MDA-MB231 Cell Lines Research Articles

1066 A simple method to prepare tumour stem cells from the human breast cancer cell line MDA-MB 231

1066 A simple method to prepare tumour stem cells from the human breast cancer cell line MDA-MB 231

ANTI-CANCER EFFECT OF ICD-85(VENOM DERIVED PEPTIDES) ON MDA-MB231 CELL LINE (IN VITRO) AND EXPERIMENTAL MICE WITH BREAST CANCER (IN VIVO)

Breast cancer has become common in developing and developed countries. Alarming increase in this disease as a leading cause of death in women is a concern of all. Despite the significant improvements in the management of breast cancer, the survival rate is not more than 20% - 25%. For this study, MDA-MB231 cell line was used and the effect of ICD-85 (biologically active peptides from venomous animals) was assayed by measuring the activity of the cytosolic enzyme lactate dehydrogenase(LDH) released into the culture medium after membrane legions. Morphological changes of cells were checked in control and cells incubated with ICD-85 as chemo preventive agent. Results showing in test groups - incubation with 10mg/ml dose of ICD-85 had decreased cytoplasmic branch. Some cells were ruptured and lost the continuity of their surrounding membranes, and number of cells was decreased. On the other hand, when ICD-85 was used as an anticancer treatment of mice with breast cancer, it was shown that ICD-85 can prevent the growth of breast tumor, increase the life duration of experimental mice and inhibit angiogenesis in breast tumor as compared with control group. It seems that ICD-85 acts at the membrane level and can prevent the cell growth as well as producing apoptosis (cell suicide) for breast cancer cells.

Modulation of the Membrane Type 1 Matrix Metalloproteinase Cytoplasmic Tail Enhances Tumor Cell Invasion and Proliferation in Three-dimensional Collagen Matrices

Increasing evidence suggests that the cytoplasmic tail of membrane type 1 matrix metalloproteinase (MT1-MMP) is subject to phosphorylation and that this modification may influence its enzymatic activity at the cell surface. In this study, phosphorylated MT1-MMP is detected using a phospho-specific antibody recognizing a protein kinase C consensus sequence (phospho-TXR), and a MT1-MMP tail peptide is phosphorylated by exogenous protein kinase C. To characterize the potential role of cytoplasmic residue Thr(567) in these processes, mutants that mimic a state of either constitutive (T567E) or defective phosphorylation (T567A) were expressed and analyzed for their functional effects on MT1-MMP activity and cellular behavior. Phospho-mimetic mutants of Thr(567) exhibit enhanced matrix invasion as well as more extensive growth within a three-dimensional type I collagen matrix. Together, these findings suggest that MT1-MMP surface action is regulated by phosphorylation at cytoplasmic tail residue Thr(567) and that this modification plays a critical role in processes that are linked to tumor progression.

Tissue Transglutaminase Is an Essential Participant in the Epidermal Growth Factor-stimulated Signaling Pathway Leading to Cancer Cell Migration and Invasion

Epidermal growth factor (EGF) exerts pleiotropic effects during oncogenesis, including the stimulation of cell migration and invasiveness. Although a number of traditional signaling proteins (e.g. Ras and Rho GTPases) have been implicated in EGF-stimulated cancer cell migration, less is known about the identity of those proteins functioning further downstream in this growth factor pathway. Here we have used HeLa carcinoma cells as a model system for investigating the role of tissue transglutaminase (TGase), a protein that has been linked to oncogenesis, in EGF-stimulated cancer cell migration and invasion. Treatment of HeLa cells with EGF resulted in TGase activation and its accumulation at their leading edges, whereas knocking down TGase expression, or treating cells with a TGase inhibitor, blocked EGF-stimulated cell migration and invasion. We show that EGF signaling through Ras and c-Jun N-terminal kinase is responsible for targeting TGase to the leading edges of cells and activating it. The requirement for EGF to properly localize and activate TGase can be circumvented by the expression of oncogenic Ras (G12V), whose ability to stimulate migration is also dependent on TGase. We further show that, in the highly aggressive breast cancer cell line MDAMB231, where EGF stimulation is unnecessary for migration and invasive activity, TGase is already at the leading edge and activated. These findings demonstrate that TGase plays a key role in cancer cell motility and invasiveness and represents a previously unappreciated participant in the EGF pathway that stimulates these processes in cancer cells.

