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or mTOR inhibition as a potential target in trastuzumab resistant breast cancer cells with MUC-4 overexpression
Abstracts Results: EGFR signaling was inhibited in pancreatic carcinoma cell lines by all the small EGFR inhibitors tested. The effects of the EGFR inhibitors AG1478, erlotinib, geficitinib and cetuximab have been determined in pancreatic carcinoma cell lines (IMIM-PC-1, IMIM-PC-2, RWP-1 and PANC-1). Our results show that all cell lines were resistant to the antiproliferative effect of cetuximab (only IMIM-PC-1 cell line shows a slightly decrease in proliferation) as determined by MTT analysis as well as by cell cycle analysis using flow cytometry. Meanwhile, all cell lines were sensitive to at least one of the EGFR tyrosin-kinase activity inhibitors (AG-1478, erlotinib and geficitinib). We have found that IMIM-PC-2 cell line was sensitive to all the EGFR-TK inhibitors, RWP-1 cells were sensitive to geficitib and erlotinib but they were quite resistant to AG-1478, IMIMPC-1 cells were sensitive to geficitinib and to a lesser extent to erlotinib and, finally PANC-1 cells were only moderately sensitive to geficitinib. The discrepancies found between the differential effects of cetuximab versus EGFR-TK inhibitors as well as the differences observed in the effects of the different TK-inhibitors upon the same cell lines, suggest that the EGFR inhibitors act in these pancreatic carcinoma cell lines not only trough inhibition of EGFR but also through differential effects on secondary targets. For identification of the putative secondary targets, we have determined by DNA arrays, the level of expression of tyrosin-kinases in the four pancreatic cell lines. Differential expression of several kinases has been found in our cell lines that could be related to the effects observed with the EGFR inhibitors in the pancreatic carcinoma cell lines. Inhibitory peptides designed against PTK6 as well as specific siRNas for this kinase, are able to potentiate the effects of EGFR inhibitors in pancreatic carcinoma cell lines. Conclusion: PTK6 and in lesser extension TNK2 kinases are able to potentiate the effects of EGFR inhibitors on pancreatic carcinoma cell lines No conflict of interest. 159 POSTER EGFR induced JAK-STAT activations inhibited by Quercetin A. Demiroglu Zergeroglu1 , H. Gul1 , Z. Ulupinar1 . 1 Gebze Technical University, Molecular Biology and Genetics, Gebze/Kocaeli, Turkey Recently several in vitro studies and in vivo treatments propose that using natural plant derived compounds are promising and alternative application to treat malignant diseases. Quercetin (QU) is known as an anti-cancer agent since its anti-proliferative effects available on several malignant cells. Signal transducers and activators of transcription (STAT) proteins provide integration point for various signalling pathways to regulate genes involved in proliferation and apoptosis. STATs are constitutively phosphorylated and thereby activated in many forms of cancer. STAT proteins can mediate the epidermal growth factor receptor (EGFR) signalling, with activation of STATs by EGFR binding and activation of STATs through Src-mediated EGFR signalling. In present study, we investigated the effect of QU on JAK-STAT pathway regulation on EGF induced cell line at a dose and timedependent manner. For this purpose, human cancer cells derived from mesothelioma were treated with 50, 75 and 100mM QU and then, protein levels determined by western blot. Next, we analysed the gene expression level of STAT molecules with time intervals by using real-time RT-PCR. Our results showed that STAT3 and STAT5 protein phosphorylations and mRNA expressions were down regulated at 12, 24, 48hs in SPC212 cells. Our results suggest that QU might be a potential agent for the treatment of EGFR and STAT overexpressed malign mesothelioma and other cancers. No conflict of interest. 160 POSTER AKT and/or mTOR inhibition as a potential target in trastuzumab resistant breast cancer cells with MUC-4 overexpression J.A. Perez-Fidalgo1 , E. Espin2 , E. Tormo2 , B. Pineda2 , J.M. Cejalvo1 , M.A. Sabbaghi3 , E. Alonso4 , A. Rovira3 , F. Rojo5 , J. Albanell6 , B. Bermejo1 , O. Burgues4 , A. Lluch1 . 1 INCLIVA − Hospital Clinico Universitario, Medical Oncology and Hematology, Valencia, Spain; 2 INCLIVA, Oncology, Valencia, Spain; 3 IMIM Hospital del Mar Medical Research Institute, IMIM, Barcelona, Spain; 4 INCLIVA, Pathology, Valencia, Spain; 5 Fundacion Jimenez Diaz, Pathology, Madrid, Spain; 6 IMIM Hospital del Mar Medical Research Institute, Medical Oncology, Barcelona, Spain Background: The membrane-associated glycoprotein MUC4 might mask HER2 by preventing it from binding effectively to trastuzumab (T). Overexpression of MUC-4 is a mechanism of resistance to this treatment in HER2+ breast cancer cells. Little is known about the best strategy to overcome this resistance mechanism. We aimed at assessing the potential role of AKT/mTOR inhibition as an alternative to HER2 blockage in a T-resistant cell line with MUC-4 overexpression.
