Therapeutic Role of Secondary Metabolites from Probiotic Strains for Ehrlich Solid Tumors in Mice.

This study aimed to screen the bioactive components in Streptococcus equinus WC1 (SE-WC1) and Limosilactobacillus reuteri GM4 (LR-GM4) and estimate the therapeutic role in Ehrlich solid tumors (EST) mice model. Forty-four male albino EST mice were assigned into 7 groups and treated daily for 2 weeks, including the EST group, the EST mice that received SE-WC1 at a low or a high dose (0.5 ml *106 or 0.5 ml *108 cfu), the EST mice that received LR-GM4 at the low or the high dose (0.5 ml *106 or 0.5 ml *108 cfu), and the EST mice that received SE-WC1 plus LR-GM4 at the low or the high dose. Tumors were harvested, weighed, examined, and used for the determination of apoptosis-related gene expression. Samples of the intestine, liver, and kidney were gathered for histological examination. The GC-MS identified 24 and 36 bioactive compounds in SE-WC1 and LR-GM4, respectively. The main compound in SE-WC1 was lupeol; however, the main compound in LR-GM4 was retinaldehyde. EST mice showed disturbances in Bcl-2, Bax, and p53 mRNA expression along with histological changes in the intestine, liver, and kidney. Administration of both bacterial strains reduced the tumor weight, alleviated the disturbances in the gene expression, and improved the histological structure of the intestine, liver, and kidney in a dose-dependent. Moreover, LR-GM4 was more effective than SE-WC1 due to its higher content of bioactive compounds. It could be concluded that these strains of probiotics are promising for the treatment of solid tumors.

Evaluation of MMR live attenuated vaccine oncolytic potential using Ehrlich ascites carcinoma in a murine model.

MMR vaccine is a common vaccine that contains oncolytic viruses (Measles, Mumps, and Rubella) and could be used as a potential anti-cancer treatment. In this study, we assessed the anti-tumor activity of the MMR vaccine against Ehrlich ascites carcinoma (EAC) solid tumor induced in mice. The in vitro assay showed that vaccine IC50 in EAC was approximately 200 CCID50. The vaccine was intratumorally administrated twice weekly in EAC-bearing mice. The antitumor response of the vaccine was measured by tumor growth, survival rate, histopathologic examination, flow cytometry analysis, and body biochemical parameters. The MMR vaccine demonstrated a substantial reduction of tumor growth and prolongation of life span as well. The proliferation marker was significantly lower in the vaccine-treated group. Moreover, the apoptosis key parameter Casp-3 was also higher in the vaccine-treated group. The vaccine somewhat restored the deterioration of the biochemical parameters (LDH, GOT, GPT, MDA, NO, and PON-1) in the tumor-bearing mice. Finally, this study indicated the potential antitumor effect of MMR vaccine via anti­proliferative, apoptotic activities, and modulating the antioxidant parameters. This study opens a new field of inquiry for future research on the vaccine's anti-cancer properties.

Combination of selol nanocapsules and magnetic hyperthermia hinders breast tumor growth in aged mice after a short-time treatment.

Short time treatment with reduced dosages of selol-loaded PLGA nanocapsules (NcSel) combined with magnetic hyperthermia (MHT) is evaluated in aged Erhlich tumor-bearing mice. Clinical, hematological, biochemical, genotoxic and histopathological parameters are assessed during 7 d treatment with NcSel and MHT, separately or combined. The time evolution of the tumor volume is successfully modeled using the logistic mathematical model. The combined therapy comprising NcSel and MHT is able to hinder primary tumor growth and a case of complete tumor remission is recorded. Moreover, no metastasis was diagnosed and the adverse effects are negligible. NcSel plus MHT may represent an effective and safe alternative to cancer control in aged patients. Future clinical trials are encouraged.

Graphene Oxide Theranostic Effect: Conjugation of Photothermal and Photodynamic Therapies Based on an in vivo Demonstration.

