Murine Mammary Adenocarcinoma Research Articles

Propranolol inhibits EMT and metastasis in breast cancer through miR-499-5p-mediated Sox6

PurposeThis study will focus on 4T1 cells, a murine mammary adenocarcinoma cell line, as the primary research subject. We aim to investigate the inhibitory effects and mechanisms of propranolol on epithelial–mesenchymal transition (EMT) in breast cancer cells, aiming to elucidate this phenomenon at the miRNA level.MethodsIn this study, the EMT inhibitory effect of propranolol was observed through in vitro and animal experiments. For the screening of potential target miRNAs and downstream target genes, second-generation sequencing (SGS) and bioinformatics analysis were conducted. Following the screening process, the identified target miRNAs and their respective target genes were confirmed using various experimental methods. To confirm the target miRNAs and target genes, Western Blot (WB), reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence experiments were performed.ResultsIn this study, we found that propranolol significantly reduced lung metastasis in 4T1 murine breast cancer cells (p < 0.05). In vitro and in vivo experiments demonstrated that propranolol inhibited the epithelial–mesenchymal transition (EMT) as evidenced by Western Blot analysis (p < 0.05). Through next-generation sequencing (SGS), subsequent bioinformatics analysis, and PCR validation, we identified a marked downregulation of miR-499-5p (p < 0.05), suggesting its potential involvement in mediating the suppressive effects of propranolol on EMT. Overexpression of miR-499-5p promoted EMT, migration, and invasion of 4T1 cells, and these effects were not reversed or attenuated by propranolol (Validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). Sox6 was identified as a functional target of miR-499-5p, with its downregulation correlating with the observed EMT changes (p < 0.05). Silencing Sox6 or overexpressing miR-499-5p inhibited Sox6 expression, further promoting the processes of EMT, invasion, and migration in 4T1 cells. Notably, these effects were not alleviated by propranolol (validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). The direct interaction between miR-499-5p and Sox6 mRNA was confirmed by dual-luciferase reporter gene assay.ConclusionThese results suggest that propranolol may have potential as a therapeutic agent for breast cancer treatment by targeting EMT and its regulatory mechanisms.

Tumor Radiosensitization by Gene Electrotransfer-Mediated Double Targeting of Tumor Vasculature.

Targeting the tumor vasculature through specific endothelial cell markers involved in different signaling pathways represents a promising tool for tumor radiosensitization. Two prominent targets are endoglin (CD105), a transforming growth factor β co-receptor, and the melanoma cell adhesion molecule (CD1046), present also on many tumors. In our recent in vitro study, we constructed and evaluated a plasmid for simultaneous silencing of these two targets. In the current study, our aim was to explore the therapeutic potential of gene electrotransfer-mediated delivery of this new plasmid in vivo, and to elucidate the effects of combined therapy with tumor irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice in the syngeneic murine mammary adenocarcinoma tumor model TS/A. Histological analysis of tumors (vascularization, proliferation, hypoxia, necrosis, apoptosis and infiltration of immune cells) was performed to evaluate the therapeutic mechanisms. Additionally, potential activation of the immune response was evaluated by determining the induction of DNA sensor STING and selected pro-inflammatory cytokines using qRT-PCR. The results point to a significant radiosensitization and a good therapeutic potential of this gene therapy approach in an otherwise radioresistant and immunologically cold TS/A tumor model, making it a promising novel treatment modality for a wide range of tumors.

Study of anti-tumorigenic actions of essential fatty acids in a murine mammary gland adenocarcinoma by micro-XRF.

In the present work, a statistical experiment based on the microscopy X-ray fluorescence technique was developed to evaluate the effect of diets rich in ω-3 and ω-6 polyunsaturated fatty acids on tumour tissues. Relative variations on the local content of P, S, Ca, Fe, Cu and Zn were analysed in the experiment. Neoplastic tissues were obtained from mammary gland adenocarcinomas inoculated in mice belonging to three different dietary groups: normal, rich in ω-3 and in ω-6 polyunsaturated fatty acids. Slices of 30 microns thick sections of these samples were scanned in the air atmosphere in areas of 5 mm × 5 mm with a spatial resolution of 50 microns using synchrotron radiation. Principal component analysis was employed to analyse the correlation between the X-ray fluorescence signals of P, S, Ca, Fe, Cu and Zn. The subsequent application of the K-means clustering was used for the automatic segmentation of the image scans. By comparison with conventional histological analysis, the clusters were positively identified as tumour parenchyma, transition and necrotic region. The calculation of the mean content of P, S, Ca, Fe, Cu, and Zn in these regions showed that dietary polyunsaturated fatty acids modify elemental content of tumour parenchyma, suggesting its involvement in the antitumour effects of chia oil and protumour effects of safflower oil.

HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells.

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape.

Combination synergy between β-carotene and lupeol against breast adenocarcinoma in vitro and in vivo using combination index

BackgroundBreast adenocarcinoma is one of the leading causes of death among women regardless of development in the field of diagnosis, treatment and prevention. Synergistic approaches are emerging as a strategy to overcome monotherapy resistance in cancer treatment. PurposeEvaluation of the combinatory activity of β-carotene (BC) and lupeol (LUP) for anti-proliferative activity by median effect equation and combination index. Validation was performed by fixed dose combination against murine mammary adenocarcinoma namely Ehrlich ascites carcinoma (EAC). MethodsCytotoxicity assay was performed using the MTT and Trypan blue methods. Isobologram and combination index analysis were used to analyze the type of interaction between β-carotene (BC) and lupeol (LUP). Based on the synergistic dose combination determination of in-vivo antitumor activity was performed using EAC cells inoculated in mice treated with BC, LUP and their combination (BC+LUP). Tumor volume, cell count, weight along with hematological, biochemical and antioxidant parameters were evaluated. ResultsThe combination (BC+LUP) exhibited the highest inhibition (IC50=54.8 ± 1.84) compare to the standard inhibitor, wher eas BC and LUP showed moderate inhibition with an IC50 of 101.5 ± 2.66, and 84.08±3.56 respectively. The hematological, biochemical, and tissue antioxidant parameters were reverted to normal levels in the treated animal groups. BC and Lupeol reduced the volume, weight, and viable cell count of the tumor; increased the life span of EAC tumor-bearing mice. Conclusionβ-Carotene and lupeol were found to be more effective in combination for the anti-proliferative activity. Thus combination techniques must be employed to find out the synergistic activity of the molecules.

PEGylation of genistein-loaded bovine serum albumin nanoparticles and its effect on in vitro cell viability and genotoxicity properties

Self-assembled bovine serum albumin nanoparticles loaded with the isoflavone genistein have shown apoptosis-mediated cytotoxicity against murine mammary adenocarcinoma F3II cells. Due to their protein nature and small particle size (13–15 nm), their parenteral administration could be affected by possible immunogenic reactions and rapid clearance from the bloodstream. To avoid these problems, PEGylation of the systems was achieved in this work by using a 30 kDa methoxy-polyethylene glycol carbonyl imidazole derivative through the reaction between the carbonyl imidazole group and the amino groups of Lys residues on the protein surface, which was confirmed by a 17% reduction in the available amino groups content measured by the o-phthaldialdehyde method. PEGylated isoforms were obtained, showing an increase of particle size from 13 to 15 nm to around 260 nm, and were purified by SEC-FPLC and characterized by SDS-PAGE, DLS and AFM techniques. The effect of PEGylation on BSAnp-Gen cytotoxicity and genotoxicity against F3II cells was evaluated in vitro by MTT assay, flow cytometry analysis and micronucleus assay. From the results, PEGylation produced an improvement of the biological properties of genistein-loaded nanoparticles in terms of cytotoxicity (lower IC50), not affecting the induction of apoptosis, decreasing the genotoxicity of the systems (less induction of micronucleus formation).

Anticancer potential of Marina Crystal Minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo

In vitro studies have shown that Marina Crystal Minerals (MCM), a crystallized mixture of minerals and trace elements from sea water, possesses apoptotic and immune modulatory effects in human breast cancer cells MDA-MB-231. The current study aimed to evaluate MCM’s anticancer effect in vivo against murine mammary adenocarcinoma cells and to explore its underlying mechanisms. Mice were inoculated intramuscularly with Ehrlich ascites carcinoma (EAC) cells, a breast adenocarcinoma. Tumors became palpable within 9 days. Tumor-bearing mice were injected with MCM intraperitoneally (IP) or intratumorally (IT) at a dose of 40 mg/kg BW for 6 days/week until day 28 post-inoculation. Tumor growth, cell cycle progression, cell cycle regulatory proteins, apoptosis, apoptotic regulatory markers, mitochondrial membrane potential (MMP), natural killer (NK) cell activity, and histopathological effects were investigated. Treatment with MCM reduced tumor volume by 49.4% for IP and 59.5% for IT injection. MCM induced cancer cell apoptosis, as indicated by a sub-G1 peak and confirmed by Annexin V/PI assay and histopathological examination. This was mediated by increased Bax expression, caspase-3 activation, decreased Bcl-2 expression, and MMP disruption. Furthermore, MCM treatment induced G1 cell cycle arrest, mediated through significantly increased expression of p53, p21, and p27 and decreased expression of cyclin D1 and PCNA in cancer cells. Finally, MCM treatment markedly enhanced NK cell cytotoxicity. MCM possesses chemopreventive potential to reduce tumor growth by suppressing cell proliferation, inducing apoptosis in EAC cells via a mitochondrial dependent pathway, and activating the immune system. Our results suggest MCM’s beneficial potential for treating breast adenocarcinoma.

Effect Of Shikimic Acid And Acetylsalicylic Acid On Biofilm Formation In Staphylococcus Epidermidis

The aim of the current study was to evaluate the activity of Shikimic acid (SA) and Acetylsalicylic acid (ASA) against S. epidermidis biofilms formation in different conditions. A total of (231) samples were collected from patients who attended different hospitals in Ramadi and Faluja cities during the period from December(2020) to the end of July (2021). Samples were cultured on blood, and manitol salt agar and identification was performed by the VITEK 2 system. Antibiofilm activity of SA was evaluated by two methods, modified crystal violet, and MTT assay. The SA was tested in five MIC (0.25 MIC, 0.5 MIC, MIC2, MIC, and 4 MIC) and was tested during different incubation periods (2 hr, 4 hr, 6 hr, 12 hr, and 24 hr), Shikimic acid gave highest percentage inhibition of biofilm formation was 93% at 12hr and ,94% at 24hr , while Acetyl salicylic acid was gave 85% at 12hr and 91% at 24hr incubation period . Then evaluated the synergistic activity of SA and ASA acid with ceftriaxone and azithromycin against biofilm formation also then determination of the antibiofilm activity of SA and ASA acid against bacteria adherence to Foley balloon latex catheter (FBLC) pieces by REMA assay.In addition, determine the efficacy of SA and ASA acid against cell adherence using murine mammary adenocarcinoma cell line (amn3)cells. The results showed high percentage inhibition of biofilm formation as the concentration of SA and ASA increased . In conclusion, SA and ASA have the potential to be further developed as a new antitumor agent due to their impressive antibiofilm action against bacteria. shikimic acid , acetyle salicylic acid were gave up regulation of atlE gene , 2 ( −ΔΔCT) was (2)for shikimic acid while (3)for acetyl salicylic acid , aap,icaA genes, all antibiofilm agents were gave down regulation result 2 ( −ΔΔCT) was (0.2)for shikimic acid while (0.1)for acetyl salicylic acid.

Cromolyn chitosan nanoparticles reverse the DNA methylation of RASSF1A and p16 genes and mitigate DNMT1 and METTL3 expression in breast cancer cell line and tumor xenograft model in mice

BackgroundDeveloping epigenetic drugs for breast cancer (BC) remains a novel therapeutic approach. Cromolyn is a mast cell stabilizer emerging as an anticancer drug; its encapsulation in chitosan nanoparticles (CSNPs) improves its effect and bioavailability. However, its effect on DNA and RNA methylation machineries has not been previously tackled. MethodsThe possible anticancer effect of cromolyn CSNPs and its potential as an epigenetic drug was investigated in vitro using MCF-7 human BC cell line and in vivo using Ehrlich ascites carcinoma-xenograft model in mice symbolizing murine mammary adenocarcinoma. Mice were injected with a single dose of Ehrlich ascites carcinoma cells subcutaneously for the induction of tumor mass, and then randomized into three groups: control, cromolyn CSNPs (equivalent to 5 mg cromolyn/kg, i.p.) and plain CSNPs twice/week for 2 weeks. ResultsCromolyn CSNPs showed prominent anticancer effect in MCF-7 cells by reducing the cell viability percent and enhancing DNA damage in the comet assay demonstrating its apoptotic actions. Mechanistically, cromolyn CSNPs influenced potential epigenetic processes through mitigating DNA methyltransferase 1 (DNMT1) expression, reversing the hypermethylation pattern of the tumor suppressor RASSF1A and p16 genes and attenuating the expression of the RNA N6-methyladenosine writer, methyltransferase-like 3 (METTL3). Cromolyn CSNPs diminished ERK1/2 phosphorylation, a possible arm influencing DNMT1 expression. In vivo, cromolyn CSNPs lessened the tumor volume and halted DNMT1 and METTL3 expression in Ehrlich carcinoma mice. ConclusionsCromolyn CSNPs have the premise as an epigenetic drug through inhibiting ERK1/2 phosphorylation/DNMT1/DNA methylation and possibly impacting the RNA methylation machinery via mitigating METTL3 expression.

