The effect of antioxidants in Ehrlich Ascites Cancer.

A fundamental goal in molecular oncology is to unravel the underlying mechanisms which cause the cell transformation. In line with this approach, genome-wide functional screening approaches have revealed exciting insights into heterogeneous nature of cancer. Rapidly expanding horizons of research have unraveled myriad of pathways which play instrumental role in carcinogenesis and metastasis. Oxidative stress has also been reported to be significantly involved in cancer onset and progression. In line with this approach, oxidative stress modulating chemicals have always been sharply divided into antioxidants and oxidative stress-inducing agents. Conceptual and experimental advancements have enabled us to critically analyze full potential of these two different groups of chemicals in cancer chemoprevention. Different antioxidants are currently being analyzed in different phases of clinical trials. Although it has been reported in the literature that antioxidant supplements reduce tumor cells in some tumors or cause volume reduction in solid tumor sizes, there is no definite consensus. Therefore, an antioxidant supplement guideline based on more detailed clinical research and as a result of these is needed to achieve the best care for cancer patients and to avoid risky treatments for cancer patients.

Thymoquinone upregulates miR-125a-5p, attenuates STAT3 activation, and potentiates doxorubicin antitumor activity in murine solid Ehrlich carcinoma.

In breast cancer, there has been evidence of atypical activation of signal transduction and activators of transcription 3 (STAT3). Thymoquinone (TQ) exerts its anti-neoplastic effect through diverse mechanisms, including STAT3 inhibition. The tumor suppressor, microRNA-125a-5p was reported to be downregulated in various breast cancer cells. Therefore, we investigated the influence of TQ and/or doxorubicin on microRNA-125a-5p and its correlation with STAT3 activation as well as tumor growth in mice bearing solid Ehrlich tumors. We found that TQ markedly suppressed inducible and constitutive phosphorylation of STAT3 in tumor tissue without affecting STAT5. Moreover, it attenuated tumor growth, downregulated STAT3 downstream target proteins, and increased the apoptotic activities of caspase-3 and -9. Interestingly, TQ-elicited synergism of doxorubicin anti-neoplastic activity was coupled with upregulation of tumoral microRNA-125a-5p. Taken together, the current findings raise the potential of TQ as a promising chemomodulatory adjuvant to augment mammary carcinoma sensitivity to doxorubicin.

Indole glucosinolates exhibit anti-inflammatory effects on Ehrlich ascites carcinoma cells through modulation of inflammatory markers and miRNAs.

BACKGROUND: Nuclear factor-κB (NF-κB) has been identified as the major link between inflammation and cancer. Natural agents that inhibit this pathway are essential in attenuating inflammation induced by cancer or chemotherapeutic drugs. High intake of Brassicaceae vegetables has been determined to modulate essential pathways related to chronic diseases. In this study, we investigated the anti-proliferative and anti-inflammatory effects of the indole glucosinolates; indole-3-carbinol (I3C) and its metabolite 3,3-diindolylmethane (DIM) on the inflammatory biomarkers and miRNAs controlling the NF-κB pathway. METHODS AND RESULTS: In our study, we inoculated Ehrlich ascites carcinoma (EAC) cells in female albino mice, which increased their packed cell volume and induced a significant increase in the levels of several cytokines and inflammatory biomarkers (NF-κB IL-6, IL-1b, TNF-α, and NO). A significant elevation in inflammatory-medicated miRNAs (miR-31 and miR-21) was also noted. Treatment with 5-fluorouracil (5-FU) significantly reduced packed cell volume and viable cell count. However, it was accompanied by a significant increase in the levels of inflammatory markers and expression of miR-31 and miR-21. Nevertheless, although treatment with indoles (I3C and DIM) significantly reduced the packed cell volume and viable cell count, their prominent effect was the marked reduction of all inflammatory biomarkers compared to both the EAC untreated group and the EAC group treated with 5-FU. Moreover, the anti-inflammatory effect of I3C or DIM was accompanied by a significant decrease in the expression of miR-31 and miR-21. CONCLUSION: Our findings have; therefore, revealed that I3C and DIM have strong anti-inflammatory effects, implying that their use as a co-treatment with chemotherapeutic drugs can effectively improve the anti-tumor effect of chemotherapeutic drugs.

A N-acetyl-D-galactosamine-binding lectin from Amaranthus gangeticus seeds inhibits biofilm formation and Ehrlich ascites carcinoma cell growth in vivo in mice.

