Erlotinib-Loaded Poly(ε-Caprolactone) Nanocapsules Improve In Vitro Cytotoxicity and Anticlonogenic Effects on Human A549 Lung Cancer Cells.

Lung cancer is the most frequent type of cancer and the leading cause of cancer-related mortality worldwide. This study aimed to develop erlotinib (ELB)-loaded poly(ε-caprolactone) nanocapsules (NCELB) and evaluated their in vitro cytotoxicity in A549 cells. The formulation was characterized in relation to hydrodynamic diameter (171 nm), polydispersity index (0.076), zeta potential (- 8 mV), drug content (0.5 mg.mL-1), encapsulation efficiency (99%), and pH (6.0). NCELB presented higher cytotoxicity than ELB in solution against A549 cells in the MTT and LIVE/DEAD cell viability assays after 24 h of treatment. The main mechanism of cytotoxicity of NCELB was the induction of apoptosis in A549 cells. Further, a significant decrease in A549 colony formation was verified after NCELB treatment in comparison with the unencapsulated drug treatment. The reduction in clonogenic capacity is very relevant as it can reduce the risk of tumor recurrence and metastasis. In conclusion, erlotinib-loaded PCL nanocapsules are promising nanoparticles carriers to increase the efficacy of ELB in lung cancer treatment.

Revitalizing the AZT Through of the Selenium: An Approach in Human Triple Negative Breast Cancer Cell Line.

Triple-negative breast cancer represents about 15% of all cases of breast cancer, and still represents a therapeutic challenge. 3'-Azido-3'-deoxythymidine (AZT) is a nucleoside reverse transcriptase inhibitor with antitumor activity. Chalcogenides compounds, such as selenium, are very important intermediates applied in organic synthesis. Our objective was to investigate the effect and the underlying cell death mechanisms of AZT and its derivatives, in human breast cancer cell lines. The inhibitory effect of AZT and derivatives (1072, 1073, and 1079) was determined by MTT assay (0.1, 1, 10, 50, and 100 µM for concentrations and times 4, 24, 48, and 72 h) and Live/Dead in tumor cell lines MCF-7, MDA-MB 231 and also in non-tumor cell line CHO. Gene expression profiles related to apoptosis were investigated by qRT-PCR and induction of apoptosis was investigated by flow cytometry. MTT and Live/Dead assays showed that AZT derivatives decreased the rate of cell proliferation at concentrations of 50 and 100 µM in tumor cell lines MCF-7 and MDA-MB 231 while the commercial AZT presented a low antitumoral potential in all strains tested. In flow cytometry analysis we demonstrated that derivatives of AZT induced apoptosis, with an increase in both initial and late stages in both tumor cell lines evaluated, especially in MDA-MB 231. Our data show that the AZT derivative 1072 increased the expression of transcripts of the genes caspase 3 and 8 in MDA-MB 231 cell line when compared to control, suggesting that the extrinsic pathway of apoptosis was activated. In conclusion, derivatives of AZT, especially 1072, induce cytotoxicity in vitro in the triple negative breast cancer cell line through activation of the extrinsic pathway of apoptosis. These compounds containing selenium in its formulation are potential therapeutic agents for breast cancer.

Cytotoxic Activity of Fatty Acids From Antarctic Macroalgae on the Growth of Human Breast Cancer Cells.

Macroalgae are a natural source of clinically relevant molecules such as polyunsaturated and monounsaturated fatty acids. The Antarctic environment, due to its cold climate, leads to high production of these bioactive molecules. Adenocystis utricularis, Curdiea racovitzae, and Georgiella confluens from three distinct islands in the Antarctic Peninsula were collected and analyzed for their fatty acid content by gas chromatography flame ionization detection. Results revealed that the algal extracts consisted of 22 fatty acids, of which 9 were saturated, 4 were monounsaturated, and 9 were polyunsaturated (PUFA). In addition, fucosterol was identified within the lipidic extracts. The cytotoxic activity of these fatty acids was evaluated in human breast cancer cell lines MCF-7 and MDA-MB-231. The most notable result was the effect of PUFA on the growth inhibition of cancer cells ranging from 61.04 to 69.78% in comparison to control cells. Significant cytotoxic activity of fatty acids from A. utricularis was observed at 48 h, resulting in an inhibition of growth of more than 50% for breast cancer cells at a concentration of 100 µg/mL. A cell viability assay showed that the fatty acids from A. utricularis significantly reduced cell viability (68.7% in MCF-7 and 89% in MDA-MB-231 after 72 h of exposure). At the same time, DAPI staining demonstrated chromatin condensation, and apoptotic bodies formed in cells that were cultured with fatty acids from A. utricularis. These data indicate that fatty acids from Antarctic macroalgae have the potential to reduce the proliferation of and induce apoptosis in breast cancer cells.

