Rat Embryo Fibroblasts Research Articles

Study of cytotoxicity of purified toxin A produced from pseudomonas aeruginosa on cell lines

Pseudomonas aeruginosa is the leading etiology of gram negative bacteria causing diseases at most medical centers . The bacterium’s virulence depends on number of cell-associated and extracellular factors. Exotoxin A among these virulence factors are toxic for a wide variety of cell line, it inhibits protein synthesis in these lines as well as in numerous organs in animals models. In vitro cytotoxic effects of purified toxin A on two cell lines Rat embryo fibroblast cell line (REF) and on tumor cell line Rhabdomyosarcoma cell line (RD) were studied. Results revealed that toxin A had significant cytotoxic effects on both cell lines. Conclusion: In vitro evaluating the cytotoxic effects of purified toxin A produced from pseudomonas aeruginosa at different concentration on normal cell line Rat embryo fibroblast cell line (REF) ) and on tumor cell line Rhabdomyosarcoma cell line (RD) revealed that purified exotoxin A has significant inhibitory effects on REF cell line which was dose and time dependent. On the other hand cytotoxic effects of purified toxin A on tumor cell line (RD) reveal that inhibition in cell viability was dependent on concentration of purified toxin A but independent on time. Key words : Toxin A, cytotoxicity assay, cell line

The Cytotoxic Activity of Punica granatum on Growth of Hela and REF Cell Lines

The crude aqeouse extracts of punica granatum peel were assessed for their cytotoxic activity assay.This study utilized two different types of cell lines, Hela (Cervical human carcinoma) and REF (Rat Embryofibroblast).The results showed that the aqueous extract of punica granatum peel exhibited high cytotoxicity against theREF cell line with an inhibition value of 97.3 μg/ml after 24 h incubation. However, punica granatum peeldisplayed less toxicity against the Hela cell line with an inhibition value 24.4 μg/ml after 24 h incubation andthis effect was dose- and time-dependent.In conclusion, the results demonstrated that punica granatum peel is potently cytotoxic against REF cell,and lesser extend against Hela cells.

The in vitro assessment of dipyridophenazine complexes in H-ras oncogene transformed rat embryo fibroblast 5RP7 cell line.

Purpose The aim of this study is to detect apoptotic and cytotoxic/antiproliferative effects of a ligand substance and its metal derivatives. The substances were investigated by using an h-ras oncogene transformed rat embryo fibroblast cell line (5RP7). Methods The cytotoxic influences of dipyrido[3,2-a:2',3'c]phenazine ligand, dipyrido[3,2-a:2',3'c] phenazine-platinum(II) complex ([Pt(dppz)Cl2]) and dipyrido[3,2-a:2',3'c] phenazine-gold(III) complex ([Au(dppz)Cl2]Cl) were determined with MTT (3[4,5-dimetiltiyazol2-yl]-2,5-difeniltetrazolyum bromid) assay on 5RP7 cells. Results Dipyrido[3,2-a:2',3'c] phenazine, dipyrido[3,2-a:2',3'c] phenazine-platinum(II) complex ([Pt(dppz)Cl2]) and dipyrido[3,2-a:2',3'c] phenazine-gold(III) complexes ([Au(dppz)Cl2]Cl) caused significant increase in cytotoxicity in a dose and time dependent manner. The effects of dipyridophenazine ligand (dppz) and its metal derivatives on apoptosis were monitorized using cytotoxic dose (10μM) DAPI fluorescent staining. It was shown that dppz and its compounds induced apoptosis. Conclusions These findings show that dpzz and its complexes can be studied as novel alternative chemotherapeutics in cancer treatment.

Targeted elimination of senescent Ras-transformed cells by suppression of MEK/ERK pathway.

