Combination of NOS- and PDK-Inhibitory Activity: Possible Way to Enhance Antitumor Effects

We have previously demonstrated a high antitumor potential of NOS inhibitor T1023 (1-isobutanoyl-2-isopropylisothiourea hydrobromide): antitumor antiangiogenic activity in several animal tumor models and its ability to synergistically enhance the antitumor effects of bevacizumab, cyclophosphamide and γ-radiation. At the same time, rather rapid adaptation of experimental neoplasias to T1023 treatment was often observed. We attempted to enhance the antitumor activity of this NOS inhibitor by supplementing its molecular structure with a PDK-inhibiting fragment, dichloroacetate (DCA), which is capable of hypoxia-oriented toxic effects. We synthesized compound T1084 (1-isobutanoyl-2-isopropylisothiourea dichloroacetate). Its toxic properties, NOS-inhibiting and PDK-inhibiting activity in vivo, and antitumor activity on the mouse Ehrlich carcinoma model (SEC) were investigated in compare with T1023 and Na-DCA. We found that the change of the salt-forming acid from HBr to DCA does not increase the toxicity of 1-isobutanoyl-2-isopropylisothiourea salts, but significantly expands the biochemical and anti-tumor activity. New compound T1084 realizes in vivo NOS-inhibiting and PDK-inhibiting activity, quantitatively, at the level of the previous compounds, T1023 and Na-DCA. In two independent experiments on SEC model, a pronounced synergistic antitumor effect of T1084 was observed in compare with T1023 and Na-DCA at equimolar doses. There were no signs of SEC adaptation to T1084 treatment, while experimental neoplasia rapidly desensitized to the separate treatment of both T1023 and Na-DCA. The totality of the data obtained indicates that the combination of antiangiogenic and hypoxia-oriented toxic effects (in this case, within the molecular structure of the active substance) can increase the antitumor effect and suppress the development of hypoxic resistance of neoplasias. In general, the proposed approach can be used for the design of new anticancer agents.

Evaluation of the antitumor and antioxidant effects of jatobá (Hymenaea courbaril) extracts / Avaliação do efeito antitumoral e antioxidante de extratos do jatobá (Hymenaea courbaril)

Ethnobotanical surveys have revealed the use of jatobá for the treatment of several diseases. This study determined the effect of plant extracts on the development of Ehrlich carcinoma. Male Swiss mice (n=6) were subcutaneously inoculated with 106 tumor cells and intragastrically administered ethanol (2 mg·mL-1, 5 mg·mL-1, or 10 mg·mL-1) or aqueous extracts of jatobá seed or bark for 90 days. Tumor development did not significantly differ between the groups studied; however, animals treated with the aqueous extract of the seed (2.205 mg·mL-1) had a reduction in tumor size compared to those treated with the aqueous extract of the bark (1.7 mg·mL-1). The treatment was not found to influence the survival of the animals studied. A new group of animals (n=7), with or without the tumor, received the aqueous extract of jatobá seed for 7, 14, and 30 days to evaluate oxidative stress. The extract reduced the thiobarbituric acid reactive substances levels at 7 days in the liver and kidneys, and 14 days in brain and renal tissue. Protein carbonylation levels were also reduced at 7 days in the liver and brain tissue and 14 days in the liver. The reduced glutathione levels diminished in animals treated for 7 and 14 days. We conclude that treatment with the aqueous extract of the jatobá seed presents promising activity in the reduction of oxidative stress.

Deep-Sea Anemones Are Prospective Source of New Antimicrobial and Cytotoxic Compounds