Efficient induction of apoptosis by doxorubicin coupled to cell-penetrating peptides compared to unconjugated doxorubicin in the human breast cancer cell line MDA-MB 231

Doxorubicin (Dox) is a commonly used drug to treat various types of cancers. Previously, we demonstrated that coupling Dox to cell-penetrating peptides (CPPs) represent a valuable strategy to overcome drug resistance in MDA-MB 231 breast cancer cells. In the present study, we evaluated the properties of these Dox conjugates (Dox–CPPs) in terms of apoptosis induction. Dox–CPPs were found to induce apoptotic death in MDA-MB 231 cells at a lower dose than that needed for unconjugated Dox. Cell death induction was associated with Bax oligomerisation, release of cytochrome c, caspase activation, chromatin condensation and internucleosomal degradation. However, whereas Bcl-2 overexpression was very potent in inhibiting apoptosis triggered by Dox, this anti-apoptotic protein was largely inefficient in preventing Dox–CPPs-induced apoptosis. These observations suggest that mitochondrial disruption is the main event in Dox-induced apoptotic signaling but that Dox–CPPs are probably able to trigger additional apoptotic pathways independent of mitochondrial events. Thus, the higher efficacy of Dox conjugated to CCPs in apoptosis induction might not be due exclusively to increased drug accumulation but also to the activation of multiple apoptotic pathways.

American Ginseng Inhibits Induced COX-2 and NFKB Activation in Breast Cancer Cells

Epidemiologic evidence suggests reduced breast cancer mortality in users of American Ginseng (AG) (Panax quinquefolium). We hypothesized that AG extract decreases proliferation of human breast cancer cells via an anti-inflammatory effect applicable to the prevention of breast and other cancers. A defined lyophilized aqueous extract of AG (LEAG) was dissolved in DMSO 1mg/mL, and serially diluted in saline. The cell lines MDA MB 231 and MCF7 were stimulated with the phorbol ester PDBu and treated with 100-500 mcg/mL LEAG. Proliferation was measured by MDA assay. Induced COX-2 expression was assayed by ELISA. Activation of NFkappaB by phosphorylation of the p65 subunit was quantified by CASE (cellular activation of signaling ELISA). Both cell lines had reduced proliferation when treated with LEAG. PDBu stimulation of MDA MB 231 increased expression of the COX-2 protein 20-fold at 48 hours (P<0.005). COX-2 protein expression remained at baseline concentrations in PDBu- treated MDA MB 231 cells exposed to 100 mcg/mL LEAG. The CASE assay showed a 4-fold increase in p65 activation 24 hours after PDBu treatment in normal medium, while phosphorylated p65 dropped below baseline in the cells treated with PDBu plus LEAG. In MDA MB 231, COX-2 was inducible with PDBu. This induced COX-2 expression was blocked by 100 microgram/mL LEAG in a time course consistent with the decline in the activated p65 subunit of NFkappaB. These results provide an anti-inflammatory mechanism for a possible anti-cancer effect of American Ginseng.

Design, preparation, in vitro and in vivo evaluation of 99mTc-N 2S 2-Tat(49–57)-bombesin: A target-specific hybrid radiopharmaceutical