The Ras-Raf-MEK-ERK pathway plays a central role in tumorigenesis and is a target for anticancer therapy. The successful strategy based on the activation of cell death in Ras-expressing cells is associated with the suppression of kinases involved in Ras pathway. However, activation of cytoprotective autophagy overcomes antiproliferative effect of the inhibitors and develops drug resistance. We studied whether cellular senescence induced by HDAC inhibitor sodium butyrate in E1a+cHa-Ras-transformed rat embryo fibroblasts (ERas) and A549 human Ki-Ras mutated lung adenocarcinoma cells would enhance the tumor suppressor effect of MEK/ERK inhibition. Treatment of control ERas cells with PD0325901 for 24 hresults in mitochondria damage and apoptotic death of a part of cellular population. However, the activation of AMPK-dependent autophagy overcomes pro-apoptotic effects of MEK/ERK inhibitor and results in restoration of the mitochondria and rescue of viability. Senescent ERas cells do not develop cytoprotective autophagy upon inhibition of MEK/ERK pathway due to spatial dissociation of lysosomes and autophagosomes in the senescent cells. Senescent cells are unable to form the autophagolysosomes and to remove the damaged mitochondria resulting in apoptotic death. Our data show that suppression of MEK/ERK pathway in senescent cells provides a new strategy for elimination of Ras-expressing cells.

In Vitro Cytotoxic Study for Purified Resveratrol Extracted from Grape Skin Fruit Vitis vinifera


 This study was conducted with the aim to extract and purify a polyphenolic compound “ Resveratrol” from the skin of black grapes Vitis vinifera cultivated in Iraq. The purified resveratrol is obtained after ethanolic extraction with 80% v/v solution for fresh grape skin, followed by acid hydrolysis with 10% HCl solution then the aglycon moiety was taken with organic solvent
 ( chloroform). Using silica gel G60 packed glass column chromatography with mobile phase benzene: methanol: acetic acid 20:4:1 a partial purified resveratrol was obtained then preperative thin layer chromatography technique yielded pure crystals identified as resveratrol (mixture of two isomers cis and trans) in relation to resveratrol standard (35 mg resveratrol crystals / 0.5 kg fresh grape skin was obtained as a result of these processes ). The study was also employed an in vitro evaluation the cytotoxic effect of pure resveratrol on some cell line including : the murine mammary adenocarcinoma AMN-3 cell line, the human laryngeal carcinoma (Hep-2) cell line and the Rat embryo fibroblast (Ref) cell line, at different concentrations and different expousure time. The cytotoxic effect of the pure resveratrol was studied in comparison with trans- resveratrol standard in concentrations of (12.5, 25, 50 and 100) µg/ml for both purified resveratrol and the standard, also the comparism included methotrexate drug in concentrations (0.05 , 0.1 , 0.2 and 0.4) µg/ml toward the growth effects of the three types of cell lines and at three exposure times (24, 48 and 72) hours. The cytotoxic inhibition effect for the purified extracted resveratrol revealed that the highest significant effect (P<0.01) was achieved after 24 hr of exposure on both AMN-3 and Ref cell lines. Hep-2 cell line responded to extracted resveratrol in different manners.
 Keywords: Resveratrol , grape skin , polyphenols, cytotoxic study

The antiapoptotic gene bcl-2 prevents reactivation of the senescence program induced by the histone deacetylase inhibitor sodium butyrate in rat fibroblasts transformed by the oncogenes E1A and c-Ha-Ras

The role of the gene bcl-2 conferring resistance to apoptosis in the activation of the cellular senescence program induced by the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBut) has been studied in transformed rat fibroblasts. The work was carried out in the rat embryo fibroblast cell line transformed by the oncogenes E1A, cHa-Ras, and bcl-2 (ERasBcl cells) resistant to apoptosis. The parent line of E1A-and cHa-Ras-transformed cells (ERas cells) was used as a control. It has been shown that NaBut suppresses the proliferation of ERasBcl cells to a much lesser extent than do the control ERas cells, in spite of the delay in cell cycle progression in the G1 phase for both lines. NaBut-induced hypertrophy is less marked in ERasBcl cells than in ERas cells due to the lower level of activation of the mTORC1 complex controlling protein synthesis and ribosome biogenesis. The activation of mTORC1 was assessed from the phosphorylation of its targets: ribosomal protein S6 and the inhibitor of translation initiation factor eIF4E (4E-BP1). Investigation of the dynamics of the level of one of the major markers of autophagosome formation (protein LC3) has shown the transient character of NaBut-induced accumulation of lipid-bound form LC3-II marking autophagosome membranes in bcl-2-expressing transformants in contrast to the ERas line. In addition, in ERasBcl cells there was no activation of the senescence-associated β-galactosidase (the marker for senescent cells). The results obtained suggest that the high level of expression of bcl-2, which blocks apoptotic cell death, prevents the implementation of the NaBut-induced tumor-suppressive cellular senescence program.