AGL, a 15-kDa lectin from Amaranthus gangeticus seeds was isolated using ion-exchange and gel filtration chromatography. AGL contained 8.55% of neutral sugar and became specifically inhibited by N-acetyl-D-galactosamine. Hemagglutination activity of the lectin was maximum over the pH range of 4.0-6.0 and temperatures of 30-60 °C though it lost the activity when treated with urea and EDTA. With an LC50 value of 250 µg/ml, AGL showed mild toxicity against Artemia nauplii. It inhibited the growth of pathogenic bacteria like Shigella boydii, Shigella dysenteriae and Staphylococcus aureus when treated for 8 and 16 h, respectively, but lost the antibacterial activity during a 24 h treatment. AGL could not inhibit the growth of Escherichia coli and mitogenic growth (7.0-9.0%) was observed instead. AGL inhibited 37.14%, 65.71% and 82.85% of biofilm formation of Escherichia coli at the concentrations of 250, 500 and 1000 µg/ml, respectively. Marked inhibition of the proliferation of Ehrlich ascites carcinoma cells was determined when treated with various doses of AGL. AGL inhibited 65.89% and 81.25% of the in vivo growth of EAC cells in mice at the doses of 2.0 and 4.0 mg/kg/day, respectively. Significant alteration of the expression of apoptosis related genes Fas, NF-kB and MAPK were observed.

Deciphering the molecular pathways of apoptosis using purified fractions from leaf extract of Basella alba through studying the regulation of apoptosis related genes.

Apoptosis plays a pivotal role in the exclusion of abnormal cells without any ruin of surrounding healthy cells. Generally, it occurs through an orderly and autonomously process which is controlled by proper function of various genes. Therefore, the current experiments detect the expression level/pattern of those genes to confirm the involvement of extrinsic and intrinsic pathway using Basella alba leaf (BAL). Several fractions after gel filtration chromatography of BAL extract have been pooled to evaluates its apoptosis induction potentiality on Ehrlich's Ascites Carcinoma (EAC) cells through conducting a number of bio-assays such as cell growth inhibition assay, fluorescence and optical microscopy, DNA fragmentation assay and gene expression analysis etc. The pooled fractions of BAL showed 12-56% inhibitory effect on EAC cell line at the concentration range of 25-400 µg/ml that was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. They also exhibited excellent cell growth inhibition at in vivo and in vitro condition when treated with 10, 20 and 40 mg/kg day. After administration of six consequent days, significant morphological features of apoptosis were observed in EAC cells under both fluorescence and optical microscope which was further supported by DNA fragmentation assay. The polymerase chain reaction amplification of bax, bcl-2 (B-cell lymphoma 2), p53, tumor necrosis factor-α, Fas, NF-kß (Nuclear factor-Kappa-B), PARP-1 (Poly (ADP-ribose) polymerase), Cyt-c cas-8, cas-9 and cas-3 revealed that the experimental sample able to induce apoptosis in both extrinsic and intrinsic pathways through altering the gene expression. The current findings suggest that sample from BAL occupy wonderful competence to induce cell apoptosis and become an ideal resource for cancer treatment.

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 inflammation and epigenetic modifications.

Anti-cancer activity of two novel heterocyclic compounds through modulation of VEGFR and miR-122 in mice bearing Ehrlich ascites carcinoma.

Metastasis in breast cancer is a leading cause of mortality among women in many countries. This study investigated the anti-cancer role of benzoimidazoquinazoline and benzimidazotriazin; two novel compounds that were designed, synthesized, structurally elucidated, and biologically evaluated as potent anti-angiogenic agents that act through inhibition of vascular endothelial growth factor receptor-2 (VEGFR2). Breast cancer was induced by inoculation of Ehrlich Ascites Carcinoma (EAC) cells. Seventy swiss albino mice were randomly divided into 7 groups, 10 animals each: (1) normal, (2) control EAC group, (3) cisplatin treated group, (4&5) benzoimidazoquinazoline treated (5 mg/kg and 10 mg/kg), (6&7) benzimidazotriazin treated (5 mg/kg and 10 mg/kg). The expression of miR-122 was assessed in the tumor tissue by quantitative PCR, and the VEGF level was determined in serum by ELISA. VEGFR2 and cluster of differentiation (CD)34 were assessed by immunohistochemistry. Serum ALT, AST, creatinine, and urea were measured. Treatment with benzoimidazoquinazoline and benzimidazotriazin decreased tumor weight and serum levels of VEGF, and down-regulated expression of VEGFR2 and CD34 in the tumor tissue. miR-122 was upregulated, particularly in the benzimidazotriazin (10 mg/kg) group. Relative to cisplatin, the novel compounds were less toxic to kidneys. Benzoimidazoquinazoline and benzimidazotriazin are promising anti-cancer agents that act through inhibition of angiogenesis and thus provide a new strategy for advancement of chemotherapy.