Abstract 691: Antineoplastic effect of marina crystal minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo

Abstract Purpose: Marina crystal minerals (MCM) is a crystallized mixture of minerals and trace elements from sea water. Earlier in vitro studies have shown that MCM possesses apoptotic and immune modulatory effects in human breast cancer cells MDA-MB-231. The current study aimed to evaluate the anticancer effect of MCM in vivo against the growth of murine mammary adenocarcinoma cells and to explore the mechanisms underlying its effect. Materials and Methods: Mice were inoculated intramuscularly with Ehrlich ascites carcinoma (EAC) cells, a breast adenocarcinoma of mouse origin. The tumors grew and became palpable within 9 days post-inoculation. Tumor-bearing mice were injected with MCM intraperitoneally (IP) or intratumorally (IT) at a dose of 40 mg/kg BW for 6 days/week. Different parameters were evaluated, including tumor growth, cell cycle progression, cell cycle regulatory proteins, apoptosis, apoptotic regulatory markers, mitochondrial membrane potential (MMP), natural killer (NK) cell activity, and histopathological effects. Results: Treatment with MCM caused a reduction in tumor volume by 49.4% for IP and 59.5% for IT injection. MCM induced apoptosis in cancer cells, as indicated by the appearance of a sub-G1 peak and confirmed by Annexin V/PI assay and histopathological examination. This was mediated by increased Bax expression, caspase-3 activation, decreased Bcl-2 expression, and MMP disruption. Furthermore, MCM treatment induced G1 cell cycle arrest which was mediated through significantly increased expression of p53, p21, and p27 and decreased expression of Cyclin D1 and PCNA in cancer cells. Finally, MCM treatment markedly enhanced NK cell cytotoxicity. Conclusion: MCM possesses chemopreventive potential to reduce tumor growth by suppressing cell proliferation, inducing apoptosis in EAC cells via a mitochondrial dependent pathway, and activating the immune system. Our results suggest that MCM may have clinical implications for the treatment of breast adenocarcinoma in humans. MCM was provided by the Foundation for Basic Research Institute of Oncology, Japan. Citation Format: Nariman K. Badr El-Din, Mai Alaa El-Dein, Mamdooh Ghoneum. Antineoplastic effect of marina crystal minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 691.

Phenotypic Changes in Mammary Adenocarcinoma (4T1) cells In Vitro after Treatment with Carcinosinum.

The present study aimed to identify possible phenotypic changes in 4T1 (murine mammary adenocarcinoma) cells in vitro, including viability, HER-2 (human epidermal growth factor receptor-type 2) expression, and metastatic potential, after treatment with Carcinosinum in different homeopathic dilutions (12cH, 30cH, 200cH) shaken mechanically in pure, sterile, water from a commercial stock dilution. Treated cells were cultured in R10 medium, using 24-well plates, 105 cells per well, and treated with vehicle, Carcinosinum 12cH, 30cH or 200cH; untreated cells were used as the baseline control. After 24 hours of treatment, the percentage of apoptotic cells was analyzed by annexin V. Cell morphology was evaluated by microscopy after hematoxylin-eosin and Giemsa staining, whilst HER-2 expression was assessed using immunocytochemistry. The metastatic potential was determined by the expression and activity of the enzyme matrix metalloproteinase 9 (MMP-9) using zymography. The cytokine profile was established using the cytometric bead array method. Treatment of 4T1 cells in vitro with Carcinosinum 30cH produced an increase in the number of annexin V-positive cells (apoptosis) and decreased expression of proactivated MMP-9. Cells treated with Carcinosinum 200cH presented hyper-expression of HER-2 on the plasma membrane, identified by immunocytochemistry. There were no differences in cytokine production among treatments. The data show promising results for Carcinosinum 30cH in vitro, but in vivo studies are also required to evaluate the role of tumor microenvironment in its effects.