Melatonin induces antiproliferative activity through modulation of apoptoticpathway in H-ras oncogene transformed 5RP7 cells

In the present study, cytotoxic and apoptotic effects of melatonin were investigated on H-ras oncogene transformed rat embryo fibroblast 5RP7 cells. Melatonin inhibited cell growth and induced apoptosis in 5RP7 cells. We showed inhibition of cell proliferation that was time- and dose-dependent in 5RP7 cells for 24 and 48 h. Melatonin was not toxic in NIH/3T3 primary mouse embryonic fibroblast cells at low doses for 24 and 48 h. The IC50 (380 mM) value of melatonin for 48 h was chosen for advanced assays. The percentages of early/late apoptotic cells to which melatonin (IC50: 380 mM) was administered were increased 6.2-fold in 5RP7 cells compared to controls for 48 h. The melatonin treatment resulted in increased caspase-3 activity and reduced the mitochondrial membrane potential in the 5RP7 cells for 48 h. The cell cycle arrest caused by melatonin was observed in G1 and G2 phases in the cell cycle analyses in 5RP7 cells for 48 h. These findings show that melatonin induces cell death and apoptosis in H-ras oncogene transformed 5RP7 cells. Melatonin may be an anticancer agent against H-ras oncogene activated cancer cells.

The role of the MEK/ERK pathway in regulation of HDACI-induced senescence of transformed rat embryo fibroblasts

A key regulator of cellular senescence, mTORC1 complex, is a target of many signaling cascades, including Ras/Raf/MEK/ERK cascade. In this paper, we investigated the role of the MEK/ERK branch of this cascade in the process of cellular senescence induced by sodium butyrate (NaBut), a histone deacetylase inhibitor (HDACI), in transformed rat-embryo fibroblasts. Suppression of MEK/ERK activity by inhibitor PD0325901 did not prevent activation of mTORC1 complex induced by NaBut treatment. Inhibition of MEK/ERK increased mTORC1 activity and activated mTORC2 complex. Activation of mTOR-containing complexes was accompanied by reorganization of the actin cytoskeleton (formation of actin stress fibers) and the appearance of cellular senescence markers. In contrast to NaBut-induced senescence, no protein accumulation was observed, probably due to increased activity of the degradation processes. Furthermore, senescence induction under suppression of MEK/ERK drastically decreased the cell viability, Thus, NaBut-induced senescence upon suppressed activity of the MEK/ERK branch of MAP kinase cascade has a more pronounced tumor-suppressing effect that is manifested by activation of both mTOR complexes, reorganization of the actin cytoskeleton and protein degradation.

Failure of cell cleavage induces senescence in tetraploid primary cells.

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

Evaluation of antimicrobial and anticancer activity of Rosemary Rosmarinus oficinalis leaves extracts on pathogenic bacteria and cancer cell lines