Synergistic and additive effects of ATRA in combination with different anti-tumor compounds.

All-trans retinoic acid (ATRA), a derivative of vitamin A, has been shown to potentiate cancer chemotherapy due to its ability to induce signals for cell differentiation or death, and inhibit cell proliferation. The combination of ATRA with taxoids, kinase inhibitors, natural compounds, retinoids, ER or HER2 inhibitors, chemotherapeutic drugs, proteasome inhibitors and nanoformulations of tretinoin have demonstrated additive or synergistic effects in anti-cancer activities. The mechanisms by which the compounds exert their synergistic effects depend on the tumor and the cell type. However, several experiments demonstrated similar mechanisms such as reduction of PCK, c-myc, E2F and Bcl-2, as well as increase of p21 and TGF-ß. When the apoptotic synergistic effect was observed, the predominant effect of ATRA was in differentiation induction. The results indicate that future combinations of ATRA and anti-tumor agents hold promise to enhance and improve anti-carcinogenic therapies.

Drug-loaded nanoemulsion as positive control is an alternative to DMSO solutions for in vitro evaluation of curcumin delivery to MCF-7 cells.

BACKGROUND: In vitro evaluation of toxicity and/or efficacy of nanostructured drug delivery systems involves the uses of different controls, including positive and negative controls, as well as a solution or dispersion of the drug in water. One of the most frequently solvent used to dilute poorly water soluble drugs to in vitro tests are dimethylsulfoxide (DMSO). However, its different specific surface area and different diffusion coefficients could make the comparative effects difficult. We proposed that a solvent-free dispersions having similar specific surface area could be a better control than drug in solution against cell lines. METHODS: We evaluate the effect of curcumin-loaded lipid-core nanocapsules, curcumin-loaded nanoemulsion and curcumin DMSO-water solution on viability and colony forming efficiency of human breast cancer cell line, MCF7. RESULTS: The cytotoxic effect of nanocapsules at 24-72h was similar to nanoemulsion and lower than drug solution. However, the nanocapsules had a superior anticancer activity when long periods (10days) were evaluated, which highlight the sustained drug release by nanocapsules. CONCLUSIONS: Our results showed a superior anticancer activity of curcumin-loaded lipid-core nanocapsules compared to curcumin-loaded nanoemulsion and curcumin dissolved in DMSO in long exposition time assay, wihch is not observed in short exposition time assays like MTT. When a poorly water-soluble drug is under investigation, the nanoemulsion prepared with the same compounds of the nanocapsules, except the polymer, could be a better control than DMSO-solution of drug.

Tretinoin-loaded lipid-core nanocapsules overcome the triple-negative breast cancer cell resistance to tretinoin and show synergistic effect on cytotoxicity induced by doxorubicin and 5-fluororacil.

Nanostructured drug delivery systems have been extensively studied, mainly for applications in cancer therapy. The advantages of these materials include protection against drug degradation and improvement in both the relative solubility of poorly water soluble drugs as in targeting of therapy, due to the enhanced permeability and retention effect on tumor sites. In this work, we evaluate the antitumor activity of tretinoin-loaded lipid core nanocapsules (TT-LNC) in a tretinoin-resistant breast cancer cell-line, MDA-MB- 231, as well as the synergistic effect of combination of this treatment with 5-FU or DOXO. The inhibition of cell growth was assayed by MTT reduction. Live/Dead assay and DAPI staining evaluated cytotoxicity. Apoptosis was evaluated by Annexin V-PE/7AAD and the effect of chronic exposure was evaluated by colony formation assay. TT-LNC reduced the cell viability even at lower concentrations (1µM) and displayed synergistic effect with 5-FU or DOXO on cytotoxicity and colony formation inhibition. Our work shows a possibility of using nanocapsules to improve the antitumoral activity of TT for its use either alone or in combination with other chemotherapeutic drugs, especially considering the chronic effect.

Evaluation of the toxicity of α-(phenylselanyl) acetophenone in mice.

