Murine Mammary Adenocarcinoma Cell Line 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.

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.

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.

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.

In Vitro Oncolytic activity of non-virulent Newcastle Disease Virus LaSota Strain against Mouse mammary adenocarcinoma

Newcastle disease virus (NDV) is a wide-spectrum anti-tumor agent. The oncolytic selectivity of NDV, a family of Paramyxoviridae, depends on the differential type of inducing different death pathways. This work was conducted to further understand the oncolytic effect of LaSota strain. A mouse breast cancer model (Murine mammary adenocarcinoma cell line AMN3) was used in this study. Methyl Thiazolyl Tetrazolium (MTT) viability assay tested different NDV multiplicity of infection (MOI) values on mouse mammary adenocarcinoma cells incubated for 72 hours post-infection. The IC50 values and anti-tumor activity of LaSota strain against AMN3 cell line were determined. Following Hematoxylin and Eosin Stain, we examined the morphological modifications of IC50 along with 10 MOI values of NDV. The induction of NDV apoptosis in AMN3 cells was investigated using the technique of staining acridine orange and propidium iodide (AO / PI). Immunocytochemistry assay was performed using anti-NDV mAbs and caspases 8 and 9 to study NDV replication and apoptosis induction mechanisms. The lentogenic LaSota NDV strain, a live vaccine, demonstrated the oncolytic effect on mammary cancer cells of the AMN3 mouse and showed that LaSota strain triggered a dose-dependent increase in infected cells’ apoptosis relative to untreated mammary cancer cells. The immunocytochemistry study showed that NDV infected cells were positive for virus infection and that caspase9 in mouse mammary cancer cells after LaSota strain infection was significantly enhanced compared to caspase 8. In conclusions, NDV LaSota strain had oncolytic effects by destroying tumor cells and triggering the intrinsic apoptosis pathways in mouse mammary cancer cells. However, the mechanisms of the in vivo anti-tumor activity need to be better understood.

Effect of vitex agnus castus fruits methanol extract against murine mammary adenocarcinoma cell line (amn3) and rat embryonic fibroblast normal cell line (ref)

Vitex agnus castus showed antiproliferative activity in several previous studies. Angiogenesis is one of the targets in the remediation of cancer. This study aimed to demonstrate the effect of methanol fruits extract of vitex agnus castus on mouse mammary gland adenocarcinoma cell line and rat embryonic fibroblast cell line. The cell lines used in this study were obtained from tissue culture unit/ Iraqi Center for Cancer and Medical Genetic Researches, Al-Mustansiriyah University was maintained in RPMI- 1640 tissue media after preparing from 10% fetal calf serum, antibiotics solution and other materials to make complete growth medium. Serial solutions of vitex agnus castus, methanol crude extract have been tested on 10*4 of AMN3 and REF in each well of 96 well plates. The results of the current study showed that the concentration that inhibits fifty percent of cell line after 72 hours of the experiment (IC50) was 129 ug/ml for AMN3 and 1324ug/ml for REF cell line. The antioxidant activity of Vitex agnus castus may indicate the proliferation inhibition activity of Vitex agnus castus methanol extract. The study concluded that this extract might be of benefit if used in combination with other anti-cancer drugs as adjuvant therapy.

Abstract 4508: Glypican-3 (GPC3) signaling pathway involved in breast cancer progression