INTRODUCTION: Cancer is the second leading cause of death globally and is responsible, where about 1 in 6 deaths in the world. Therefore, there is a need to develop effective antitumor agents that are targeted only to the specific site of the tumor to improve the efficiency of cancer diagnosis and treatment and, consequently, limit the unwanted systemic side effects currently obtained by the use of chemotherapeutic agents. In this context, due to its unique physical and chemical properties of graphene oxide (GO), it has attracted interest in biomedicine for cancer therapy. METHODS: In this study, we report the in vivo application of nanocomposites based on Graphene Oxide (nc-GO) with surface modified with PEG-folic acid, Rhodamine B and Indocyanine Green. In addition to displaying red fluorescence spectra Rhodamine B as the fluorescent label), in vivo experiments were performed using nc-GO to apply Photodynamic Therapy (PDT) and Photothermal Therapy (PTT) in the treatment of Ehrlich tumors in mice using NIR light (808 nm 1.8 W/cm2). RESULTS: This study based on fluorescence images was performed in the tumor in order to obtain the highest concentration of nc-GO in the tumor as a function of time (time after intraperitoneal injection). The time obtained was used for the efficient treatment of the tumor by PDT/PTT. DISCUSSION: The current study shows an example of successful using nc-GO nanocomposites as a theranostic nanomedicine to perform simultaneously in vivo fluorescence diagnostic as well as combined PDT-PTT effects for cancer treatments.

Pro-apoptotic and anti-neoplastic impact of luteolin on solid Ehrlich carcinoma.bearing mice exposed to gamma radiation.

BACKGROUND: Cancer remains a major health issue and the second foremost root of morbidity worldwide behind cardiovascular diseases. Apoptosis had linked to the eradication of possibly malignant cells, hyperplasia, and tumor progression. OBJECTIVE: The present study is an endeavor to evaluate the influence of luteolin, a modifier to apoptotic regulator on the tumor growth and the tumor cell sensitivity to ionizing radiation in Ehrlich solid tumor-bearing mice (E). MATERIALS AND METHODS: Mice were immunized with Ehrlich carcinoma cells (2.5 × 106 cells/mouse), received consecutive equal doses of luteolin, 1.25 mg/mouse/day and exposed to 6.5 Gy of whole-body gamma irradiation (0.46 Gy/min). RESULTS: Luteolin markedly suppresses the developing of tumor in E mice group or mice which bearing tumor with exposure to radiation (E + R group) which has collimated with significant inhibition in protein expression of inflammatory molecules cyclooxygenase 2 and the concentration of (prostaglandin E2). Also, matrix metalloproteinase-2, 9 proteins concentrations significantly decreased with amelioration in apoptotic regulators (Caspase-3 and Granzyme-B activities). The expression of signal transducer and activator of transcription (STAT) and tumor necrosis factor-alpha genes meliorated significantly. Besides, the level of oxidant/antioxidant (reduced glutathione/malondialdehyde) markedly improved. Obviously, the most reduction of changes in all measured parameters has appeared in tumor bearing mice, injected with luteolin and exposed to gamma radiation (E + Luteolin + R group). CONCLUSION: It could be suggested that luteolin has a potential beneficial effect against cancer. This could be due to its ability on the induction of apoptosis, inhibition of inflammatory response, downregulation of angiogenic factors as well as increase sensitivity of tumor cells to gamma radiation.

Cytotoxicity and genotoxicity of gold nanorods assisted photothermal therapy against Ehrlich carcinoma in-vivo.

AIM: Preparation of pegylated gold nanorods (PEG-AuNRs) that are capable of converting near infrared (NIR) light into heat. Evaluation of cancer therapeutic efficacy and long-term toxicity of the proposed photothermal therapy in comparison with other conventional modalities. MATERIALS AND METHODS: Prepared PEG-AuNRs were characterized by measuring their absorption spectra, zeta potential, and transmission electron microscope (TEM). Cancer therapeutic efficacy was assessed by monitoring tumor growth, measuring DNA damage and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in addition to examining tumor histopathology. Further analysis concerning the toxicity of all the proposed treatment modalities was also assessed by evaluating the cytotoxicity and genotoxicity in liver and kidney tissues. KEY FINDINGS: The results demonstrated that both photothermal therapy (PEG-AuNRs + NIR laser) and chemotherapy (cisplatin) have higher efficacy in diminishing Ehrlich tumor growth with significance DNA damage over the other treatment modalities. Concerning the biosafety issue, mice treated photothermally exhibited lower MDA level and higher SOD activity in liver and kidney tissues compared with other treated groups. DNA damage represented by tail moment and olive moment of kidney tissues exhibited lower values for photothermal treated group and higher values for cisplatin treated group. SIGNIFICANCE: Photothermal therapy (PEG-AuNRs + NIR laser) potentiates higher efficacy in treating Ehrlich tumor with minimum toxicity in comparison with other conventional treatment modalities.