The gastrin-releasing peptide receptor (GRP-r) is over-expressed in various human tumors. Recently, 99mTc-EDDA/HYNIC-Lys 3-bombesin ( 99mTc-BN) was reported as a radiopharmaceutical with specific cell GRP-r binding and images in breast cancer patients demonstrated distinct radioactivity accumulation in malignant tissue. The HIV Tat-derived peptide has been used to deliver a large variety of cargoes into cells. Therefore, a new hybrid radiopharmaceutical of type 99mTc-N 2S 2-Tat(49–57)-Lys 3-bombesin ( 99mTc-Tat-BN) would increase cell uptake. The aim of this research was to prepare and assess in vitro and in vivo uptake kinetics in cancer cells of 99mTc-Tat-BN and to compare its cellular internalization with that of 99mTc-BN. Structures of N 2S 2-Tat-BN and Tc(O)N 2S 2-Tat-BN were calculated by an MM procedure. 99mTc-Tat-BN was synthesized and stability studies carried out by HPLC and ITLC-SG analyses in serum and cysteine solutions. In vitro internalization was tested using human prostate cancer PC-3 cells and breast carcinoma cell lines MDA-MB231 and MCF7. Biodistribution was determined in PC-3 tumor-bearing nude mice. Results showed a minimum energy of 271 kcal/mol for N 2S 2-Tat-BN and 300 kcal/mol for Tc(O)N 2S 2-Tat-BN. 99mTc-Tat-BN radiochemical purity was >90%. In vitro studies demonstrated stability in serum and cysteine solutions, specific cell receptor binding and internalization in three cell lines was significantly higher than that of 99mTc-BN ( p < 0.05). The tumor-to-muscle radioactivity ratio was 8.5 for 99mTc-Tat-BN and 7 for 99mTc-BN. Therefore, this hybrid is potentially useful in breast and prostate cancer imaging.

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

Activity and mechanism of action of histone deacetylase inhibitors in trastuzumab resistant breast cancer.

Abstract Abstract #3060 Introduction: The Her2 antibody trastuzumab (T) is the first biological therapy that has proven the value of Her2 blockade in breast cancer. However, not all patients respond to trastuzumab and relapses occur after this treatment. Several studies suggest that histone deacetylase inhibitors (HDACis) are active in Her2-amplified breast cancer but none of them have addressed the role of HDACis in trastuzumab-resistant breast cancer. HDACis act by inhibiting the deacetylation of histones and other non-histone proteins such as Hsp90. Our goal is to study the activity, optimal sequencing and mechanism of action of HDACis in Her2-amplified cell lines, which demonstrate either de novo or acquired resistance to trastuzumab.&amp;#x2028; Methods: T-sensitive and Her2-overexpressing BT474 &amp; SkBr3, T-resistant BT474 and de novo T-resistant cell lines MDA-MB361, MDA-MB453, UACC812 and UACC893 were used for all experiments. The HDACi TSA (0-1uM) and trastuzumab at 100ug/mL were used for proliferation assays applying WST1 reagent (Roche). Western-immunoblotting was performed using cell lysates from untreated and treated cells. Proteolytic cleavage of caspase-3 and PARP was analyzed as indicator for apoptosis, Akt expression and phosphorylation for survival, Her2 and Erk1/2 expression and phosphorylation for proliferation and Hsp90 and Hsp70 were analyzed as key enzymes for chaperone activity.&amp;#x2028; Results: TSA inhibited proliferation in all cell lines [IC50 100-300 nM]. The addition of trastuzumab was antagonistic in de novo T-resistant cell lines MDA MB 453, UACC 812 and 893 but did not change the effect induced by TSA in acquired T-resistant cells (BT474_H100). Her2 expression decreased in TSA and TSA+T treated cells but not in cells treated with trastuzumab alone. Erk1/2 phosphorylation decreased in TSA and TSA+T treated cells but was not affected by trastuzumab alone. Similar phosphorylation patterns were observed regarding Akt. Of note, Akt, Erk1/2, Hsp90 and Hsp70 levels did not change. Finally, TSA induced caspase-3 and PARP cleavage when given as single agent or in combination with trastuzumab.&amp;#x2028; Discussion: This study suggests that HDACis are active in both trastuzumab-sensitive and de novo or acquired trastuzumab-resistant cell lines. However, use of trastuzumab together with TSA may be antagonistic in tumors that are resistant against trastuzumab. Further studies are required in these cell lines in order to elucidate the mechanism of resistance against trastuzumab and the mechanism of the here observed antagonistic effect between trastuzumab and TSA. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3060.

Orally active gnRH-antagonist AEZS-115 inhibits growth of human ovarian and breast cancers in vitro.