Abstract GPC3 is a proteoglycan downregulated in breast tumors. Employing LM3 murine mammary adenocarcinoma cell line (ER-, PR-), we have previously shown that GPC3 acts as a metastasis suppressor and induces a reversion of the epithelial to mesenchymal transition (EMT). GPC3 overexpressing LM3 cells presented an inhibition of canonical Wnt and Akt pathways. On the other hand, non-canonical Wnt (JNK) and p38 signaling were activated. However, the hierarchical sequence through which GPC3 modulates those pathways has not been determined yet.The aim of this work was to study the molecular mechanism involved in the GPC3 effect on breast tumor progression, focusing on canonical Wnt pathway. We investigated the receptor complex with which GPC3 is interacting and the relationships among the pathways modulated by this glypican. We determined by IP that GPC3 could physically interact with the Wnt receptor Fz. Our results suggested that GPC3 binds to Fz and blocks the binding of the canonical Wnt factors. We also confirmed by the determination of cytoplasmic β-Catenin levels through WB and their transcriptional activity employing gene reporter assay, that GPC3 is inhibiting this pathway. We showed by qPCR array that GPC3 also modulates canonical Wnt signaling in a genomic way. Of 84 examined genes, only Wnt5b (non-canonical factor) was upregulated in LM3-GPC3 cells, 66 genes were downregulated (several Fzds, LRP5/6, DVL1 and DVL2, Frat1, Ccnd1, Ccnd2, Ccnd3, Csnk1d, jun and myc, among others), and the expression of 17 genes was not modified (including several Wnt factors). Finally, LM3-GPC3 cells were treated with SB203589, SP600125 (p38 and JNK pharmacological inhibitors respectively), LiCl (canonical Wnt activator) or transduced with a constitutively active variant of Akt1 (CA Akt). The effect of the treatments on the other pathways activity was evaluated by WB. We showed that the Akt inhibition found in LM3-GPC3 cells is required for the activation of the non-canonical Wnt pathway, as well as for the inhibition of the canonical one, but it had no effects on p38 signaling. p38 activation was necessary for non-canonical Wnt upregulation and for Akt and canonical Wnt inhibition. Canonical Wnt inhibition was essential for Akt downregulation as well as for p38 and non-canonical Wnt signaling activation. We demonstrated that in addition to the significant role of JNK in the non-canonical Wnt pathway, it also regulates canonical signalling. Non-canonical Wnt activity also participates in p38 activation but has no effect on Akt activity.In sum, we have described the receptor complex with which GPC3 interacts, as well as the canonical Wnt signaling inhibition induced by GPC3 - both genomic and non-genomic - and finally several interactions among Wnt, Akt and p38 pathways. We believe that our results provide additional information regarding the molecular mechanism of GPC3 involved in metastasis suppression. Citation Format: Dolores Fernandez, Magali Cercato, Macarena Guereño, Maria G. Peters. Glypican-3 (GPC3) signaling pathway involved in breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4508.

Treatment with Src inhibitor Dasatinib results in elevated metastatic potential in the 4T1 murine mammary carcinoma model.

The src inhibitor Dasatinib has been widely studied as an anti-metastatic agent. The aims of this study were to examine the effect of Src inhibition on the metastatic potential of the 4T1 murine mammary carcinoma. Src is a non-receptor tyrosine kinase well-known to contribute to the metastatic potential of tumour cells. It does so through alteration of signalling pathways important to metastasis. Elevated levels of Src are common in many cancer types, and have been correlated with tumour progression and poor patient prognosis. This study examined whether disruption of the Src signalling pathway could inhibit metastases formation. The Src inhibitor Dasatinib was evaluated in vitro and in vivo using the highly metastatic 4T1 murine mammary adenocarcinoma cell line. In vitro assays included growth curve, western blot, migration, and invasion assays. In vivo assays included intradermal and tail vein injection models. In vitro data were analysed using one-way ANOVA with Dunnett's multiple comparisons in GraphPad Prism 6.0. In vivo data were analysed using GraphPad Prism 6.0, using the Wilcoxon matched pairs test. Dasatinib is effective at inhibiting in vitro phosphorylation of Src, migration and invasion in the 4T1 cell line, as well as angiogenesis in vivo. In vitro treatment with Dasatinib impaired the metastatic ability of tumour cells as assessed by a tail vein injection model. However, both the syngeneic BALB/c and the athymic nu/nu mice receiving oral doses of the drug developed significantly higher numbers of 4T1 lung metastases. This effect was not seen in a different breast carcinoma cell line, the MDA-MB-231-4175-LM2, nor was this effect seen in the murine fibrosarcoma KHT cell line. The 4T1 cell line is not an appropriate model to study Src inhibition.

Myoepithelial and luminal breast cancer cells exhibit different responses to all-trans retinoic acid.