A novel kefir product (PFT) inhibits Ehrlich ascites carcinoma in mice via induction of apoptosis and immunomodulation.

BACKGROUND: The popularity of fermented foods such as kefir, kuniss, and tofu has been greatly increasing over the past several decades, and the ability of probiotic bacteria to exert anticancer effects has recently become the focus of research. While we have recently demonstrated the ability of the novel kefir product PFT (Probiotics Fermentation Technology) to exert anticancer effects in vitro, here we demonstrate its ability to inhibit Ehrlich ascites carcinoma (EAC) in mice. METHODS: Mice were inoculated intramuscularly with EAC cells to develop solid tumors. PFT was administered orally (2 g/kg/day) to mice 6 days/week, either 2 days before tumor cell inoculation or 9 days after inoculation to mice bearing solid tumors. Tumor growth, blood lymphocyte levels, cell cycle progression, apoptosis, apoptotic regulator expression, TNF-α expression, changes in mitochondrial membrane potential (MMP), PCNA, and CD4+ and CD8+ T cells in tumor cells were quantitatively evaluated by flow cytometry or RT-PCR. Further studies in vitro were carried out where EAC cells along with several other human cancer cell lines were cultured in the presence of PFT (0-5 mg/mL). Percent cell viability and IC50 was estimated by MTT assay. RESULTS: Our data shows that PFT exerts the following: 1) inhibition of tumor incidence and tumor growth; 2) inhibition of cellular proliferation via a marked decrease in the expression of tumor marker PCNA; 3) arrest of the tumor cell cycle in the sub-G0/G1 phase, signifying apoptosis; 4) induction of apoptosis in cancer cells via a mitochondrial-dependent pathway as indicated by the up-regulation of p53 expression, increased Bax/Bcl-2 ratio, decrease in the polarization of MMP, and caspase-3 activation; and 5) immunomodulation with an increase in the number of infiltrating CD4+ and CD8+ T cells and an enhancement of TNF-α expression within the tumor. CONCLUSIONS: PFT reduces tumor incidence and tumor growth in mice with EAC by inducing apoptosis in EAC cells via the mitochondrial-dependent pathway, suppressing cancer cell proliferation, and stimulating the immune system. PFT may be a useful agent for cancer prevention.

Magnetic fields enhance the anti-tumor efficacy of low dose cisplatin and reduce the nephrotoxicity.

The present work was to examine a combination of therapy for a low dose of cisplatin and a magnetic field (MF) on Ehrlich carcinoma-bearing mice. In this study, a total of 50 BALB/C female mice were equally distributed into five groups. Mice from the control group did not receive MF or cisplatin. The low and high dose cisplatin groups were injected intraperitoneal (i.p.) with 3 and 6 mg/kg cisplatin, respectively, on the experimental days (1, 4, and 8). Mice group of cisplatin + MF was injected with a low dose of cisplatin followed by MF exposure (50 Hz, 50 mT), and the MF group was exposed to MF only. The impact of MF and cisplatin on the tumor and kidney were evaluated by measuring superoxide dismutase (SOD) activity, malondialdehyde (MDA) and glutathione (GSH) levels, DNA injury (comet assay), histopathological investigation of tissues, and tumor progress. The results suggested that the combination of a low dose of cisplatin with MF was significantly elevated in MDA levels, reduced SOD activity, and GSH levels. Furthermore, it caused a rise in comet parameters and inhibition in tumor growth. These results showed that MF enhances the therapeutic efficacy of low cisplatin doses and reduces nephrotoxicity.

Fatty Acid Content and Tumor Growth Changes in Mice After Exposure to Extremely High-Frequency Electromagnetic Radiation and Consumption of N-3 Fatty Acids.