The peculiarities of the survival and adaptation of deep-sea organisms raise interest in the study of their metabolites as promising drugs. In this work, the hemolytic, cytotoxic, antimicrobial, and enzyme-inhibitory activities of tentacle extracts from five species of sea anemones (Cnidaria, orders Actiniaria and Corallimorpharia) collected near the Kuril and Commander Islands of the Far East of Russia were evaluated for the first time. The extracts of Liponema brevicorne and Actinostola callosa demonstrated maximal hemolytic activity, while high cytotoxic activity against murine splenocytes and Ehrlich carcinoma cells was found in the extract of Actinostola faeculenta. The extracts of Corallimorphus cf. pilatus demonstrated the greatest activity against Ehrlich carcinoma cells but were not toxic to mouse spleen cells. Sea anemones C. cf. pilatus and Stomphia coccinea are promising sources of antimicrobial and antifungal compounds, being active against Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and yeast Candida albicans. Moreover, all sea anemones contain α-galactosidase inhibitors. Peptide mass fingerprinting of L. brevicorne and C. cf. pilatus extracts provided a wide range of peptides, predominantly with molecular masses of 4000–5900 Da, which may belong to a known or new structural class of toxins. The obtained data allow concluding that deep-sea anemones are a promising source of compounds for drug discovery.

Nifuroxazide Mitigates Angiogenesis in Ehlrich’s Solid Carcinoma: Molecular Docking, Bioinformatic and Experimental Studies on Inhibition of Il-6/Jak2/Stat3 Signaling

Nifuroxazide is an antidiarrheal medication that has promising anticancer activity against diverse types of tumors. The present study tested the anticancer activity of nifuroxazide against Ehrlich’s mammary carcinoma grown in vivo. Furthermore, we investigated the effect of nifuroxazide on IL-6/jak2/STAT3 signaling and the possible impact on tumor angiogenesis. The biological study was supported by molecular docking and bioinformatic predictions for the possible effect of nifuroxazide on this signaling pathway. Female albino mice were injected with Ehrlich carcinoma cells to produce Ehrlich’s solid tumors (ESTs). The experimental groups were as follows: EST control, EST + nifuroxazide (5 mg/kg), and EST + nifuroxazide (10 mg/kg). Nifuroxazide was found to reduce tumor masses (730.83 ± 73.19 and 381.42 ± 109.69 mg vs. 1099.5 ± 310.83) and lessen tumor pathologies. Furthermore, nifuroxazide downregulated IL-6, TNF-α, NFk-β, angiostatin, and Jak2 proteins, and it also reduced tumoral VEGF, as indicated by ELISA and immunohistochemical analysis. Furthermore, nifuroxazide dose-dependently downregulated STAT3 phosphorylation (60% and 30% reductions, respectively). Collectively, the current experiment shed light on the antitumor activity of nifuroxazide against mammary solid carcinoma grown in vivo. The antitumor activity was at least partly mediated by inhibition of IL-6/Jak2/STAT3 signaling that affected angiogenesis (low VEGF and high angiostatin) in the EST. Therefore, nifuroxazide might be a promising antitumor medication if appropriate human studies will be conducted.

Phytochemical profile, toxicological evaluation of Rhipsalis baccifera (Sol.) Stearn (Cactaceae) extract and their antitumor activity in Ehrlich carcinoma-bearing mice

<i>Rhipsalis baccifera</i> (Sol.) Stearn is a typical cactus from tropical regions with wide geographic distribution, and its therapeutic potential is not yet fully understood, such as antitumoral property. Thus, this study evaluated the cytotoxic ethanolic extract of <i>R. baccifera</i> (EERB) and its antitumor activity against Erlich's tumor in mice. The EERB was obtained, and its phytochemical profile was filed by thin-layer chromatography. The toxicity was evaluated in vitro and in vivo using the microcrustacean<i> Artemia salina</i> Leach and mice. The lethal dose was determined after implantation of a tumor cell suspension, with subsequent treatment with EERB (200 mg/kg and 100 mg/kg) 48h after implantation. These values represent the tenth part of the DL₅₀ and CL₅₀, respectively. The presence of phenols, tannins and triterpenes were demonstrated in the phytochemical results. Toxicity was dose-dependent, and the tumor inhibition was 84.1% and 75.8% at doses of 200 mg/kg and 100 mg/kg, respectively. We can highlight that the growth of Erlich's carcinoma suffered inhibitory effects against the EERB. EERB was found to have low acute toxicity and a high potential for use in antitumor therapy. Thus, new studies involving pre-clinical and clinical analyses of the extract are essential to determine the safe dose.