Breast cancer is the leading cause of death among women worldwide. The exact role of luminal epithelial (LEP) and myoephitelial (MEP) cells in breast cancer development is as yet unclear, as also how retinoids may affect their behaviour. Here, we set out to evaluate whether retinoids may differentially regulate cell type-specific processes associated with breast cancer development using the bi-cellular LM38-LP murine mammary adenocarcinoma cell line as a model. The bi-cellular LM38-LP murine mammary cell line was used as a model throughout all experiments. LEP and MEP subpopulations were separated using inmunobeads, and the expression of genes known to be involved in epithelial to mysenchymal transition (EMT) was assessed by qPCR after all-trans retinoic acid (ATRA) treatment. In vitro invasive capacities of LM38-LP cells were evaluated using 3D Matrigel cultures in conjunction with confocal microscopy. Also, in vitro proliferation, senescence and apoptosis characteristics were evaluated in the LEP and MEP subpopulations after ATRA treatment, as well as the effects of ATRA treatment on the clonogenic, adhesive and invasive capacities of these cells. Mammosphere assays were performed to detect stem cell subpopulations. Finally, the orthotopic growth and metastatic abilities of LM38-LP monolayer and mammosphere-derived cells were evaluated in vivo. We found that ATRA treatment modulates a set of genes related to EMT, resulting in distinct gene expression signatures for the LEP or MEP subpopulations. We found that the MEP subpopulation responds to ATRA by increasing its adhesion to extracellular matrix (ECM) components and by reducing its invasive capacity. We also found that ATRA induces apoptosis in LEP cells, whereas the MEP compartment responded with senescence. In addition, we found that ATRA treatment results in smaller and more organized LM38-LP colonies in Matrigel. Finally, we identified a third subpopulation within the LM38-LP cell line with stem/progenitor cell characteristics, exhibiting a partial resistance to ATRA. Our results show that the luminal epithelial (LEP) and myoephitelial (MEP) mammary LM38-P subpopulations respond differently to ATRA, i.e., the LEP subpopulation responds with increased cell cycle arrest and apoptosis and the MEP subpopulation responds with increased senescence and adhesion, thereby decreasing its invasive capacity. Finally, we identified a third subpopulation with stem/progenitor cell characteristics within the LM38-LP mammary adenocarcinoma cell line, which appears to be non-responsive to ATRA.

439. Oncolytic Newcastle Disease Virus Iraqi Virulent Strain Induce Apoptosis In Vitro through intrinsic Pathway and Association of Both Intrinsic and Extrinsic Pathways In Vivo

Newcastle disease virus is very promising antitumor agent. Iraqi virulent strain showed pronounced oncolytic activity. This study aimed to investigate the specific pathway of apoptosis induced by Iraqi strain of Newcastle disease virus (NDV) on tumor cells In vitro and In vivo. Several cell lines were tested (Human Rhabdomyosarcoma cell line (RD), Human Glioblastoma multiforme culture (AMGM) and Murine mammary adenocarcinoma cell line (AMN3) for ability of Iraqi NDV strain to induce apoptosis and to give deep look about the main pathway that induce apoptosis in tumor cell lines by the virus. The cell lines were infected with Newcastle disease virus and investigated for apoptotic pathway by using immunocytochemistry for caspase 3, 8, and 9. The result of this in vitro experiment revealed that NDV induce apoptosis in tumor cell lines when compared to control group with marked increase in the percentage of cells expressing caspase 9 with low percentage of cells expressing caspase 8 in NDV treated group at 24,48hr. These results indicated the role of NDV Iraqi strain in inducing apoptosis through intrinsic mitochondrial pathway in vitro. The in vivo experiment included female mice transplanted with mammary adenocarcinoma transplantable tumor line (AM3), after tumor growth; tumor bearing mice randomly divided into groups, first group treated by NDV by intratumoral injection, second group treated by NDV intrapretonially. Caspase 3, caspase 8, and caspase 9 expression in tumor sections were checked after 24hrs, 48hrs, 72hrs, 7 and 14 days of treatment with NDV. The results revealed that NDV induce apoptosis significantly in treated tumors by both ways IT and IP when compared with control group. There is significant (p<0.05) marked increase in the mean percentage of cells expressing caspase 9 in NDV treated groups compared with mean percentage of cells expressing caspase 8 and untreated control group at 24,48,72, 1week, 2 weeks post single injection treatment. Moreover, Caspase-8 expression in IT and IP groups was significantly higher than control untreated group. These results revealed that NDV had a powerful effect on inducing mitochondrial (intrinsic) apoptotic pathway in vivo in association of extrinsic pathway. This association can explained by NDV induction for antitumor immune response and this immune response can trigger tumor killing by extrinsic pathway. The current work suggest that Iraqi oncolytic virulent strain of NDV induce apoptosis through intrinsic pathway directly as the results of in vitro study and induce extrinsic pathway indirectly through immune stimulation properties of NDV.