The utilities of pyrazolines encouraged synthesis of a new pyrazoline derivative via ring closure of chalcone, for optimistic neurodegenerative applications

Pyrazolines and their derivatives have been extensively studied as coordinated ligands of high potential applications in diverse chemical and biological systems. This work explores some methods of synthesis of pyrazolines, such as the preparation of pyrazole derivatives via chalcones. It also demonstrates that 2-pyrazoline complexes were biologically active and have had a range of clinical applications. Palladium (II) complex of pyrazole was active as antitumor when tested against murine mammary adenocarcinoma (LM3). Copper (II) and Cobalt (II) complex are biologically active in the living system as biomolecules or co factors. Based on these information, we tried here to synthesis a new 2-pyrazoline from (E)-3-(4-bromophenyl)-1-(pyridin-2-yl)prop-2-en-1-one. The newly synthesized pyrazoline was characterized using mass spectroscopy, nucleic magnetic resonance spectroscopy NMR and Fourier transform infrared spectroscopy FTIR. The characterization results showed that 2-pyrazoline has successfully synthesized. The microanalyses (C.H.N.S), GC-MS, H and 13C NMR and FT-IR spectra confirmed the formation of 2-pyrazoline ring with substitution at N1,C-3 and C-5 and the spin-spin coupling constants (J) for the multiple peaks at H NMR spectra pointed to the de shielded aromatic protons in α and β protons of the prepared chalcone. Interestingly, some new family of pyrazoles that have isosteres of Zonisamide have previously showed activity toward treating neuroglial disorders such as epilepsy and autism. Thus, our synthesized pyrazole could have the potency to moderate some neurodegenerative disorders. It could negatively interact with some neurotransmitters through hydrogen bonding and electrostatic interactions with the amino and carboxylic ends of the functional ends of the neurotransmitters. This promising trend by the promising candidate 2- pyrazoline should have further investigations.

Investigation of the Lethal Effect of Purified Arginine Deiminase Purified from Lactobacillusplantarum p5 on Murine Mammary Adenocarcinoma and Vero cell Lines.

Cancer is one of the most serious diseases facing humanity; accordingly, it is urgent to find a cure that is rarely harmful to the patient as much as possible. It has been approved that arginine deiminase (ADI) can hydrolyze the plasma arginine to citrulline. This hydrolysis activity and reduction in the amount of intercellular arginine suppress lipopolysaccharide-induced nitric oxide synthesis. On the other hand, arginine depletion arrests the cell cycle at the G1 phase; therefore, ADI has been considered a powerful anticancer agent. The current study aimed to investigate the lethal effects of ADI purified from the Lactobacillus plantarum p5 strain on murine mammary adenocarcinoma and Vero cell lines. Anti-proliferative activity of ADI against murine mammary adenocarcinoma) AMN3) cell line was evaluated after different incubation times (3, 6, 24, 48, and 72 h) of exposure to 1 µg/mL of ADI, compared to Vero (non-cancer cell line) transformed cell line with same conditions. The autophagy process in cancer cells was recognized after three hours of incubation with ADI which was clearly observed in the AMN3 cell line under an inverted microscope. The first stages of the programmed cell death (apoptosis) pathway were only observed in AMN3 cells after 24 h of incubation with ADI, and this process continued with the time until they reached the last stages of apoptosis after 72 h of incubation. The results of the current study showed that the AMN3 cell line was auxotrophic for arginine because it could not produce it in the presence of enzyme which had a robust activity to kill these cancer cells; however, Vero non-cancer cell line survived in the presence of ADI because it had the ability to produce arginine.

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.

