2-Deoxyglucose Glycolysis Inhibitor Augment Oncolytic Virotherapy to Induce Oxidative Stress and Apoptosis in Breast Cancer (Part Ⅲ)

One of the "hallmarks of cancer" is altered energy metabolism, which is increased glycolysis in cancer cells, the primary source of energy that uses this metabolic pathway to generate ATP. Oncolytic virotherapy with aerobic glycolysis inhibitor smart therapeutic approach to induce apoptosis in cancer cells. The current study aimed to use the 2-Deoxyglucose (2DG), a specific glycolysis inhibitor, to enhance the Newcastle disease virus (NDV). In this study, a mouse model of breast cancer allograft with mammary adenocarcinoma tumor cells (AN3) was used and treated with 2DG, NDV, and a combination of both. Anti-tumor efficacy and glycolysis analysis (hexokinase -1 (HK-1), pyruvate, and ATP) were determined. The induction of oxidative stress was investigated by reactive oxygen species (ROS) and total glutathione assay examination. Apoptosis induction was investigated using immunohistochemistry (cleaved Caspase-3) and histopathology. The result showed that combination therapy enhances anti-tumor efficacy (decrease in relative tumor volume and increase in tumor growth inhibition) of NDV against breast cancer. This effect was accompanied by a reduction in HK-1 concentration, pyruvate, and ATP (glycolysis products). Moreover, NDV+2DG therapy induces oxidative stress (decreases total glutathione and increases ROS). Immunohistochemistry and histopathological examination showed the apoptotic area in tumor tissues in treated groups. In conclusion, the present study found that the combination therapy could be considered as an effective cancer therapy through induction of glycolysis inhibition, oxidative stress, and apoptosis selectively in cancer cells.

Combination Treatment of Retinoic Acid Plus Focal Adhesion Kinase Inhibitor Prevents Tumor Growth and Breast Cancer Cells Metastasis

All-trans retinoic acid (RA) is the primary metabolite of vitamin A and controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, known as retinoids, are promising agents in treating and chemoprevention different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with the metastatic behavior of tumors. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that RA plus FAKi (RA+FAKi) approach could improve inhibition of tumor progression. By in silico analysis, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. In metastatic BC cells, we reveal that genes encoding proteins that FAK directly or indirectly modulates are deregulated compared to normal cells. We showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. We also evidenced that RA effects are extrapolated to other cancer cells, including the human cervical carcinoma cells HeLa. In an orthotopic assay of LM3 tumor growth, RA and FAKi administered separately reduce tumor growth; however, the combined treatment induces the more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduces metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance would reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbated these effects suggesting that the sensibility to RA therapies could be increased with FAKi coadministration in BC tumors.

Extracts of Jordanian Date Palm Fruit (Phoenix Dactylifera L.) Inhibit Human Mammary Adenocarcinoma (MCF-7) Cells In Vitro by Inducing Cell Viability

Worldwide, and in Jordan specifically, date palm fruit production has been steadily increasing. Recently, various dates extracts have been used functionally as antioxidants or anticancer agents. In addition, Jordanian date palm fruit of Barhi variety at two maturity stages (Rutab) and (Tamr) inhibited chemically-induced mammary cancer in animal model. The aim of this study is to strengthen the scientific evidence on the effect of dates on mammary cancer via assessing the effect of different concentrations of water extracts of three varieties of dates grown in Jordan (Barhi, Belle Huwaimil, and Medjool) at two maturity stages (Rutab and Tamr) on the viability of MCF-7 breast cancer cell line. The effects of different extracts on MCF7 inhibition/proliferation was analyzed using MTT assay. Percentage of inhibition was calculated. The most effective concentration was 100 mg/ml for all varieties. At this concentration, Medjool variety at the Tamr stage exhibited the highest inhibition. At the 12.5 and 25 mg/ml concentrations of the extracts, the most effective date palm fruit varieties were Belle Huwaimil and Barhi at the Tamr stage respectively. At lower concentrations (3.125 and 6.25 mg/ml), Medjool at the Tamr stage and Belle Huwaimil respectively were the most effective extracts were Barhi at the Tamr and Rutab stages were the least effective (P=0.000**). It is concluded that water extract of palm fruit reduced MCF-7 cell viability.