The topical problem is to find new, more effective and safe treatments for cancer. The purpose of the present work was to study the combined effects of low-intensity extremely high-frequency electromagnetic radiation (EHF EMR) and consumption of n-3 polyunsaturated fatty acids (PUFAs) on tumor growth and the content of FAs in the thymus and tumor tissue in mice. Fatty acid composition was determined using gas chromatography. Exposure of tumor-bearing mice with solid Ehrlich carcinoma to EHF EMR with effective parameters (42.2 GHz, 0.1 mW/cm2, 20 min daily for 5 consecutive days beginning on the first day after the tumor inoculation) led to delaying the tumor growth and restored the content of almost all FAs in thymic tissue to the level of intact animals. Animal intake of the preparation enriched with n-3 PUFAs increased the content of n-3 PUFAs in thymic tissue significantly, but did not affect the tumor growth, even in combination with EHF EMR exposure. Combined action of EHF EMR exposure and n-3 preparation promoted recovery of thymus weight in tumor-bearing animals. The data obtained assume a complex interaction between the immune system and the tumor, and the important role of FAs in the regulation of this interaction.

The effect of peritoneal dialysis with alkaline dialysate in peritonitis carcinomatosis: an experimental study in mice.

OBJECTIVE: The aim of this study was to neutralize acidic pH using an alkaline dialysate for continuous ambulatory peritoneal dialysis (CAPD) in mice with peritoneal carcinomatosis (PC) and to investigate the change of the pH level in the acidic fluid along with its effects on liver oxidative stress, liver and kidney histopathology and the lifespan of the body. MATERIALS AND METHODS: A total of 38 mice were randomly divided into 4 groups.PC development was inhibited by intraperitoneal injection of Ehrlich tumor cells in all mice in each group. RESULTS: In the group-1 receiving CAPD, the pH levels of acidic liquid were higher; and the levels of liver TBARS were lower with higher reduced glutathione levels. Histopathological damage in group-1 was less than in group-2. In Group 3 receiving CAPD, the average lifespan extended by 10.4%. The average lifespan extended by 26.1%. CONCLUSION: This study indicated that applying CAPD with alkaline dialysate in PC contributed to the neutralization of acidosis of the intraperitoneal acid structure;had favorable effects on oxidative stress markers in liver tissue; prevented histopathological injury in liver and kidney tissues, and extended the life span of the body in mice. As this is a simple, inexpensive, and easily available method, larger studies are warranted to evaluate its effects.

[Reprogrammed M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 extends lifespan of mice with experimental carcinoma].

Objective. Reprogramming of M1 macrophage phenotype with inhibited M2 phenotype transcription factors, such as STAT3, STAT6 and SMAD and assess their impact on the development of Ehrlich carcinoma (EC) in vitro and in vivo. Methods. Tumor growth in vitro was initiated by addition of EC cells in RPMI-1640 culture medium and in vivo by intraperitoneal of EC cell injection into mice. Results. It was found that M1-STAT3/6- SMAD3 macrophages have a pronounced anti-tumor effect in vitro, and in vivo, which was greater than anti-tumor effects of M1, M1-STAT 3/6, M1-SMAD3 macrophages and cisplatin. Conclusion. M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 effectively restrict tumor growth. The findings justify the development of new anti-tumor cell therapy technology.

Assessment of electrochemotherapy effects on the development of Ehrlich solid tumor in swiss mice using a novel electroporator device / Avaliação dos efeitos da eletroquimioterapia sobre o desenvolvimento do tumor sólido de Ehrlich em camundongos Swiss utilizando um novo eletroporador

Electrochemotherapy is a local anticancer treatment in which non-permeant chemotherapeutic drugs are associated with electric pulses of well-established parameters. The electric pulses cause pores to open on the plasma membrane and facilitate drug transport, enhancing cytotoxicity and reducing side effects. Assessment of electrochemotherapy effects on Ehrlich solid tumor development in this work aims to evaluate in vivo usage of the electroporator device developed by the Department of Electrical Engineering of Engineering School of UFMG. Therefore, 40 Swiss mice were inoculated with Ehrlich tumor cells, and developed the tumor in solid form. After 21 days, mice were subjected to specific treatment protocols (control, bleomycin, electric pulses and electrochemotherapy); 17 days later they were euthanized and the tumors collected for histopathology analysis. Electrochemotherapy induced discrete weight loss and an inflammatory response in the tumor, which was not seen on the other treatment groups. Bleomycin alone induced necrosis. Both groups showed lower cellular proliferation rates. From this study, it was concluded that the animals tolerated electrochemotherapy treatment under anesthesia and the electroporator device developed by the Engineering School of UFMG was adequate when used in an electrochemotherapy protocol.(AU)