Tumorigenic effect mediated by ROS/eicosanoids and their regulation on TP53 expression in a murine mammary gland adenocarcinoma

The aim of this study was to investigate the in vivo and in vitro effects of dietary ω-6 and ω-3 polyunsaturated fatty acids (PUFAs) and their derivatives on the expression of TP53 and their relationship with cellular proliferation and death in a murine mammary adenocarcinoma model. BALB/c mice were divided in three diet groups: chia oil (ChO) rich in ω-3, corn oil (CO) rich in ω-6/ω-3 and safflower oil (SO) rich in ω-6 and subcutaneously inoculated with LMM3 mammary tumor cell line. Results demonstrated that diets with higher concentration of omega-6 PUFAs induced an increment of linoleic and arachidonic acid on tumor cell membranes increasing ROS liberation, 12(S)-HHT generation, TP53, Ki67 expression and cell proliferation. However, diets enriched with high content in omega-3 PUFAs induced higher tumor cell apoptosis and tumor infiltration of CD3+ lymphocytes, lowest cell viability and proliferation. Dietary omega-3 PUFAs nutritional intervention can be used as a potential preventative strategy to inhibit the molecular signaling pathways involved in the mammary tumor growth process as the TP53.

Nanodispersions of magnetic poly(vinyl pivalate) for biomedical applications: Synthesis and in vitro evaluation of its cytotoxicity in cancer cells

The development of polymeric nanomaterials, combining properties of polymers and superparamagnetic nanoparticles, has potential for biomedical applications, such as treatment of tumors through intravascular embolization and hyperthermia. Therefore, this work aims at development of a new magnetopolymeric fluid formed by magnetic nanoparticles of iron oxide (Fe3O4) coated with chemically modified oleic acid and which were uniformly dispersed in spherical nanoparticles of poly(vinyl pivalate) through in situ polymerization process via miniemulsion, synthesized using from 5.0 to 20.0 wt% of magnetic oxide in relation to vinyl pivalate content. Samples were characterized by infrared absorption vibration spectroscopy, thermogravimetric analysis, dynamic light scattering, differential scanning calorimetry, X-ray diffraction, transmission electron microscopy, Mössbauer spectroscopy, Raman spectroscopy, vibrating sample magnetometer and magnetic induction heating test. Cytotoxicity was evaluated in vitro in B16F10 melanoma cancer cells, murine mammary adenocarcinoma (4T1-luciferase), NIH-3T3 fibroblast cells and human keratinocytes (HaCat). The magnetopolymeric fluids exhibited maximum conversions of approximately 82 %, hydrodynamic diameter of 180 nm, spherical morphology, preserved superparamagnetic behavior and glass transition temperature ranging from 56.2 to 62.5 °C. Cell viability tests demonstrated that nanocomposites presented low cytotoxicity to tumor and non-tumor cells at concentrations studied. These results suggest potential of magnetopolymeric fluids as an embolic agent for selective and complete obstruction of blood vessels of different sizes and capacity for treatment by hyperthermia.

Molecular changes associated with the anticancer effect of sulforaphane against Ehrlich solid tumour in mice.

The anticancer effect of sulforaphane (SFN) is mediated by several signalling pathways. However, little is known regarding the underlying mechanism in Ehrlich solid tumours (ESTs) in mice. This study was conducted to determine molecular changes associated with the anticancer effect of SFN and to compare its preventive (cotreatment) and therapeutic (posttreatment) effects. Ehrlich (murine mammary adenocarcinoma) solid tumour was selected and changes in the gene expression were determined in tumour tissues by the real-time polymerase chain reaction. The results showed that SFN increased the expression of the oxidative stress gene NrF2 and its downstream targets (HO1 and CAT). Conversely, SFN administration decreased the expression of the epigenesis-related genes (HDAC1 and DNMT1)and inflammation-related genes (TNFa, NFkB and Cox2). Overall, SFN cotreatment presented notable molecular changes than the posttreatment strategy. These data suggest that molecular changes associated with the anticancer effects of SFN against EST involved induction of oxidative stress, inhibition of inflammationand epigenetic modifications.