Abstract Abstract #3065 Introduction: AEZS-115 is an orally active peptidomimetic antagonist of GnRH, which has been shown to block the pituitary GnRH receptor in nanomolar range. Recently an increasing number of human cancers have been demonstrated to express receptors for GnRH, which mediate growth promoting signals. Accordingly, we tested the antitumor activity of AEZS-115 in in vitro models of human breast and ovarian cancer.&amp;#x2028; Material and Methods: Human ovarian SKOV3, Ovcar 3 as well breast cancer cell line MDA-MB 468 were analyzed for GnRH receptor expression by immunocytochemistry. To screen for a putative anti-tumor effect, these cell lines were incubated with increasing concentrations of AEZS-115, peptidic GnRH-antagonist Cetrorelix and GnRH-agonist Triptorelin (1, 10, 100 µm) for 48 hours and the number of viable cells was determined by crystal violet staining as well as by ATP-dependent luminometric assay.&amp;#x2028; Results: Cell lines SKOV3, OVCAR3 and MDA-MB 468 expressed GnRH I receptors as demonstrated by immunehistochemistry. Both GnRH-antagonists dose dependently inhibited growth of all three cell lines, while GnRH-agonist Triptorelin showed marginal growth inhibition. At 10 µM AEZS-115 inhibited cell growth by 40-60%, at 100 µM growth inhibition was 60-80%. Inhibition with Cetrorelix at 100 µM ranged from 20-40%, while only minor effects on cell growth were seen at 10 µM. These results obtained by crystal violet staining were confirmed by additional luminometric evaluation of the ATP content after treatment with the respective substances.&amp;#x2028; Conclusions: To our knowledge here we report for the first time in vitro anti-cancer activity for a peptidomimetic GnRH antagonist.&amp;#x2028; AEZS-115 showed substantial anti-tumor activity in human ovarian and breast cancer cell lines, while GnRH-agonist Triptorelin showed only minor growth inhibition. Surprisingly AEZS-115 was somewhat more potent than peptidic GnRH-antagonist Cetrorelix. Due to the good antitumor-activity and the possible oral administration AEZS-115 should be a promising candidate for in vivo studies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3065.

Development of a breast cancer brain metastases model to study 131I radioablative therapy.

Abstract Abstract #2011 Background: An increasing number of women develop brain metastases (BM) after breast cancer (BC) treatment. A large proportion of these are estrogen/progesterone receptor-negative (ER-/PR-) and/or Her-2/neu overexpressing tumors. 131I radioablative therapy may provide a therapeutic alternative to treat metastases at this anatomic sanctuary since over 70% of invasive breast cancers, including a majority of ER- tumors and some brain metastases (unpublished data) express the sodium-iodide symporter (NIS). This approach relies on the success of radioiodide as a targeted treatment for thyroid cancers. To test this concept, we developed a BC BM model using tumor cells engineered to express NIS.&amp;#x2028; Methods: MDAMB231 and SKBr3 cell lines were transduced with a lentiviral vector carrying a bicistronic cassette with NIS and the firefly luciferase (Fluc) genes separated by an internal ribosomal entry site. Single cell clones were selected and characterized for iodide uptake and bioluminescence. NIS-Fluc-MDAMB231 or NIS-Fluc SKBr3 cells (2.5 x 106 cells) were implanted subcutaneously (sc) in the mammary fat pad (mfp) of nude mice (NCr nude; 5-6 weeks old; n=5). NIS-Fluc mfp tumor xenografts were then explanted, 1x1 mm pieces excised and inserted stereotactically into the basal ganglia of the animal. All tumor development was monitored by serial in vivo bioluminescent imaging. Once established, brain tumors were excised, dissociated, established in tissue culture and re-implanted sc in the mfp of a new set of mice. Successive passages in the mfp then in the brain were performed in an attempt to increase tumor take. A second strategy tested with MDAMB231 cells consisted of direct implantation of cells into the basal ganglia. NIS expression was evaluated on tissue sections with a polyclonal antibody raised against the C-terminus of the human NIS.&amp;#x2028; Results: All mice survived and were healthy in appearance. Intracranial implantation of mfp xenografts was highly successful with 66% take in both MDAMB231 (after two passages) and SKBr3 (after first passage). Bioluminescent imaging revealed sustained growth of tumors for more than 4 weeks. Microscopically, the explanted brain tumors had a cellular appearance without stromal cell or lymphocytic infiltration and were congruent with the histology of mfp xenografts. However, the tumor cell population was heterogeneous as NIS expression was present with plasma membrane staining in about 50% of SKBr3 and 15% of MDAMB231 cells. Direct cell implantations failed as no discernible bioluminescence was noted over a period of 3 weeks and no visible tumor at necropsy.&amp;#x2028; Conclusions: A BCBM model has been developed by implanting intracranially mfp xenografts obtained with ER-/PR- +/- Her-2/neu overexpressing cells. Using this model, it will be possible to evaluate the effects of 131I on NIS-expressing BCBM. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2011.