Eletroquimioterapia é uma modalidade de tratamento local contra o câncer em que a administração de quimioterápicos não penetrantes à membrana plasmática é associada à aplicação de pulsos elétricos com parâmetros bem estabelecidos, que abrem poros na membrana plasmática e facilitam a entrada desses fármacos nas células, aumentando sua citotoxicidade e reduzindo efeitos colaterais. A avaliação dos efeitos da eletroquimioterapia sobre o desenvolvimento do tumor sólido de Ehrlich em camundongos Swiss neste trabalho teve como objetivo testar o uso in vivo do aparelho eletroporador desenvolvido pelo Departamento de Engenharia Elétrica da Escola de Engenharia da UFMG. Para tanto, foram utilizados 40 camundongos fêmeas da linhagem Swiss, nos quais foram inoculadas células de tumor de Ehrlich, para o desenvolvimento do tumor na forma sólida. Após 21 dias, os camundongos foram submetidos ao protocolo de tratamento específico (controle, bleomicina, pulsos elétricos e eletroquimioterapia); 17 dias depois foram eutanasiados e seus tumores coletados para análise histopatológica e imuno-histoquímica. A eletroquimioterapia induziu perda de peso discreta e uma resposta inflamatória no tumor que não foi observada nos outros grupos. O grupo bleomicina apresentou maior porcentagem de necrose. Ambos os grupos apresentaram menor índice de proliferação celular. Com este estudo, pode-se concluir que o tratamento sob anestesia foi bem tolerado pelos animais e que o aparelho eletroporador desenvolvido pela Escola de Engenharia da UFMG é adequado para utilização em um protocolo de eletroquimioterapia.(AU)

Combination of bacteriolytic therapy with magnetic field for Ehrlich tumour treatment.

Referable to the limited response of the current available cancer treatment modalities, new effective cancer fighting treatments are needed. This work investigates the efficiency of intratumoural injection of Pseudomonas aeruginosa bacteria combined with a local tumour exposure to extremely low frequency square pulsed magnetic field (ELF SPMF) in the mouse Ehrlich tumour. 64 Ehrlich ascites tumour-implanted female albino BALB/C mice were equally split up into 4 groups. Group 1 (GP1) was the positive control group. Group 2 (GP2) received a single intratumoural injection of P. aeruginosa bacteria. Group 3 (GP3) was exposed to ELF SPMF for tumour local exposure. Group 4 (GP4) was treated with P. aeruginosa intratumoural injection followed by local exposure of the tumour to ELF SPMF. Treatment monitoring was evaluated using ultrastructural examination, flow cytometry analysis in addition to the measurement of tumour dielectric properties. Tumour cell apoptosis was obvious in GP2 and GP4, but, with higher severity and percentages in GP2. Tumour biophysical properties revealed a significant increase in static conductivity σS of GP2, and decreases in dielectric increment Δɛ´of both GP2 and GP4 compared to the GP1. Unfortunately, GP2 mice showed severe signs of toxicity. We advocate the utilization of the combination of P. aeruginosa and SPMF to yield the most effective antitumour agent with less bacteria-related toxicity.

M3 Macrophages Stop Division of Tumor Cells In Vitro and Extend Survival of Mice with Ehrlich Ascites Carcinoma.