Effect of Curcumin Extract (Curcuma Longa) on Cancer Cell Lines in Vitro

Objectives: This study aims to detect the active compounds found in ethanolic crude extract of Curcuma longa rhizome and evaluate the cytotoxic effect of ethanolic extract on two malignant cell lines; Human laryngeal Carcinoma (HEP-2) cell line, murine mammary adenocarcinoma (AMN-3) cell line and one transformed cell line of Rat Embryogenic Fibroblast (REF-3). Methodology: Administrative Arrangement: This study was conducted in Iraqi Centre for Cancer and Medical Genetic Research (ICCMGR), Baghdad, Iraq into the period (1 / 1 / 2011 to 1 / 1 / 2012), periods of exposure of cell lines Different concentrations were prepared from ethanolic extract starting in (1 to 10000) µg/ml to were measured at) 24, 48, and 72 (hrs. in a microtitration plate under complete sterile conditions. Results: Chemical detection of extract appeared that the ethanolic extract contain flavonoid, saponin, phenols, resins, volatile oils, tannins, terpins and steroids while glycosids, alkaloids and cumarines gave negative results appeared the ethanolic extract of curcuma longa showed inhibition effect at 48 hr. period for all concentration on cancer cell lines. And amount the inhibition percentage of (AMN-3) cell line (88.3%) for treatment in concentration (6 µg/ml) after (24hr). of exposure time, and inhibition percentage begin in lower concentration on (Hep-2) cancer cell line and gave inhibition percentage (77.1%) after treatment in (2000 µg/ml) after (48hr). of exposure time and appeared the ethanolic extract on REF transformed cell line and gave higher inhibition percentage (93%) after treatment in (100 µg/ml) of exposure time. Conclusion: The ethanolic crude extract of Curcuma longa Which contain flavonoid, saponin, phenols, resins, Volatile Oils, tannins, terpins and steroids while alkaloids, glycosids, and cumarines gave negative results, In the in vitro study, The ethanolic crude extract of C. longa has a potent cytotoxic activity against REF transformed cell line, AMN-3 and Hep-2 cell lines in a concentration and time dependent manners (in vitro), The cell lines REF and AMN-3 were the most sensitive to the inhibitory effects of ethanolic extract of C. longa rhizomes, The cell line Hep-2 showed less response at low concentration while the extract had cytotoxic effect at highest concentration after exposure 48 hr. Recommendations: Using further different cancer cell lines as well as normal cell lines to study the cytotoxicity of Curcuma longa extract such as oral, stomach and colon cell lines. Using the comet test technique to detect the effects of plants extracts on DNA, use different solvent to extract of Curcuma longa for example methanol.

In Vitro CYTOTOXIC STUDY FOR PARTIAL PURE LIGNAN EXTRACT FROM FLAX SEED (Linum usitatissirnurn) ON RHABDOMYOSARRCOMA (RD) AND MURINE MAMMARY ADENOCARCINOMA (MMA) CELL LINES

The lignan (Secoisolaricinesinal diglucoside, SDG), was obtained from flaxseed ( Linum usitatissirnurn) appear to act as both anti-estrogens and weak estrogens. The anti-estrogenic potential of lignans has been postulated to protect against hormone sensitive cancers such as breast, prostate and colon cancer. The aim of this study was to examine the cytotoxic effect of partial pure lignan on some cell lines including, the Murine Mammary Adenocarcinoma, Rhabdomyosarcoma RD, L20B and normal cell line (Rat Embryo Fibroblast REF). Four concentrations of partial pure SDG which obtained by using alcohol hydrolysis dioxin / ethanol flaxseed extraction and liquid/liquid partition, have been prepared 5, 10, 15, 20 μM with different of exposure periods 24, 48, 72 h. These parameters have been applied on all cancer and normal cell line. The highest percentage of inhibition appears in 5, 10,15 μM at 24 h (60.11, 49.99, 50.28 %) respectively on RD cell line when treated with partial pure. and the with same concentrations this percentage decreases without any significant difference. In case of L20B cell line in 72 h, the inhibitory effect of partial pure at 5,10 μM, 57.67 and 52.11 % respectively. While the cytotoxicity of partial pure SDG on ANM3 cell line appeared in 24h at 15um which was 64.88%. The lignans are growth inhibitors of colon tumor cells and suggest act through mechanisms other than anti-estrogenic activity.