An Unusual Pathway of Mitoptosis Found in Ehrlich Carcinoma Cells.

An integrated microscopic study of the destruction of mouse Ehrlich ascites carcinoma (EAC) cells under starvation conditions has been carried out. It has been found that, in addition to apoptosis, necrosis, and apoptotic necrosis, already known for EAC, cell destruction can also occur through mitochondrial autolysis, which is proposed to be considered a new kind of mitoptosis. A mitoptosis in EAC is characterized by the appearance of many autolyzing mitochondria, the fusion of which leads to rupture of the cell membrane and the ejection of the nucleus from the cell. It is assumed that the polymorphism of EAC destruction patterns is explained by the different physiological state of the cells, which determines the "choice" of the cell death mechanism. This situation poses a challenge for researchers to develop complex inducers with the ability to stimulate all possible types of cancer cell death.

Therapeutic effect of Arthrocnemum machrostachyum methanolic extract on Ehrlich solid tumor in mice.

BACKGROUND: The anti-cancer effect of the halophyte Arthrocnemum indicum, a member of Arthrocnemum family of salt-tolerant plants, was evaluated against colorectal cancer cell, CaCo2. However, the anti-cancer effect of another halophyte Arthrocnemum machrostachyum was not investigated yet. Herein, the anticancer effect of A. machrostachyum methanolic extract (AME) was evaluated against Ehrlich solid tumor (EST) in mice and the potential mechanism of action was also studied. METHODS: Male Swiss albino mice (n = 28) were randomly divided into 4 groups (n = 7/group). Group 1 (negative control group); group 2 (EST) injected intramuscularly by 0.2 mL Ehrlich ascitic carcinoma (2 × 106 cells); and groups 3 and 4 injected intratumorally with AME (180 and 360 mg/kg body weight, respectively) at D12 trice weekly for 2 weeks. Gene expression, protein expression, DNA damage, and TNFa level in tumors were determined by real-time PCR, western blot, comet assay, and Elisa, respectively. RESULTS: Treatment with AME induced anti-tumor effects against EST as indicated by 1) notable reduction in tumor size; 2) elevation in tissue necrosis and apoptosis, as confirmed histologically; 3) increased DNA fragmentation; 4) decreased expression of the apoptotic genes (p53, Bax and caspase 3), and increased expression of the anti-apoptotic marker Bcl2; 5) significantly upregulated cell cycle regulatory genes Cdc2 and connexin26, and; 6) decreased TNFa levels in tumor tissues. Interestingly, a high dose of AME exhibited a more potent anti-tumor effect against EST. CONCLUSION: These findings indicate that AME has a potent antitumor effect against EST and could be used as an adjuvant to anticancer drugs to combat tumor, but after application of further confirmatory clinical trials.

Targeting MDR-1 gene expression, BAX/BCL2, caspase-3, and Ki-67 by nanoencapsulated imatinib and hesperidin to enhance anticancer activity and ameliorate cardiotoxicity.

There is a great demand to introduce new approaches into cancer treatment field due to incidence of increased breast cancer all over the world. The current study was designed to evaluate the role of imatinib mesylate (IM) and/or hesperidin (HES) nanoparticles alone or in combination in enhancing the anticancer activity and to investigate the ability of nanoencapsulation to reduce cardiotoxicity of IM in solid Ehrlich carcinoma (SEC)-bearing mice. IM and HES were loaded into PLGA (poly(lactic-co-glycolic acid) polymer. SEC was induced in female albino mice as a model for experimentally induced breast cancer. Mice were randomly divided into eight groups (n = 10). On day 28 from tumor inoculation, mice were sacrificed and blood samples were collected in heparinized tubes for hematological studies, biochemical determination of lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (SGOT) levels. In addition, tumor and cardiac tissues were utilized for histopathological examination as well as determination of MDR-1 gene expression. Immunohistochemical staining of BAX and BCL-2 was done. Nano IM- and/or Nano HES-treated groups showed a significant reduction in tumor volume, weight, hematological, cardiac markers, and tumor MDR-1 gene downregulation compared to free conventional treated groups. In conclusion, the use of HES as an adjuvant therapy with IM could improve its cytotoxic effects and limit its cardiac toxicity. Furthermore, nanoencapsulation of IM and/or HES with PLGA polymer showed a remarkable anticancer activity.