BACKGROUND M1 macrophages target tumor cells. However, many tumors produce anti-inflammatory cytokines, which reprogram the anti-tumor M1 macrophages into the pro-tumor M2 macrophages. We have hypothesized that the problem of pro-tumor macrophage reprogramming could be solved by using a special M3 switch phenotype. The M3 macrophages, in contrast to the M1 macrophages, should respond to anti-inflammatory cytokines by increasing production of pro-inflammatory cytokines to retain its anti-tumor properties. Objectives of the study were to form an M3 switch phenotype in vitro and to evaluate the effect of M3 macrophages on growth of Ehrlich ascites carcinoma (EAC) in vitro and in vivo. MATERIAL AND METHODS Tumor growth was initiated by an intraperitoneal injection of EAC cells into C57BL/6J mice. RESULTS 1) The M3 switch phenotype can be programed by activation of M1-reprogramming pathways with simultaneous inhibition of the M2 phenotype transcription factors, STAT3, STAT6, and/or SMAD3. 2) M3 macrophages exerted an anti-tumor effect both in vitro and in vivo, which was superior to anti-tumor effects of cisplatin or M1 macrophages. 3) The anti-tumor effect of M3 macrophages was due to their anti-proliferative effect. CONCLUSIONS Development of new biotechnologies for restriction of tumor growth using in vitro reprogrammed M3 macrophages is very promising.

Noninvasive Microsurgery Using Aptamer-Functionalized Magnetic Microdisks for Tumor Cell Eradication.

Magnetomechanical cell disruption using nano- and microsized structures is a promising biomedical technology used for noninvasive elimination of diseased cells. It applies alternating magnetic field (AMF) for ferromagnetic microdisks making them oscillate and causing cell membrane disruption with cell death followed by apoptosis. In this study, we functionalized the magnetic microdisks with cell-binding DNA aptamers and guided the microdisks to recognize cancerous cells in a mouse tumor in vivo. Only 10 min of the treatment with a 100 Hz AMF was enough to eliminate cancer cells from a malignant tumor. Our results demonstrate a good perspective of using aptamer-modified magnetic microdisks for noninvasive microsurgery for tumors.

Assessment of DNA damage in Ehlrich carcinoma after treatment with doxorubicin encapsulated in nanoscales thermosensitive liposomes in combination with localized hyperthermia.

Nanoscales thermosensitive liposomes (TSL) composed of synthetic lipids (dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine), were used for doxorubicin encapsulation with 70% encapsulated efficiency. The liposomes were characterized by dynamic light scattering, transmission electron microscopy and turbidity method. Additionally, the liposomes exhibited a significant release of doxorubicin (Dox) by 60% within 5 min at 42°C. To assess the therapeutic efficacy of Dox in combination with hyperthermia, Dox free and encapsulated TSL were administered directly to Ehrlich tumor bearing mice at 1 mg/kg dose. Immediately after the drug administration, hyperthermia was applied to mention the temperature inside the tumor site at 42°C either for 5 min and 30 min. The results indicate a significant increase in the percent of apoptotic and necrotic cells in the treated group. Moreover, disrupts the integrity and the amount of intact DNA in tumor cells. In conclusion, Dox and hyperthermia may serve as a useful targeted drug delivery system for management of Ehrlich carcinoma.

Features of the Antitumor Effect of Vaccinia Virus Lister Strain.

Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, "natural" oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells.

Polarization of macrophages towards M1 phenotype by a combination of 2-deoxy-d-glucose and radiation: Implications for tumor therapy.

2-Deoxy-d-glucose (2-DG) has been found to enhance the cytotoxicity of ionizing radiation and chemotherapeutic drugs in several tumor cell lines in vitro. Systemic administration of 2-DG together with localized irradiation of the tumor leads to tumor regression and cure (disease free survival), which correlate with the differential levels of anti-tumor immunity observed in Ehrlich ascites tumor (EAT) bearing mice. Macrophages being a major player of the innate immune system, we investigated the activation status of splenic macrophages during radio-sensitization of EAT in mice as well as in peritoneal macrophages ex vivo and macrophagic cell line (Raw 264.7) in vitro. Results suggest that under in vivo conditions, the combined treatment (2-DG+radiation) restores the M1 phenotype in spleen that correlated with the tumor response. However, 2-DG neither induced significant cytotoxicity nor enhanced radiation-induced cell death in peritoneal macrophages and the macrophage cell line (Raw 264.7). Further, increased arborization and enhanced functional status (expression of MHC class II, CD80, CD86 and phagocytosis) were observed after the combined treatment. Besides this activation, the combined treatment also skewed the macrophages towards M1 phenotype as evidenced by the enhanced secretion of IL-12, IL-2, TNF-α and IFN-γ. These observations suggest that 2-DG not only preserves the survival of normal macrophages during irradiation, but also activates macrophages by polarizing towards M1 phenotype, which is known to be tumoricidal in nature. This study for the first time sheds light on a potential antitumor immune activation by 2-DG involving macrophagic stimulation during in vivo radio-sensitization of tumors, besides the other known antitumor effects of this glucose analogue.