The present study was carried out to detect the active constitutions extracted from Rosmarinus oficinalis leaves by 70% methanol, the dried weight of aqueous extract (F1) were 12%(w:w) contain polyphenols represented by phenols, flavonoids and coumarins as a major compounds , while the dried weight of organic extract, [chloroform(F2)] was 2.8%(w:w) represented as terpenoids, tannins and coumarins detected by qualitative chemical analysis. Antimicrobial activity of Polyphenols and terpenoids were tested against four pathogenic bacteria isolates including: Escherichia coli; Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus cereus ,which grown on Mueller Hinton agar (MHA) plates and then treated with different concentrations of rosemary extract (F1) and (F2) ranged from 0.15-1.2mg/ml, the results revealed that the higher inhibition zone obtained by F1 were 35mm in S. aureus, 24mm in B. cereus and lowest inhibition were 16mm in E. coli and P. aeruginosa at concentration 1.2mg/ml, and the inhibition were depends on extract concentrations. The MIC value of F1 was 600 µg/ml against S. aureus, 1200 µg/ml against B. cereus and 2400 µg/ml against E. coli and P. aeruginosa, while the MICS values of F2 were 2400 µg/ml against S. aureus and P. aeruginosa, and 4800 µg/ml against E. coli and B. cereus. Furthermore calculating index fractional inhibitory concentration (∑FIC) of rosemary extracts (F1 or/and F2), the ∑FICA ranged from synergistic to additional on E. coli and P. aeruginosa respectively, antagonistic on S. aueras and B. cereus, while the ∑FICB index showed synergistic effects on all tested organisms except the additional effect on P. aeurginosa on the basis of resultant zone of inhibition. The anticancer effects of (F1) were used on three cell lines, human larynx epidermoid carcinoma (Hep-2), mammary adenocarcinoma (AMN-3), rhabdomyosarcoma (RD) and one normal cell line, Rat embryo fibroblast (REF) at different concentrations ranged from 62.5-1000 µg/ml. The highest inhibition rate (IR) was 82.41%, 85% on RD, 52.62%, 75.48% on Hep2, 39.14% and 49.75% on AMN-3 at concentrations 500 and 1000µg/ml after 72h exposure time respectively, while IR of Ref cell line exhibit low inhibition rate ranging from 5.8 to 15.7% after 72h in the same conditions.

Abstract A176: Total oligomeric flavonoids (TOF) of the herb tubers Cyperus rotundus induce growth inhibition and apoptosis in some cancer cell lines, a preliminary study.

Abstract In Iraq, just like other eastern countries, local folklore medicinal plants represent an unlimited resource popularly known to treat wide spectrum of diseases, including cancer. Traditionally these plants are used without having any fundamental understanding for their biological action; the tubers of Cyperus rotundus herb are one of such resources. This study evaluates and estimates the nature of the anticancer properties of total oligomeric flavonoids (TOF) extracted from Cyperus rotundus tubers and its main effector mechanism against four different cell lines. The cell lines invested for this purpose were human glioblastoma cells (AMGM), mice mammary adenocarcenoma cells (AMN3), rat embryo fibroblast cells (REF) and African green monkey kidney cells (Vero). Total oligomeric flavonoids (TOF) were extracted from the dry rhizomes of locally harvested Cyprus rotundus herb. The treated cells were cultured in the presence of TOF extracts at different concentrations extended from 50 µg/ml to 500 µg/ml with 50 µg/ml increment each time for 24 hr. Cells viability was determined by MTT assay and calculated as a percentage of control untreated cells. Apoptosis induction in the TOF treated cells was detected using cell membrane integration assay in adherent cells, DNA fragmentation assay, acridine orange/propidium iodide staining in adherent cells, and disruption of mitochondrial membrane potential. The prepared TOF extract was a yellowish crystalline powder that can be totally dissolved in distilled water without any remission. The extract inhibited both AMGM and AMN3 cancer cells proliferation by 67.09% and 52.41% at concentration of 350 µg/ml (IC50) during incubation time of 24 hr. Both REF and Vero cell lines were totally resistant to TOF at 500 and even for 1000 µg/ml which are the highest concentrations tested. Cell membrane integrity was destructed in AMGM, AMN3, REF, and Vero cell lines by 54.50%, 53.67%, 3.5%, and 11.5%, respectively after 24 hr of TOF treatment at IC50. Both AMGM and AMN3 cells possessed a smear of DNA fragmentation extended from 3000 to 400. REF cells did not show any fragments of their DNA with MW lower than 10,000. Cells and nucleus morphological changes for apoptosis were clearly evident in both AMGM and AMN3 cancer cells when treated with IC50 for 24 hr as assessed using acridine orange and propidium iodide staining. The distraction of mitochondrial membrane potential for AMGM and AMN3 cells treated with IC50 of TOF for 24 hr indicated that 65.33% and 75.76% of the cells were apoptotic cells respectively. The treatment with 1000 µg/ml increased the apoptotic cells to 89.5% and 79.89% respectively. These results indicate that TOF of Cyperus rotundus have apparent antiproliferative activity against the tested cancer cells due to the induction of apoptosis and can be claimed to have a therapeutic potential for cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A176. Citation Format: Amer T. Al-Saeedi. Total oligomeric flavonoids (TOF) of the herb tubers Cyperus rotundus induce growth inhibition and apoptosis in some cancer cell lines, a preliminary study. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A176.