S17 Methods: The JIMT-1 is a T-resistant, ER and PR negative and HER2+ cell line with an overexpression of MUC-4 as main mechanism of resistance. JIMT-1 cells have been exposed in a first step to a control agent and T. Thereafter JIMT-1 cells were treated with three AKT or mTOR inhibitors: MK-2206 (AKT inhibitor), GDC-0980 (dual PI3K and mTOR inhibitor) and everolimus (mTOR inhibitor) which were administered either in monotherapy and in combination with T. Cell viability (CV) was determined with the colorimetric assay MTT. Differences in the mean between both groups were assessed with T-student test. Results: No differences were found in cell viability when comparing between exposure to control versus T treatment alone confirming the novoresistance of JIMT-1 to T exposure. When compared with T monotherapy (mean CV 1.04) all three AKT-mTOR inhibitors showed a decrease in cellularity with a mean CV after exposure to MK-2206, GDC-0980 and everolimus of 0.76, 0.66 and 0.72 (p values <0.001, <0.01 and <0.01 respectively). No differences were found between either AKT-mTOR inhibitor alone versus the respective combination with T (MK-2206 vs MK-2206+T 0.76 vs 0.77, GDC-0980 vs GDC-0980+T 0.66 vs 0.68 and everolimus vs everolimus+T 0.72 vs 0.90) suggesting no enhanced effect of trastuzumab activity by AKT-mTOR inhibition in this cell line. Conclusions: Our results suggest that downstream inhibition of AKT could be a potential strategy to overcome HER2 resistance due to MUC-4 overexpression. However this strategy failed to show an enhancement of trastuzumab effect if MUC-4 overexpression is present. No conflict of interest. 161 POSTER Newly synthesized Zinc/Gold and Zinc/Silver complexes with Schiff-base ligands as potential anticancer agents R. Alexandrova1 , T. Zhivkova1 , A. Abudalleh1 , L. Dyakova2 , D. Dinev1 , P. Mitrenga1 , D. Ivanov1 , R. Toshkova1 , I. Gavrilov3 , M. Georgieva4 , G. Miloshev4 , D. Culita5 , G. Marinescu5 , L. Patron5 , R. Kalfin2 . 1 Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences; 2 Institute of Neurobiology, Bulgarian Academy of Sciences, Acad. Georgi Bonchev St.; 3 Specialized Hospital for Active Treatment in Oncology, Plovdivsko Pole St. No.6, Sofia, Bulgaria; 4 Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. Georgi Bonchev St.; 5 Institute of Physical Chemistry “Ilie Murgulescu”, Splaiul Independentei St. No. 202, Bucharest 060021, Romania Background: The aim of our study was to evaluate the putative antitumor activity of newly synthesized Zn/Ag and Zn/Au complexes in vitro. Materials and Methods: The total of 16 complexes of Zn/Ag and Zn/Au with Schiff-base ligands (H2Salen, HSalampy, HSaldmen as well as Schiff bases obtained by condensation reactions involving 2,6-diformyl-p-cresol) were investigated. They were applied at a concentration range of 0.05– 100 mg/ml. A wide range of cell cultures were used as model systems: i) permanent cell lines from human (MCF-7, MDA-MB-231, A549, HT29, HepG2, HeLa, 8 MGBA, K562), rat (LSR-SF-SR) and chicken (LSCC-SFMc29) cancers, non-tumor human (Lep-3, MRC-5) cell lines; ii) primary cultures from human invasive ductal breast carcinomas; from Ehrlich ascites tumor in mouse, hepatoma of Zajdela in rat, Graffi myeloid tumor in hamster as well as bone-marrow cells and spleen lymphocytes of the same tumor-bearing animals; iii) sensitive human cell line A431 and its multidrug resistant clones expressing mdr1, mrp1 or abcg2 gene. Short- (4−72 h, with 2D cell cultures) and long- (16−18 days, with 3D colonies) term