Dual-targeted therapeutic strategy combining CSC-DC-based vaccine and cisplatin overcomes chemo-resistance in experimental mice model.

BACKGROUND AND PURPOSE: Emerging evidence suggests that one of the main reasons of chemotherapy treatment failure is the development of multi-drug resistance (MDR) associated with cancer stem cells (CSCs). Our aim is to identify a therapeutic strategy based on MDR-reversing agents. MATERIALS AND METHODS: CSC-enriched Ehrlich carcinoma (EC) cell cultures were prepared by drug-resistant selection method using different concentrations of cisplatin (CIS). Cell cultures following drug exposure were analyzed by flow cytometry for CSC surface markers CD44+/CD24-. We isolated murine bone marrow-derived dendritic cells (DCs) and then used them to prepare CSC-DC vaccine by pulsation with CSC-enriched lysate. DCs were examined by flow cytometry for phenotypic markers. Solid Ehrlich carcinoma bearing mice were injected with the CSC-DC vaccine in conjunction with repeated low doses of CIS. Tumor growth inhibition was evaluated and tumor tissues were excised and analyzed by real-time PCR to determine the relative gene expression levels of MDR and Bcl-2. Histopathological features of tumor tissues excised were examined. RESULTS AND CONCLUSION: Co-treatment with CSC-DC and CIS resulted in a significant tumor growth inhibition. Furthermore, the greatest response of downregulation of MDR and Bcl-2 relative gene expression were achieved in the same group. In parallel, the histopathological observations demonstrated enhanced apoptosis and absence of mitotic figures in tumor tissues of the co-treatment group. Dual targeting of resistant cancer cells using CSC-DC vaccine along with cisplatin represents a promising therapeutic strategy that could suppress tumor growth, circumvent MDR, and increase the efficacy of conventional chemotherapies.

Arabinoxylan rice bran (MGN-3/Biobran) enhances radiotherapy in animals bearing Ehrlich ascites carcinoma†.

This study examines the ability of arabinoxylan rice bran (MGN-3/Biobran) to enhance the anti-cancer effects of fractionated X-ray irradiation of Ehrlich solid tumor-bearing mice. Swiss albino mice bearing tumors were exposed to the following: (i) Biobran treatment (40 mg/kg/day, intraperitoneal injections) beginning on day 11 post-tumor cell inoculation until day 30; (ii) ionizing radiation (Rad) 2 Gy at three consecutive doses on days 12, 14 and 16; or (iii) Biobran + Rad. Final tumor weight was suppressed by 46% for Biobran, 31% for Rad and 57% for the combined treatment (Biobran + Rad) relative to control untreated mice. Biobran and Rad also arrested the hypodiploid cells in the sub-G1-phase, signifying apoptosis by +102% and +85%, respectively, while the combined treatment induced apoptosis by +123%, with similar results in the degree of DNA fragmentation. Furthermore, Biobran + Rad upregulated the relative gene expression and protein level of p53 and Bax in tumor cells, down-regulated Bcl-2 expression, and increased the Bax/Bcl-2 ratio and caspase-3 activity, with the combined treatment greater than for either treatment alone. Additionally, the combined treatment modulated the decrease in body weight, the increase in liver and spleen weight, and the elevation of liver enzymes aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transferase to be within normal values. We conclude that Biobran enhances radiation therapy-induced tumor regression by potentiating apoptosis and minimizing toxicities related to radiation therapy, suggesting that Biobran may be useful in human cancer patients undergoing radiotherapy and warranting clinical trials.

Salvia lachnostachys Benth has antitumor and chemopreventive effects against solid Ehrlich carcinoma.