Expression of MDA7/IL-24 human breast cancer and the molecular impact.

Abstract Abstract #2060 INTRODUCTION. MDA-7 (melanoma differentiation associated gene-7), also known as IL-24, was initially identified from cancer cells and was found to be up-regulated in melanoma cells. Forced expression of MDA7 in cancer cells was found to be growth inhibitory. MDA7/IL-24 operates in cells via its receptor, MDA7R/IL-24R, which includes at least the IL-20Rα and IL-20Rβ complex and the IL-22R and IL20Rβ complex. The present study investigated the impact of MDA7 on the growth and motility of breast cancer cells and the clinical relevance of MDA7 expression in mammary tumour tissues.&amp;#x2028; MATERIALS AND METHODS. Human breast cancer cell lines MDA MB-231 and MCF-7 were used for in vitro testing. The response of cancer cells to rhMDA7 in growth and cellular motility was determined using growth assay and ECIS (electric cell impedance sensing, and confirmed by wounding assay). Localisation of MDA7 in mammary issues was assessed using conventional immunohistochemical method. Levels of MDA7 transcript in fresh frozen breast tissues were determined using quantitative PCR analysis and analysed against clinical and pathological information.&amp;#x2028; RESULTS. Recombinant human MDA-7 only had a marginal effect on the in vitro growth of the breast cancer cell lines used. However, rhMDA had a significant effect on the migration of breast cancer cells. Cells treated with rhMDA-7 showed a slower rate of migration (electrical resistance 80.1±24.3Ω), when compared with control cells (130±24.3Ω) (p=0.024). Normal mammary epithelial cells showed a good degree of staining of MDA-7, a staining greatly reduced in cancer cells. Using the Nottingham Prognostic Index as a predictor of prognosis, tumours from patients with poor prognosis showed a significantly lower level of MDA-7 transcript compared with tumours from those with good prognosis (p=0.049). A significant difference was seen between tumours from patients who died of breast cancer and tumours from patients who remained disease free (p=0.035), the later showing a higher levels of MDA7 transcript. Using the Kaplan-Meier survival analysis, it was revealed that low levels of MDA-7 were significantly correlated with a shorter disease free survival (mean survival for low level group 121.7 (108.5-134.9) months compared with high levels 140.4 (133.7 – 147.1, 95% CI) months, p=0.0287. ER positive tumours showed a lower but levels of MDA-7 expression compared with ER-negative tumours, although this was not statistically significant (p=0.094).&amp;#x2028; CONCLUSIONS. MDA-7 is a potential regulator of cellular motility of breast cancer cells. The aberrant expression of the molecule is linked to the prognosis and long term survival of patients, indicating the potential clinical implication of MDA-7 in cancer therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2060.

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.