Phytolacca decandra 30 CH dilution as an anticancer agent in murine mammary adenocarcinoma model

Homeopathy is an effective and safe therapy that provides better quality of life and reduces the adverse effects of conventional therapy used in different diseases. However, there are few published studies showing effects of homeopathic medicines in cancer models. Considering the principle "like cures like" the Phytolocca decandra causes similar symptoms to those presented in cancer subjects. Therefore, the aim of this study was to evaluate the effects of homeopathic preparations of Phytolacca decandra in the development of murine mammary tumor. For this, 4T1 mammary adenocarcinoma cells were inoculated subcutaneously in the inguinal breast of female BALB/c mice. Then, mice were blind treated with water containing only vehicle (control) or vehicle with Phytolacca decandra dilutions (6CH, 12CH, 30CH or 200cH). Tumor growth was monitored in alternated days during 21 days after tumor cells inoculation. Then, mice were euthanized and tumor, spleen, and lungs were removed to histological analyses. Results showed that animals treated with 30CH when compared with other groups exhibited tumor growth delay and smaller tumor weight, less vascularization and smaller relative weight of the spleen and pulmonary metastases. Together, results obtained in this pilot study demonstrated that treatment with Phytolacca decandra 30CH dilution induces delayed of breast tumor growth suggesting that this dilution may be a promising alternative therapy in the treatment of breast cancer.

Study of the Effects of Homeopathic Medicine Carcinosinum on Mammary Adenocarcinoma (4T1 cells) in vitro.

Background: There are few published researches about the exclusive use of Carsinosinum in several potencies to treat cancer. The name Carcinosinum refers to any homeopathic preparation of epithelial cancerous tissues and is especially indicated when there are any hereditary and familial antecedents of cancer, tuberculosis, diabetes, pernicious anemia or a combination of two or more of these diseases. Homeopathic complexes which include Conium Maculatum, Sabal Serrulata, Thuja Occidentalis and Carcinosinum can reduce in 23% the incidence of prostate cancer in vivo and in 38% the tumor volume, compared to untreated groups. Another in vivo study revealed reduction of symptoms and increase of survival time in mice bearing Ehrlich ascitic carcinoma, after treatment with Carcinosinum 200cH. In vitro, Carcinosinum 200cH can increase the expression of the pro-apoptotic gene p53. However, mice treated with Carcinosinum 6cH had the highest percentage and diversity of symptoms compared to other treatments, which demonstrate the importance of homeopathic potency in pro or anti-carcinogenic action. Considering that the literature on this subject is still rare and focused on genotypic and clinical effects, the present study was proposed, with the aim of identifying the possible phenotypic changes, including viability, HER-2 expression and metastatic skills, using 4T1 cells in vitro as a model, after treatment with Carcinosinum in different homeopathic working dilutions (12cH; 30cH; 200cH), prepared mechanically (Denise Machine, Autic®) in our laboratory using sterile pure water, from a commercial matrix (HN Cristiano, São Paulo, Brazil) stocked in 70% hydro-alcoholic solution. The final dilutions were inserted in the culture medium in a volume equal to 10%, at the time of cell seeding. The same succussioned vehicle used to prepare the medicines (70% hydro-alcoholic solution), from the same batch and diluted 1:100 in sterile pure water, was used as control. All treated cells were cultivated in bottles of 25ml with cell density of 5 x 105 cells / ml and, after 24 hours of treatment, they were analyzed for the apoptosis index using the Annexin V kit and measured by the Countess® system. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. The results obtained up to now show that the treatment with Carcinosinum 12cH produced a different pattern of cell death compared to the other treatments, with significant reduction in apoptosis index (one-way ANOVA, p=0.01) and clear hydropic degeneration phenotypic pattern. The analysis of HER-2 expression and metastatic skill will be the next step of this research.