Effects of Phytolacca decandra on the viability of murine breast adenocarcinoma (4T1 cells) in vitro

Background: Comparative studies in cancer patients using conventional and alternative therapy have demonstrated that Phytolacca decandra in homeopathic potencies increases survival and improves quality of life of patients bearing breast cancer. In vitro studies show the induction of apoptosis pathways in MCF-7, a human breast cancer cells lineage, after treatment with Phytolacca decandra in different homeopathic dilutions (from 30C to 10M). Recently, we observed significant growth reduction of Ehrlich carcinoma in mice treated with Phytolacca decandra 30cH. Aims: To evaluate Phytolacca decandra effect in different homeopathic dilutions on the phenotypic features, apoptosis index, and cell morphology of 4T1 cells (murine carcinoma cell lineage) in vitro. Method: The potencies 6, 12, 30 and 200 CH prepared in sterile pure water were studied. Dynamized sterile pure water was used as control. The cytotoxicity was evaluated after different cell treatments in culture bottles (25ml) with the homeopathic medicines (equal to 10% of total medium volume). Cells were cultured in a cell density of 5 x 105 cells / ml, treated with the respective potency and, after 24 hours, analyzed for the apoptosis index using Annexin V kit and measured using the Countess® System. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. Cells were previously adhered to a glass cover slip and fixed with absolute methanol. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. Results and discussion: The results obtained up to now show that the treatment with Phytolacca decandra 200cH induced increase of apoptosis index in relation to the control. Moreover, morphological changes were observed in the respective cell smears: the presence of multinucleated cells, some of them presenting up to 8 nuclei and the increase of eosinophilic staining pattern of cytoplasm, even in mononucleated cells. Conclusion: The increase in apoptosis index reproduced the results described in the literature with other cell lineages, but the changes in morphology still deserve further evaluation.

Vochysia tucanorum Mart. butanol fraction presents antitumoral activity in vivo and prevents the installation of cachexia in solid Ehrlich tumor model

Background Cancer is a multifactorial disease caused by uncontrolled proliferation of cells. About 50–80% of cancer patients develop cachexia, a complex metabolic syndrome associated with an increase of mortality and morbidity. However, there are no effective therapies in medical clinic for cancer cachexia. Vochysia tucanorum Mart. is a common three of the Brazilian “Cerrado”. The butanolic fraction of V. tucanorum (Fr-BuVt), very rich in triterpenes with various biological activities, might be interesting in being tested in cancer cachexia syndrome. Hence, the present study was undertaken to investigate the antitumoral activity of Fr-BuVt and its potential against cachexia development. Methods Ehrlich tumor was used as model of cancer cachexia. Ascitic Ehrlich tumor cells were collected, processed and inoculated subcutaneously in saline solution (1 × 107/100 μl; ≥95% viability) for the obtention of solid Ehrlich carcinoma. After inoculation, solid Ehrlich carcinoma-bearing mice were treated by 14 consecutive days by gavage with Fr-BuVt (200 mg/kg). Body weight and tumor volume were measure during the treatment period. Tumors were removed, weighed and properly processed to measure the content and phosphorylation levels of key-proteins involved to apoptotic and proliferation process by Western Blot. Muscles and adipose tissues were removed for weighed. Serum was collected to cytokines levels and energetic blood markers measurements. Results The treatment with the Fr-BuVt (200 mg/kg, 14 days) decreased the solid Ehrlich tumor volume and weight besides increased the expression of the pro-apoptotic proteins caspase-3 and BAX, but also decreased the expression of the proteins involved in proliferation NFκB, mTOR and ERK. In addition, our data shows that the administration of Fr-BuVt was able to prevent the installation of cancer cachexia in Ehrlich carcinoma-bearing mice, since prevented the loss of body weight, as well as the loss of muscle and adipose tissue. Moreover, an improvement in some blood parameters such as decrease in cytokines TNF-α and IL-6 levels is observed. Conclusions The study revealed that Fr-BuVt has antitumoral activity and prevent installation of cancer cachexia in Ehrlich model. Therefore, Fr-BuVt may represent an alternative treatment for cancer cachexia.