CoMFA and docking studies of 2-phenylindole derivatives with anticancer activity

Three-dimensional (3D) quantitative structure-activity relationship (QSAR) and docking studies of 43 tubulin inhibitors, 2-phenylindole derivatives with anticancer activity against human breast cancer cell line MDA-MB 231, have been carried out. The established 3D-QSAR model from the comparative molecular field analysis (CoMFA) in training set shows not only significant statistical quality, but also satisfying predictive ability, with high correlation coefficient value (R(2)=0.910) and cross-validation coefficient value (q(2)=0.705). Moreover, the predictive ability of the CoMFA model was further confirmed by a test set, giving the predictive correlation coefficient (R(2)(pred)) of 0.688. Based on the CoMFA contour maps and docking analyses, some key structural factors responsible for anticancer activity of this series of compounds were revealed as follows: the substituent R(1) should have higher electronegativity; the substituent R(2) should be linear alkyl with four or five carbon atoms in length; and the substituent R(3) should be selected to OCH(3)-kind group whereas should not be selected to CF(3)-kind group. Meanwhile, the interaction information between target and ligand was presented in detail. Such results can offer some useful theoretical references for understanding the action mechanism, designing more potent inhibitors and predicting their activities prior to synthesis.

Maurocalcine as a Non Toxic Drug Carrier Overcomes Doxorubicin Resistance in the Cancer Cell Line MDA-MB 231

The aim of this study is to overcome tumour cell resistance that generally develops after administration of commonly used anti-cancer drugs, such as doxorubicin. Recently, cell penetrating peptides have been used for their ability to deliver non-permeant compounds into cells. One such cell penetrating peptide, maurocalcine, has been isolated from the venom of a Tunisian scorpion. Herein, we report the effects of doxorubicin covalently coupled to an analogue of maurocalcine on drug-sensitive or drug-resistant cell lines MCF7 and MDA-MB 231. We demonstrated the in vitro anti-tumoral efficacy of the doxorubicin maurocalcine conjugate. On a doxorubicin-sensitive cancer cell line, the maurocalcine-conjugated form appears slightly less efficient than doxorubicin itself. On the contrary, on a doxorubicin-resistant cancer cell line, doxorubicin coupling allows to overcome the drug resistance. This strategy can be generalized to other cell penetrating peptides since Tat and penetratin show similar effects. We conclude that coupling anti-tumoral drugs to cell penetrating peptides represent a valuable strategy to overcome drug resistance.

The preparation and in vitro antiproliferative activity of phthalimide based ketones on MDAMB-231 and SKHep-1 human carcinoma cell lines

The ‘one pot’ condensation reaction for the synthesis and potent antiproliferative inhibition of α-phthalimide based ketones is reported here. 2-Phthalimide-1-(4-fluoro-phenyl)ethanone ( 5) showed the best growth inhibition on human MDAMB-231 breast carcinoma and SKHep-1 hepatoma cell lines. Preliminary studies showed that the reported bioactivity may be due to the presence of strong electronegative fluorine group at the para-position of the aryl ring.

Combination of imatinib and vinorelbine enhances cell growth inhibition in breast cancer cells via PDGFR β signalling

Introduction Imatinib mesylate is a tyrosine kinase receptor inhibitor targeted against PDGFR α and β, c-kit and bcr-abl. These receptors regulate cellular processes such as proliferation, differentiation, and survival. This study was performed to evaluate the effects of imatinib on breast cancer cell lines with respect to the activity of PDGFR β and Akt: a downstream modulator of cell growth and survival. Methods Expression of imatinib targets was analyzed with reverse transciptase PCR and immunoblotting assays in the breast cell lines MDA MB 231, MCF 7, ZR 75-1, and T 47-D. Changes on receptor expression and phosphorylation status under imatinib were evaluated using drug concentrations of 2 to 10 μM. The anti-proliferative and pro-apoptotic effects of imatinib alone and in combination with vinorelbine were investigated with an MTT and TUNEL assay. Results Imatinib inhibited growth and induced apoptosis of all cell lines examined. This effect was increased when combined with vinorelbine. A dose-dependent inhibitory effect on the phosphorylation of PDGFR β and Akt was detected. Conclusions The growth inhibitory effect of imatinib on breast cell lines may be caused by inhibiting the activity of the tyrosine kinases PDGFR β and Akt. Imatinib is a promising novel drug for targeted therapy of breast cancer patients.