Hyaluronate Functionalized Multi-Wall Carbon Nanotubes Loaded with Carboplatin Enhance Cytotoxicity on Human Cancer Cell Lines

Cancer is a major global public health problem and conventional chemotherapy has several adverse effects and deficiencies. As a valuable option for chemotherapy, nanomedicine requires novel agents to increase the effects of antineoplastic drugs in multiple cancer models. Since its discovery, carbon nanotubes (CNTs) are intensively investigated for their use as carriers in drug delivery applications. This study shows the development of a nanovector generated with commercial carbon nanotubes (cCNTs) that were oxidized (oxCNTs) and chemically functionalized with hyaluronic acid (HA) and loaded with carboplatin (CPT). The nanovector, oxCNTs–HA–CPT, was used as a treatment against HeLa and MDA–MB-231 human tumor cell lines. The potential antineoplastic impact of the fabricated nanovector was evaluated in human cervical adenocarcinoma (HeLa) and mammary adenocarcinoma (MDA-MB-231). The oxCNTs–HA–CPT nanovector demonstrate to have a specific antitumor effect in vitro. The functionalization with HA allows that nanovector bio–directed towards tumor cells, while the toxicity effect is attributed mainly to CPT in a dose-dependent manner.

The Potential Protective Effect of Spirulina Nanoparticles Against Ehrlich Solid Tumor Bearing Mice Induced Liver Toxicity, Tumor Markers, DNA Fragmentation, Oxidative Stress and Monooxygenase Variations

Ehrlich solid tumor (EST) is a spontaneous murine mammary adenocarcinoma, undifferentiated, making mainly beneficial for tumors studies. The current research aim to describe the antineoplastic activity of spirulina nanoparticles extract (SP NPs) against EST induced alteration in liver functions, oxidative stress, tumor markers, monooxygenase and DNA damage in female mice. 40 female mice were arbitrarily doled out into 4 gatherings (1st, Control; 2nd, SP NPs; 3rd, EST; 4th, EST + SP NPs). Results revealed significant elevation in the levels of AST, ALT, GGT, ALP, AFP, CEA, DNA damage; thiobarbituric acid reactive substances (TBARS), and glutathione peroxidase (GPx) in liver homogenate and depletions in the levels of albumin, total proteins, cytochromes (CYP450 & cyt b5), reduced glutathione (GSH), glutathione reductase (GR), glutathione S transferase (GST) and superoxide dismutase (SOD) activity in liver homogenate compared to control. EST Treatment with SP NPs (EST + SP NPs) improved and modulates these variations in liver functions, tumor markers, DNA damage, cytochromes, enzymatic and non-enzymatic, and oxidative stress as compared to mice bearing EST. These results suggest that SP NPs may have hepatic protective, antioxidant, and anti-cancer properties; in addition to inhibition of the EST development, and liver DNA damage induced by EST.

Novel Meso-substituted Porphyrin Derivatives and its Potential use in Photodynamic Therapy of Cancer

Background: Photodynamic therapy (PDT) is an anticancer treatment that utilizes the interaction of light and a photosensitiser (PS), promoting tumour cell death mediated by generation of reactive oxygen species. In this study, we evaluated the in vitro photoactivity of four meso-substituted porphyrins and a porphyrin coupled to a fullerene. Methods: The cell line employed was the LM3 mammary adenocarcinoma, and the PS with the best photokilling activity was administered to mice bearing the LM3 subcutaneously implanted adenocarcinoma. The TEMCP4+ porphyrin and its analogue TEMCC4+ chlorine contain four identical carbazoyl substituents at the meso positions of the tetrapyrrolic macrocycle and have A4 symmetry. The TAPP derivative also has A4 symmetry, and it is substituted at the meso positions by aminopropoxy groups. The DAPP molecule has ABAB symmetry with aminopropoxy and the trifluoromethyl substituents in trans positions. The TCP-C604+ dyad is formed by a porphyrin unit covalently attached to the fullerene C60.Results: The PSs are taken up by the cells with the following efficiency: TAPP> TEMCP4+= TEMCC4+> DAPP >TCP-C604+, and the amount of intracellular PS correlates fairly with the photodamage degree, but also the quantum yields of singlet oxygen influence the PDT outcome. TAPP, DAPP, TEMCC4+ and TEMCP4+ exhibit high photoactivity against LM3 mammary carcinoma cells, being TAPP the most active. After topical application of TAPP on the skin of mice bearing LM3 tumours, the molecule is localized mainly in the stratum corneum, and at a lower extent in hair follicles and sebaceous glands. Systemic administration of TAPP produces a tumour: normal skin ratio of 31.4, and high accumulation in intestine and lung.Conclusion: The results suggest a potential use of topical TAPP for the treatment of actinic keratosis and skin adnexal neoplasms. In addition, selectivity for tumour tissue after systemic administration highlights the selectivity of and potentiality of TAPP as a new PS.