Macrophages Reprogrammed In Vitro Towards the M1 Phenotype and Activated with LPS Extend Lifespan of Mice with Ehrlich Ascites Carcinoma.

BACKGROUND The majority of tumors trigger macrophage reprogramming from an anti-tumor M1 phenotype towards a pro-tumor M2 phenotype. The M2 phenotype promotes tumor growth. We hypothesized that increasing the number of M1 macrophages in a tumor would limit carcinogenesis and extend the lifespan of the tumor host. The aim of this study was to verify this hypothesis in Ehrlich ascites carcinoma (EAC). The objectives were to evaluate effects of 1) EAC on a macrophage phenotype and NO-producing macrophage activity in vivo; 2) ascitic fluid from mice with EAC on a macrophage phenotype and NO-producing macrophage activity in vitro; and 3) in vitro reprogrammed M1 macrophages on lifespan of mice with EAC. MATERIAL AND METHODS The study was conducted using C57BL/6J mice. RESULTS Concentration of nitrite, a stable NO metabolite and an index of NO production, was measured spectrophotometrically. Shifts of macrophage phenotype were assessed by changes in NO production as well as by amounts of CD80, a marker of M1 phenotype, and CD206, a marker of M2 phenotype. The CD markers were measured by flow cytometry. Macrophages were reprogrammed towards the M1 phenotype using two reprogramming factors: 0% FBS and 20 ng/ml IFN-γ. The study results showed that 1) EAC inhibited the macrophage NO production in vivo and reprogrammed macrophages towards the M2 phenotype; 2) ascitic fluid of mice with EAC inhibited the macrophage NO production in vitro and reprogrammed macrophages towards the M2 phenotype; and 3) injection of in vitro reprogrammed M1 macrophages into mice with EAC significantly increased the lifespan of mice. CONCLUSIONS These findings suggest that promising biotechnologies for restriction of tumor growth could be developed based on the in vitro macrophage reprogramming.

Synergic carcinostatic effects of ascorbic acid and hyperthermia on Ehrlich ascites tumor cell.

AIM: In this study, we evaluated the carcinostatic effects of combined ascorbic acid (AsA) and a capacitive-resistive electric transfer (CRet) hyperthermic apparatus-induced hyperthermic treatment on Ehrlich ascites tumor (EAT) cells. MATERIALS AND METHODS: EAT cells were exposed to various AsA (0-10 mM) concentrations for 1 h; they subsequently underwent CRet treatment for 15 min at 42 °C. Cell viability was assessed by the WST-8 assay 24 h after the combined treatment. Reactive oxygen species involvement was evaluated using catalase and tempol; caspase-3/7 activation was determined by their fluorescent substrates; cell proliferation were estimated by time-lapse observation. The effect on the cell cycle was analyzed by flow cytometry. RESULTS: Combined AsA and CRet treatment synergistically suppressed cell viability compared with either treatment alone, and these synergistically carcinostatic effects were evident even at noncytotoxic concentrations of AsA alone (≤ 2 mM). The carcinostatic effects of combined AsA and CRet treatment were attenuated in a dose-dependent manner by catalase addition, but not by the superoxide anion radical scavenger tempol. Time-lapse observation revealed that combined AsA and CRet treatment activated caspase-3/7 at 10-24 h after treatment, accompanied by significant cell growth suppression. Cell cycle analysis revealed that the rate of sub-G1-phase (apoptotic) cells was drastically increased at 12 h and 24 h, and that the G2/M-phase cells gradually increased at 6-24 h after treatment. CONCLUSION: These results indicate that combined AsA and CRet treatment synergistically inhibits EAT cell growth through G2/M arrest and apoptosis induction via H2O2 generation at lower AsA concentrations; this carcinostatic effect cannot be exerted by AsA alone.