermal inoculation of 5T33 MM tumor cells followed by Epo treatment induced tumor regression in 60% of mice. The common clinical manifestation of myeloma bone disease in patients with multiple-myeloma was established in these myeloma models. Epo administration to these tumor-bearing mice markedly prolonged their survival and reduced mortality. Therefore, erythropoietin seems to act as an antitumor therapeutic agent in addition to its red blood cell-stimulating activity.

In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 102-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.

Metabolic tug-of-war in tumors results in diminished T cell antitumor immunity.

NlmCategory="UNASSIGNED">Glycolytic activity in T cells governs T cell responses by controlling various cellular processes, including proliferation, survival, and effector functions. We recently showed that the tumor microenvironment diminishes T cell antitumor responses by depriving glucose to infiltrating T cells. Moreover, metabolic rewiring tumor-reactive T cells sustain T cell metabolic fitness and antitumor responses.