Salvia lachnostachys is an herbaceous plant with anti-inflammatory, analgesic and cytotoxic properties. This study investigated the antitumor effect of an ethanolic extract of Salvia lachnostachys leaves (EES) in a solid Ehrlich carcinoma model. Ehrlich cells were inoculated subcutaneously in the right pelvic member (2 × 106 cells) in female Swiss mice. The animals were treated with vehicle (10 mL kg-1, p.o.), EES (30 and 100 mg kg-1, p.o.), or methotrexate (2.5 mg kg-1, i.p.) for 21 days (early treatment) or 14 days (late treatment) after tumor inoculation, or 10 days before tumor inoculation and continued for 21 days after tumor inoculation (chemopreventive treatment). The acute toxicity test was performed according OECD guidelines Late treatment with EES had no antitumor effect. Early treatment with 100 mg kg-1 EES prevented tumor development, increased tumor necrosis factor-α (TNF-α) levels and decreased tumor superoxide dismutase (SOD) activity, interleukin-10 (IL-10) levels and Cyclin D1 expression, and tumor cell necrosis was observed. Chemopreventive treatment with EES for 10 and 31 days prevented tumor development in the same manner. EES treatment for 31 days decreased hepatic and tumor SOD activity, tumor IL-10 levels and Cyclin D1 expression, and increased tumor reduced glutathione, N-acetylglucosaminidase, reactive oxygen species, lipid peroxidation, TNF-α levels and Nrf2 expression. No toxicity was observed in the acute toxicity assay. In conclusion, EES had an antitumor effect by inhibiting Cyclin D1 expression and increasing inflammation with early and chemopreventive treatment. Modulation of the antioxidant system also contribute for the antitumor effects of EES.

Dietary baker's yeast sensitizes Ehrlich mammary adenocarcinoma to paclitaxel in mice bearing tumor.

Baker's yeast, Saccharomyces cerevisiae, has been shown to sensitize a variety of breast cancer cell (BCC) lines to paclitaxel chemotherapy in vitro. The present study evaluated the ability of S. cerevisiae to sensitize BCCs to paclitaxel in animals bearing Ehrlich ascites carcinoma (EAC). Mice bearing EAC were intratumorally injected with dead S. cerevisiae (1x107 cells/ml) in the presence or absence of low- and high-dose paclitaxel [paclitaxel-L, 2 mg/kg body weight (BW) and paclitaxel-H, 10 mg/kg BW, respectively]. At 30 days post tumor inoculation, co-treatment with yeast plus paclitaxel-L showed improvements over paclitaxel-H alone, as measured by tumor weight (-64 vs. -53%), DNA damage (+79 vs. +62%), tumor cell apoptosis (+217 vs. +177%), cell proliferation (-56 vs. -42%) and Ki-67 marker (+95 vs. +40%). Histopathology and ultra-structural examinations showed that yeast plus paclitaxel-L enhanced apoptosis in EAC more than paclitaxel-H alone and caused comparable tumor necrosis. We conclude that baker's yeast may be used with low-dose chemotherapy to achieve the same potency as high-dose chemotherapy in mice bearing EAC. This suggests that baker's yeast may be an anticancer adjuvant and may have clinical implications for the treatment of breast cancer.

Trehalose enhances the antitumor potential of methotrexate against mice bearing Ehrlich ascites carcinoma.

Methotrexate (MTX) is commonly used as a standard chemotherapy for many cancers, however its usage required high doses thereby leading to severe adverse effects. In a trial to find a suitable neoadjuvant therapy to decrease MTX dosage without lowering its chemotherapeutic efficacy, we investigated the antitumor effect of trehalose (TRE) on mice bearing Ehrlich ascites carcinoma (EAC) and checked whether TRE can enhance the antitumor potential of MTX. Treatment with TRE induced anti-tumor effects against EAC as reveled by a remarkable decrease in body weight, tumor volume, count of viable tumor cells, expression of the anti-apoptotic gene Bcl2 as well as by a significant increase in mean survival time, life span and expression of the apoptotic gene caspase-3. TRE also caused a significant decrease in autophagic activity of EAC cells as evident by reduction in the expression of the autophagic gene Beclin 1 (Bec1) and the fluorescence intensity of autophagosome marker. Additionally, TRE restored the altered hematological and biochemical parameters and improved the disrupted hepatic tissues of EAC-bearing mice. Interestingly, co-administration of TRE and MTX showed highest anti-tumor effect against EAC. These data indicate that TRE enhances the antitumor potential of MTX and could be used as neoadjuvant drug to increase the efficacy of the antitumor drug, MTX.

Metformin augments doxorubicin cytotoxicity in mammary carcinoma through activation of adenosine monophosphate protein kinase pathway.