Selenium is an essential micronutrient with several biological roles in the human body, but supra nutritional consumption can cause toxic effects. The potential deleterious effects of organoselenium compounds are controversial. The compound α-(phenylselanyl) acetophenone (PSAP) exhibits antioxidant, antidepressant-like and glutathione peroxidase-like activity, which makes important the elucidation of any toxic effects. Hence, the present study aims to investigate the in vitro toxicity of PSAP in Chinese Hamster ovary cells (through MTT assay) and analyse its genotoxicity using the comet assay in mice leukocytes after acute or chronic treatments, alongside with biochemical analyses. Our results demonstrate that the oral administration of PSAP in acute (1, 5, 10, 50, 200 mg/kg) and chronic (1, 10, 50, 200 mg/kg) doses did not cause genotoxicity. The compound presented cytotoxic effect in the MTT assay just at 500 µM after 24 h of administration and at 250 and 500 µM after 48 and 72 h of administration. According to biochemical analysis, PSAP presented a minor toxic effect by altering δ-ALA-D activity in liver and catalase activity in kidney at the highest tested concentration. Taking together, these data indicate that PSAP has low toxic effects after chronic administration in mice.

New organochalcogen multitarget drug: synthesis and antioxidant and antitumoral activities of chalcogenozidovudine derivatives.

In this article we present the synthesis, characterization, and in vitro biological and biochemical activities of new chalcogenozidovudine derivatives as antioxidant (inhibition of TBARS in brain membranes and thiol peroxidase-like activity) as well as antitumoral agents in bladder carcinoma 5637. A prominent response was obtained for the selected chalcogenonucleosides, showing effective antioxidant and antitumoral activities.

Brazilian red propolis induces apoptosis-like cell death and decreases migration potential in bladder cancer cells.

Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 µg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 µg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 µg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

Methotrexate diethyl ester-loaded lipid-core nanocapsules in aqueous solution increased antineoplastic effects in resistant breast cancer cell line.

Breast cancer is the most frequent cancer affecting women. Methotrexate (MTX) is an antimetabolic drug that remains important in the treatment of breast cancer. Its efficacy is compromised by resistance in cancer cells that occurs through a variety of mechanisms. This study evaluated apoptotic cell death and cell cycle arrest induced by an MTX derivative (MTX diethyl ester [MTX(OEt)2]) and MTX(OEt)2-loaded lipid-core nanocapsules in two MTX-resistant breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231. The formulations prepared presented adequate granulometric profile. The treatment responses were evaluated through flow cytometry. Relying on the mechanism of resistance, we observed different responses between cell lines. For MCF-7 cells, MTX(OEt)2 solution and MTX(OEt)2-loaded lipid-core nanocapsules presented significantly higher apoptotic rates than untreated cells and cells incubated with unloaded lipid-core nanocapsules. For MDA-MB-231 cells, MTX(OEt)2-loaded lipid-core nanocapsules were significantly more efficient in inducing apoptosis than the solution of the free drug. S-phase cell cycle arrest was induced only by MTX(OEt)2 solution. The drug nanoencapsulation improved apoptosis induction for the cell line that presents MTX resistance by lack of transport receptors.

Encapsulation in lipid-core nanocapsules overcomes lung cancer cell resistance to tretinoin.

Tretinoin is a retinoid derivative that has an antiproliferative effect on several kinds of tumours. Human lung adenocarcinoma epithelial cell lines (A549) exhibit a profound resistance to the effects of tretinoin. Nanocarriers seem to be a good alternative to overcomecellular resistance to drugs. The aim of this study was to test whether tretinoin-loaded lipid-core nanocapsules exert anantitumor effect on A549 cells. A549 cells were incubated with free tretinoin (TTN), blank nanocapsules (LNC) and tretinoin-loaded lipid-core nanocapsules (TTN-LNC). Data from evaluation of DNA content and Annexin V binding assay by flow cytometry showed that TTN-LNC induced apoptosis and cell cycle arrest at the G1-phase while TTN did not. TTN-LNC showed higher cytotoxic effects than TTN on A549 cells evaluated by MTT and LIVE/DEAD cell viability assay. Gene expression profiling identified up-regulated expression of gene p21 by TTN-LNC, supporting the cell cycle arrest effect. These results showed for the first time that TTN-LNC are able to overcome the resistance of adenocarcinoma cell line A549 to treatment with TTN by inducing apoptosis and cell cycle arrest, providing support for their use in applications in lung cancer therapy.

Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro.

BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ∆leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ∆leuD strain. The inhibitory effect of BCG ∆leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ∆leuD/Ag85B treatment was evaluated by Western blotting. BCG ∆leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ∆leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ∆leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ∆leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.