The isolation of novel mesenchymal stromal cell chemotactic factors from the conditioned medium of tumor cells

Bone marrow-derived mesenchymal stromal cells (MSCs) localize to solid tumors. Defining the signaling mechanisms that regulate this process is important in understanding the role of MSCs in tumor growth. Using a combination of chromatography and electrospray tandem mass spectrometry we have identified novel soluble signaling molecules that induce MSC chemotaxis present in conditioned medium of the breast carcinoma cell line MDA-MB231. Previous work has employed survey strategies using ELISA assay to identify known chemokines that promote MSC chemotaxis. While these studies provide valuable insights into the intercellular signals that impact MSC behavior, many less well-described, but potentially important soluble signaling molecules could be overlooked using these methods. Through the less directed method of column chromatography we have identified novel candidate MSC chemotactic peptides. Two proteins, cyclophilin B and hepatoma-derived growth factor were then further characterized and shown to promote MSC chemotaxis.

Graded hypoxia modulates the invasive potential of HT1080 fibrosarcoma and MDA MB231 carcinoma cells

Spatial and temporal oxygen heterogeneity exists in most solid tumour microenvironments due to an inadequate vascular network supplying a dense population of tumour cells. An imbalance between oxygen supply and demand leads to hypoxia within a significant proportion of a tumour, which has been correlated to the likelihood of metastatic dissemination in both rodent tumour models and human patients. Experimentally, it has been demonstrated that near-anoxic in vitro exposure results in transiently increased metastatic potential in some tumour cell lines. The purpose of this study was to examine the effect of graded low oxygen conditions on the invasive phenotype of human tumour cells using an in vitro model of basement membrane invasion, in which we measured oxygen availability directly at the invasion surface of the transwell chamber. Our results show a relationship between culture vessel geometry and time to achieve hypoxia which may affect the interpretation of low oxygen experiments. We exposed the human tumour cell lines, HT1080 and MDA MB231, to graded normobaric oxygen (5% O(2)-0.2% O(2)) either during or prior to in vitro basement membrane invasion to simulate conditions of intravasation and extravasation. A secondary aim was to investigate the potential regulation of matrix metalloproteinase activity by oxygen availability. We identified significant reductions in invasive ability under low oxygen conditions for the HT1080 cell line and an increase in invasion at intermediate oxygen conditions for the MDA MB231 cell line. There were differences in the absolute activity of the individual matrix metalloproteinases, MMP-2, -9, -14, between the two cell lines, however there were no significant changes following exposure to hypoxic conditions. This study demonstrates cell line specific effects of graded oxygen levels on invasive potential and suggests that intermediate levels of low oxygen may increase metastatic dissemination.

Increased oxidative stress created by adenoviral MnSOD or CuZnSOD plus BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) inhibits breast cancer cell growth

Superoxide dismutases (SODs) have been found to decrease tumor formation and angiogenesis. SOD gene therapy, as with many other gene transfer strategies, may not completely inhibit tumor growth on its own. Thus, concomitant therapies are necessary to completely control the spread of this disease. We hypothesized that intratumoral injection of Ad SOD in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy would synergistically inhibit breast cancer growth. Our data indicate that BCNU when combined with SOD overexpression increased oxidative stress as suggested by elevated glutathione disulfide (GSSG) production in one of three breast cancer cell lines tested, at least in part due to glutathione reductase (GR) inactivation. The increased oxidative stress caused by BCNU combined with adenovirally expressed SODs, manganese or copper zinc SOD, decreased growth and survival in the three cell lines tested in vitro, but had the largest effect in the MDA-MB231 cell line, which showed the largest amount of oxidative stress. Delivery of MnSOD and BCNU intratumorally completely inhibited MDA-MB231 xenograft growth and increased nude mouse survival in vivo. Intravenous (iv) BCNU, recapitulating clinical usage, and intratumoral Ad MnSOD delivery, to provide tumor specificity, provided similar decreased growth and survival in our nude mouse model. This cancer therapy produced impressive results, suggesting the potential use of oxidative stress-induced growth inhibitory treatments for breast cancer patients.