Effect of bioactive compounds in ginger extract on cancer cell lines In vitro.

his study was designed to detect the active compounds found in ethanolic crude extract of Zingeber officinale rhizome. Chemical detection of extract appeared that the ethanolic extract contain flavonoid, saponin, glycosids, phenols, resins, volatile oils, tannins, terpins and steroids while alkaloids and cumarines gave negative results. Different concentrations were prepared from ethanolic extract starting from (1-1000) µg/ml to evaluate the cytotoxic effect of ethanolic extract on two malignant cell lines, human laryngeal carcinoma (HEP-2) cell line and murine mammary adenocarcinoma (AMN-3) cell line, exposure periods of cell lines were measured at (24, 48, 72)hr in a microtitration plate under complete sterile conditions. Results showed that, Ethanolic crude extract exhibited time-dependent cytotoxic effect of all concentrations after exposure for 24hr on cancer and transformed cells lines, and the 100 µg/ml gave the highest inhibition rate for AMN-3 86.2% after 24hr of exposure time and the cytotoxic effect of the extract started at lowest concentrations on HEP-2 cell line after 48hr of exposure reached which the best concentration 600 µg/ml gave the highest inhibition cell growth 73.4% after 48hr of exposure time.

Palladium(II) complexes incorporating phenylazo arylmethine ancillary ligands: Synthesis, spectral and antitumor activity

A series of azoimine palladium(II) complexes of the general type cis-[PdCl2L] (1–6) {L=PhNNC(COMe)NC6H4X, where X=H (L1), CH3 (L2), Cl (L3), Br (L4), naphthyl (L5) and OMe (L6)} have been prepared by the reaction of cis-[PdCl2(NCPh)2] and the ligands (L) in acetone at room temperature. These complexes have been characterized by spectroscopic (IR-, UV–Vis and NMR) and electrochemical techniques. In addition, their biological activity has been studied via MTT test and by detecting the inhibition of clonal growth in three tumor cell lines, namely T47D human ductal breast carcinoma, MTLn3 murine mammary adenocarcinoma and RAW 264.7 murine leukemic monocyte macrophage. All of the tested palladium(II) complexes showed a noticeable antitumor activity. Complexes 4 and 5 showed a higher cytotoxic activity against human T47D breast tumor cell line than cisplatin, while comparable activity was observed against murine mammary MTLn3 adenocarcinoma cell line.

18F]-fluoroestradiol quantitative PET imaging to differentiate ER+ and ERα-knockdown breast tumors in mice

The purpose of this study was to develop a noninvasive model in tumor-bearing mice to investigate the use of 16α-[(18)F]fluoro-17β-estradiol (FES) positron emission tomography (PET) imaging as a tool to discriminate between tumors having different estrogen receptor (ER) α status. MC7-L1 and MC4-L2 murine mammary adenocarcinoma cell lines (ER+) received a small hairpin RNA targeting the ERα gene by lentiviral infection. In vitro assessment of ERα levels of the new cell lines (MC7-L1 and MC4-L2 ERα-knockdown; ERαKD), compared to the parental cell lines, was performed by immunoblotting (-75% ERα protein) and binding assays (-50% estrogen binding). These cell lines were implanted subcutaneously in Balb/c mice and allowed to grow up to a volume of at least 20 mm(3). FES and [(18)F]fluorodeoxyglucose (FDG) PET images were acquired to measure FES and FDG uptake in the various tumors. FES uptake as assessed by PET imaging was 1.06±0.21 percent injected dose per gram of tissue (%ID/g) for MC7-L1 tumors and 0.47±0.08 %ID/g for MC7-L1 ERαKD tumors. MC4-L2 tumors had a FES uptake of 1.03±0.30 %ID/g, whereas its ERαKD equivalent was 0.51±0.19 %ID/g. Each ERαKD tumor had a significantly lower %ID/g value, by ~50%, than its ER+ counterpart. Biodistribution studies confirmed these findings and gave %ID/g values that were not significantly different from PET imaging data. FDG PET showed no significant uptake difference between the ER+ and ERαKD tumors, indicating that the metabolic phenotype of the ERαKD cell lines was not altered. FES PET imaging was able to reliably differentiate between tumors having differences in their ERα expression in vivo, in a mouse model. Quantitative data obtained by FES PET were in concordance with biodistribution studies and in vitro assays. It is concluded that FES PET imaging can likely be used to monitor subtle ER status changes during the course of hormone therapy.