Cytomegalovirus Expresses the Chemokine Homologue vXCL1 Capable of Attracting XCR1 + CD4 − Dendritic Cells

Cytomegaloviruses (CMV) have developed various strategies to escape the immune system of the host. One strategy involves the expression of virus-encoded chemokines to modulate the host chemokine network. We have identified in the English isolate of rat CMV (murid herpesvirus 8 [MuHV8]) an open reading frame encoding a protein homologous to the chemokine XCL1, the only known C chemokine. Viral XCL1 (vXCL1), a glycosylated protein of 96 amino acids, can be detected 13 h postinfection in the supernatant of MuHV8-infected rat embryo fibroblasts. vXCL1 exclusively binds to CD4(-) rat dendritic cells (DC), a subset of DC that express the corresponding chemokine receptor XCR1. Like endogenous rat XCL1, vXCL1 selectively chemoattracts XCR1(+) CD4(-) DC. Since XCR1(+) DC in mice and humans have been shown to excel in antigen cross-presentation and thus in the induction of cytotoxic CD8(+) T lymphocytes, the virus has apparently hijacked this gene to subvert cytotoxic immune responses. The biology of vXCL1 offers an interesting opportunity to study the role of XCL1 and XCR1(+) DC in the cross-presentation of viral antigens.

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

Mitochondrial Activity of the Locally Established Rat Embryo Fibroblast Cell Line Through Different Passages

Background: Mitochondria are the cell’s powerhouse, the site where the vast majority of ATP is synthesized. Mitochondrial activity represent a central checkpoint for detection of the difference between cancer cells and normal cells at the metabolic profile.Objective: To find out if there is a correlation between in vitro transformation and mitochondrial activity, by measuring the activity during clonal evolution of the locally established rat embryo fibroblast (REF) cell line throughout studying different passages.Materials & Methods: The (REF) cell line, was in vitro cultured. Mitochondria were isolated by differential centrifugation following Mitochondria Isolation Kit. Enzymatic activity of intact mitochondria has been measured using Cytochrome C Oxidase Activity Assay Kit. The decrease in absorbance at 550nm as horse heart ferrocytochrome c was oxidized has been monitored.Results: Depending on the conducted colorimetric assay, cytochrome c oxidase activity was measured, for the three different passages (42, 72 and 91) of REF cell line. At 550 nm, spectral data were valued (0.247, 0.723 and 1.318) unit/ml for three passages of REF cells (42, 72 and 91) respectively.Conclusion: There was a significant relationship between the mitochondrial activity and the age (passage number) for these in vitro cultured cells. These variations probably due to the transformational events that have been occurred during long-term continuous subculture

Absence of a robust innate immune response in rat neurons facilitates persistent infection of Borna disease virus in neuronal tissue.

Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-α. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-α. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-α. Intriguingly, in contrast to rat neurons, IFN-α treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.

IN VITRO EFFECTS OF CEFTRIAXONE ON GROWTH OF HUMAN RHABDOMYOSARCOMA (RD) AND ON RAT EMBRYO FIBROBLASTS (REF) CELL LINES

Many reports demonstrated that antibiotics like cefazoline, ciprofloxacin, trimethoprime - sulfamethoxazo le and others have the ability to inhibit growth of various cell lines. Thus, this study was designed to investigate whether or not ceftriaxone may possess cell growth inhibition using In vitro study and utilizing two cell lines ( human rhabdomyosarcoma (RD ) and rat embryo fibroblasts (REF). Various concentrations of Ceftriaxone (62.5, 125, 250, 500 and 1000 mcg/ml) were utilized in this study. The drug relatively caused concentration - dependent inhibition on growth of the intended cell lines used in this stu dy, where, the growth inhibition induced by the drug was statistically significant at 125mcg/ml and above. The results obtained from this work encourage further study of the possibility of clinical application of ceftriaxone to prevent the occurrence of di fferent tumors.

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.

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

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.

Podocytes require the engagement of cell surface heparan sulfate proteoglycans for adhesion to extracellular matrices

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.