Analysis of phosphorous content in cancer tissue by synchrotron micro-XRF

In the present work, an analytical procedure for spatial quantification of phosphorous in cancerous tissues by micro-XRF was developed. This new methodology based on fundamental parameter approach consists of evaluating the incoming microbeam excitation x-ray spectrum on the sample and the matrix effects. The former was determined through a spectral reconstruction technique, based on the scattering pattern of a thin standard plastic material and the latter with the assistance of two transmission monitor ionization chambers. The procedure was applied on fifteen samples of murine gland mammary adenocarcinoma fixed in paraffin. Slices of 30 μm of these samples were scanned at air atmosphere in areas of 5 mm × 5 mm with a spatial resolution of 20 μm. The acquisition time per pixel was 5s allowing a detection limit for phosphorous of 240 ppm w/w. The results showed that the technique is able to detect the elevation of the phosphorous content in tumour tissues, as it was previously reported in the literature using other techniques. In addition, the spatial resolution of the technique allowed associating this increment with areas composed mainly by active tumour cells that were previously identified by Haematoxylin staining. In these areas, phosphorous concentrations showed spatial variations probably associated with tumour metabolism. Thus, micro-XRF offers valuable information of P spatial distribution that could be useful to identify active tumour cells as well as to study of tumour growth and progression.

Enhanced in vivo targeting of estrogen receptor alpha signaling in murine mammary adenocarcinoma by nilotinib/rosuvastatin novel combination

The development of resistance to endocrine therapy of estrogen receptor alpha (ERα)-positive breast cancer is inevitable, necessitating the introduction of alternative treatment strategies. Therefore, the current study was carried out to investigate the in vivo efficacy and tolerability of nilotinib/rosuvastatin novel combination against ERα-positive breast carcinoma. Results showed that treatment of tumor-bearing mice with nilotinib/rosuvastatin exerted a significant antitumor activity. Mechanistically, the combination treatment efficiently inhibited the in vivo ERα protein expression, whereas ERα mRNA levels were unaffected suggesting a posttranslational regulation. In addition, the combination treatment markedly downregulated the expression of two ERα downstream target genes: C3 and pS2 confirming the inhibition of ERα signaling in vivo. Further, nilotinib/rosuvastatin combination strongly induced apoptosis evidenced by a marked caspase-3 cleavage and downregulation of tumor nitric oxide levels. Moreover, histopathology showed significant declines in mitotic figures and tumor giant cells implying the in vivo capability of the combination treatment to interfere with cancer cell proliferation and persistence. Of note, the combination treatment abrogated nilotinib-induced hypercholesterolemia and did not adversely affect the liver function or body weight. Overall, the present study provided evidences that warrant further assessment of nilotinib/rosuvastatin combination as an alternative therapeutic modality for ERα-positive breast cancer.

In vitro Treatment of a Murine Mammary Adenocarcinoma Cell Line with Recombinant Trypanosoma cruzi Calreticulin Promotes Immunogenicity and Phagocytosis

American Trypanosomiasis, a parasitic disease produced by Trypanosoma cruzi (T. cruzi), endemic in Latin America, infects about 6 million people. During the chronic stage of the infection, approximately 30% of infected people will develop Chagas Disease, the clinical manifestation. Few decades ago it was reported that, during the chronic stage, the parasite interferes with the development of solid tumors. However, the identification of parasite molecules responsible for such effects remained elusive. Years later, we described T.cruzi Calreticulin (TcCalr), an endoplasmic reticulum resident chaperone that infective trypomastigotes translocate to the parasite exterior, where it displays anticomplement activities. Most likely, at least some of these activities are related with the antitumor properties of TcCalr, as shown in in vitro, ex vivo, in ovum, and in vivo models. In this context we, we have seen that in vivo subcutaneous peritumoral inoculation of rTcCalr enhances local infiltration of T cells and slows tumor development. Based on these precedents, we propose that in vitro treatment of a mammary adenocarcinoma (TA3 cell line) with rTcCalr, will enhance tumor immunogenicity. In agreement with this proposal, we have shown that: i). rTcCalr binds to TA3 cells in a concentration-dependent fashion, ii). C1q binds to TA3 cells in an rTcCalr-dependent fashion, confirmed by the reversion attained using anti-TcS (a central TcCalr domain that binds C1) F(ab’)2 antibody fragments, iii). incubation of TA3 cells with rTcCalr, promotes cell phagocytosis by murine macrophages and, iv). rTcCalr decreases the membrane expression of MHC class II, m-Dectin-1, Galectin-9 and PD-L1, while increasing the expression of Rae-1γ. In synthesis, herein we show that in vitro treatment of a murine mammary adenocarcinoma with rTcCalr enhances phagocytosis and modulates the expression of a variety of membrane molecules that correlates with increased tumor immunogenicity.