Abstract B151: Src inhibitor dasatinib reduces the metastatic phenotype of breast cancer cells.

Abstract Subsequent to escaping the primary tumor, a successful metastatic tumor cell has to survive in the bloodstream, invade normal tissue at a secondary site, proliferate, and induce angiogenesis. Src is a non-receptor tyrosine kinase that is frequently over-expressed in malignances, and increased Src activity may be associated with poor patient prognosis. Src is well known to contribute to the metastatic phenotype of tumor cells through conferral of increased motility, migration, and invasion. Since these cellular functions are key components of the metastatic cascade, Src may be a novel target for breast cancer therapy. The present studies were undertaken to investigate whether molecular intervention in Src associated signaling could impact the metastatic phenotype of tumor cells. Specifically, we evaluated the in vitro and in vivo efficacy of the potent, selective and orally available Src inhibitor Dasatinib (BMS 354825) in the highly metastatic 4T1 murine mammary adenocarcinoma cell line. Dasatinib was evaluated in vitro for the ability to inhibit migration and invasion in the 4T1 cell line. The results showed that Dasatinib treatment (50–100 nM) significantly inhibited both migration and invasion in a dose dependent manner (6- to 10-fold for the doses studied). To examine the effect of Dasatinib on tumor cell-induced angiogenesis in vivo, an intradermal angiogenesis assay was used. Briefly, 4T1 tumor cells were inoculated intradermally on the ventral surface of athymic nude (Ncr nu/nu) mice and 3 days later the mice were euthanized and skin flaps were removed for visual quantification of the number of blood vessels intersecting each inoculate. Mice received daily doses of either Dasatinib (5 to 15 mg/kg) or vehicle by oral gavage. The results showed that Dasatinib treatment inhibited 4T1 tumor cell-induced angiogenesis by up to 60%. In summary, Dasatinib was found to significantly inhibit multiple cell functions critical to the successful dissemination of 4T1 breast cancer cells. The results suggest that Dasatinib may be useful in the reduction of breast cancer metastasis, and studies examining its impact on 4T1 metastases formation are currently ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B151.

Curcumin induces apoptosis in a murine mammary gland adenocarcinoma cell line through the mitochondrial pathway

Curcumin, a phenol in turmeric (Curcuma longa), has been studied for the last decade as a potential anticancer drug. It has been shown to reduce viability of the highly malignant, metastatic rat mammary gland cell line ENU1564 in culture and reduce metastasis of these cells injected into nude mice. The purpose of this study was to identify the mechanisms by which curcumin induces apoptosis in these ENU1564 cells in vitro, and to examine its effects on mitochondrial membrane potential and mitochondrial Ca2+ homeostasis. The results show that curcumin induced apoptosis in ENU1564 cells through the intrinsic pathway of apoptosis, as evident by an increase in mitochondrial Ca2+ accumulation and a decrease in mitochondrial membrane potential. However, treatment of the ENU1564 cells with the mitochondrial uniporter inhibitor RU-360 prior to curcumin treatment partially inhibited the curcumin effects. SKF-96365, a store-operated Ca2+ channel blocker, suppressed the curcumin effect on mitochondrial Ca2+. In addition, curcumin down-regulated the expressions of Bcl-2 and procaspase-3 and increased the production of reactive oxygen species in ENU1564 cells. These data suggest that the mitochondrial Ca2+ is the leading factor by which curcumin induced apoptosis in ENU1564 cells, followed by reactive oxygen species production and inhibition of Bcl-2 oncoprotein.

Cytotoxic effect of the aqueous extract of Portulaca Oleracea L. on some cell lines

he cytotoxic effect of the aqueous extract of Portulaca oleracea L. was tested in vitro against the cancer cell AMN3 (Murine Mammary Adenocarcinoma cell line) and the normal cell REF (Rat Embryo Fibroblasts). Proliferation was significantly reduced at high concentrations (300-1000 µg/ml) during the first 24 hrs. of contact on AMN3. while the extract showed no significant effect during different period time of exposure as compared with control against REF cells.