Since the incidence of breast cancer increases dramatically all over the world, the search for effective treatment is an urgent need. Metformin has demonstrated anti-tumorigenic effect both in vivo and in vitro in different cancer types. This work was designed to examine on molecular level the mode of action of metformin in mice bearing solid Ehrlich carcinoma and to evaluate the use of metformin in conjunction with doxorubicin as a combined therapy against solid Ehrlich carcinoma. Ehrlich ascites carcinoma cells were inoculated in 60 female mice as a model of breast cancer. The mice were divided into four equal groups: Control tumor, metformin, doxorubicin, and co-treatment. Metformin (15 mg/kg) and doxorubicin (4 mg/kg) were given intraperitoneally (i.p.) for four cycles every 5 days starting on day 12 of inoculation. The anti-tumorigenic effect of metformin was mediated by enhancement of adenosine monophosphate protein kinase activity and elevation of P53 protein as well as the suppression of nuclear factor-kappa B, DNA contents, and cyclin D1 gene expression. Metformin and doxorubicin mono-treatments exhibited opposing action regarding cyclin D1 gene expression, phosphorylated adenosine monophosphate protein kinase, and nuclear factor-kappa B levels. Co-treatment markedly decreased tumor volume, increased survival rate, and improved other parameters compared to doxorubicin group. In parallel, the histopathological findings demonstrated enhanced apoptosis and absence of necrosis in tumor tissue of co-treatment group. Metformin proved chemotherapeutic effect which could be mediated by the activation of adenosine monophosphate protein kinase and related pathways. Combining metformin and doxorubicin, which exhibited different mechanisms of action, produced greater efficacy as anticancer therapeutic regimen.

The Antioxidative Fraction of White Mulberry Induces Apoptosis through Regulation of p53 and NFκB in EAC Cells.

In this study, the antioxidative fraction of white mulberry (Morus alba) was found to have an apotogenic effect on Ehrlich's ascites carcinoma cell-induced mice (EAC mice) that correlate with upregulated p53 and downregulated NFκB signaling. The antioxidant activities and polyphenolic contents of various mulberry fractions were evaluated by spectrophotometry and the ethyl acetate fraction (EAF) was selected for further analysis. Strikingly, the EAF caused 70.20% tumor growth inhibition with S-phase cell cycle arrest, normalized blood parameters including red/white blood cell counts and suppressed the tumor weight of EAC mice compared with untreated controls. Fluorescence microscopy analysis of EAF-treated EAC cells revealed DNA fragmentation, cell shrinkage, and plasma membrane blebbing. These characteristic morphological features of apoptosis influenced us to further investigate pro- and anti-apoptotic signals in EAF-treated EAC mice. Interestingly, apoptosis correlated with the upregulation of p53 and its target genes PARP-1 and Bax, and also with the down-regulation of NFκB and its target genes Bcl-2 and Bcl-xL. Our results suggest that the tumor- suppressive effect of the antioxidative fraction of white mulberry is likely due to apoptosis mediated by p53 and NFκB signaling.

Albendazole as a promising molecule for tumor control.

This work evaluated the antitumor effects of albendazole (ABZ) and its relationship with modulation of oxidative stress and induction of DNA damage. The present results showed that ABZ causes oxidative cleavage on calf-thymus DNA suggesting that this compound can break DNA. ABZ treatment decreased MCF-7 cell viability (EC50=44.9 for 24h) and inhibited MCF-7 colony formation (~67.5% at 5µM). Intracellular ROS levels increased with ABZ treatment (~123%). The antioxidant NAC is able to revert the cytotoxic effects, ROS generation and loss of mitochondrial membrane potential of MCF-7 cells treated with ABZ. Ehrlich carcinoma growth was inhibited (~32%) and survival time was elongated (~50%) in animals treated with ABZ. Oxidative biomarkers (TBARS and protein carbonyl levels) and activity of antioxidant enzymes (CAT, SOD and GR) increased, and reduced glutathione (GSH) was depleted in animals treated with ABZ, indicating an oxidative stress condition, leading to a DNA damage causing phosphorylation of histone H2A variant, H2AX, and triggering apoptosis signaling, which was confirmed by increasing Bax/Bcl-xL rate, p53 and Bax expression. We propose that ABZ induces oxidative stress promoting DNA fragmentation and triggering apoptosis and inducing cell death, making this drug a promising leader molecule for development of new antitumor drugs.

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.

Piper nigrum ethanolic extract rich in piperamides causes ROS overproduction, oxidative damage in DNA leading to cell cycle arrest and apoptosis in cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation. AIMS OF THE STUDY: The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity. METHODS: The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV-vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice. RESULTS: Extraction yielded 64mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC50=27.1±2.0 and 80.5±6.6µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-xL and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice. CONCLUSION: The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction that induced oxidative stress affecting key proteins involved in cell cycle arrest at G1/S and triggering apoptosis. Finally, the overall data from this study are well in line with the traditional claims for the antitumor effect of Piper nigrum fruits.