Abstract 548: Interaction between mammary adenocarcinoma cells and macrophages in a three-dimensional model of tumor spheroids

Abstract Solid tumors contain heterogeneous cellular subpopulation due to a limit supply of oxygen, nutrients and wastes. This microenvironment affects local cell growth, survival and overall therapeutic efficacy. Three-dimensional (3D) cell model reflect this situation and is a good model to study of the interaction with the immune system. Spheroids of the murine LM3 and human MCF-7 mammary adenocarcinoma cell lines were cultured with RAW 264.7 macrophages and human monocytes isolated from peripheral blood from normal volunteers respectively Spheroids were maintained in culture dishes pre-treated with 1.5% agar. In both tumor models, macrophages/monocytes were added after 10-13 days spheroids growth. Control cultures were maintained as monolayers in normoxic and hypoxic conditions (hypoxic chamber 1% O2 saturated during 4-6h). A growth curve of the spheroids were performed, reaching a peak of &amp;gt;300um (+/-) at day 15. In MCF-7 model, cell viability, monocyte distribution inside spheroids and protein expression were determined by hematoxiline-eosine staining and immunohitochemistry with specific antibodies. Results showed that 3D growth followed a concentric organization of proliferant (Ki67+), quiescent (Ki67-/Bcl-2+) and dead cells (CAS+) from periphery towards the hypoxic core (HIF-1+). In LM3 spheroids an enhanced arginase activity (140,64±2,05 vs. 18,17±1,22, p&amp;lt;0,05) and nitric oxide (NO) levels (10,21±1,15 vs 0,55±0,50, p&amp;lt;0,05) were also detected compared to LM3 cells growing as monolayer. It was also detected a decrease NO levels during spheroids growth (4 days: 38,7±8,3 vs. 13 days: 10,21±1,15). On the other hand, arginase activity and NO levels of spheroids cocultured with macrophages were lower than the spheroids alone (96,41±3,63 vs.317,7048±8,82 and 4,1±1,56 vs. 10,22±1,15; p&amp;lt;0,05 respectively). These results show that arginine metabolic pathways, either by NOS or arginase, are both more activated in spheroids than in tumor cell monolayer and coculture spheroids. In MCF7 and LM3 models, HIF-1alpha and iNOS expression was detected by western blot using hypoxic monolayers as a control. Culture system in 3D appears to be an ideal model to analyze the role of hypoxia and the mechanisms that modulate interactions between tumor and immune cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 548.

In vivo delivery of small interfering RNA to tumors and their vasculature by novel dendritic nanocarriers

New targets for RNA interference (RNAi)-based cancer therapy are constantly emerging from the increasing knowledge on key molecular pathways that are paramount for carcinogenesis. Nevertheless, in vivo delivery of small interfering RNA (siRNA) remains a crucial challenge for therapeutic success. siRNAs on their own are not taken up by most mammalian cells in a way that preserves their activity. Moreover, when applied in vivo, siRNA-based approaches are all limited by poor penetration into the target tissue and low silencing efficiency. To circumvent these limitations, we have developed novel polymerized polyglycerol-based dendrimer core shell structures to deliver siRNA to tumors in vivo. These cationic dendrimers can strongly improve the stability of the siRNA, its intracellular trafficking, its silencing efficacy, and its accumulation in the tumor environment owing to the enhanced permeability and retention effect. Here, we show that our dendritic nanocarriers exhibited low cytotoxicity and high efficacy in delivering active siRNA into cells. With use of human glioblastoma and murine mammary adenocarcinoma cell lines as model systems, these siRNA-dendrimer polyplexes silenced the luciferase gene, ectopically overexpressed in these cells. Importantly, significant gene silencing was accomplished in vivo within 24 h of treatment with our luciferase siRNA-nanocarrier polyplexes, as measured by noninvasive intravital bioluminescence imaging. Moreover, our siRNA-nanocarriers show very low levels of toxicity as no significant weight loss was observed after intravenous administration of the polyplexes. We show a proof of concept for siRNA delivery in vivo using a luciferase-based model. We predict that in vivo silencing of important cell growth and angiogenesis regulator genes in a selective manner will justify this approach as a successful anticancer therapy.