experiments were performed using methods with various molecular/cellular targets and mechanisms of action: MTT test, neutral red uptake cytotoxicity assay, crystal violet staining, trypan blue dye exclusion technique, double staining with acridine orange and propidium iodide, FACS, annexin V binding assay, TUNEL test, Comet assay and colony-forming method. Results: The compounds examined were proved to induce apoptosis and to decrease significantly viability and growth of the treated cancer cells in a time- and concentration-dependent manner. Zn/Au complexes were found to be the most active cytotoxic/cytostatic agents being more effective as compared to the ligands alone, corresponding Zn/Ag analogues and commercially available antitumor agents cis-platin, 5-fluorouracil and daunorubicin. Cell specific response was observed due to different origin of cells, histological type of cancers and tumor heterogeneity. Non-tumor cells, especially embryonic and bone-marrow cells, were found to be also sensitive to the cytotoxic/cytostatic action of the compounds that could be explained by their undifferentiated state, intensive proliferation, low expression of P-glycoprotein (in the case of bone marrow cells). Conclusions: The tested Zn/Au and Zn/Ag complexes with Schiff bases significantly decrease viability and proliferation of cultured human, rat, hamster, mouse and chicken cells obtained from cancers with different etiology, biology and behavior. Special attention deserves promising antitumor activity of Zn/Au complexes that could be administered with
S17 Methods: The JIMT-1 is a T-resistant, ER and PR negative and HER2+ cell line with an overexpression of MUC-4 as main mechanism of resistance. JIMT-1 cells have been exposed in a first step to a control agent and T. Thereafter JIMT-1 cells were treated with three AKT or mTOR inhibitors: MK-2206 (AKT inhibitor), GDC-0980 (dual PI3K and mTOR inhibitor) and everolimus (mTOR inhibitor) which were administered either in monotherapy and in combination with T. Cell viability (CV) was determined with the colorimetric assay MTT. Differences in the mean between both groups were assessed with T-student test. Results: No differences were found in cell viability when comparing between exposure to control versus T treatment alone confirming the novoresistance of JIMT-1 to T exposure. When compared with T monotherapy (mean CV 1.04) all three AKT-mTOR inhibitors showed a decrease in cellularity with a mean CV after exposure to MK-2206, GDC-0980 and everolimus of 0.76, 0.66 and 0.72 (p values <0.001, <0.01 and <0.01 respectively). No differences were found between either AKT-mTOR inhibitor alone versus the respective combination with T (MK-2206 vs MK-2206+T 0.76 vs 0.77, GDC-0980 vs GDC-0980+T 0.66 vs 0.68 and everolimus vs everolimus+T 0.72 vs 0.90) suggesting no enhanced effect of trastuzumab activity by AKT-mTOR inhibition in this cell line. Conclusions: Our results suggest that downstream inhibition of AKT could be a potential strategy to overcome HER2 resistance due to MUC-4 overexpression. However this strategy failed to show an enhancement of trastuzumab effect if MUC-4 overexpression is present. No conflict of interest. 161 POSTER Newly synthesized Zinc/Gold and Zinc/Silver complexes with Schiff-base ligands as potential anticancer agents R. Alexandrova1 , T. Zhivkova1 , A. Abudalleh1 , L. Dyakova2 , D. Dinev1 , P. Mitrenga1 , D. Ivanov1 , R. Toshkova1 , I. Gavrilov3 , M. Georgieva4 , G. Miloshev4 , D. Culita5 , G. Marinescu5 , L. Patron5 , R. Kalfin2 . 1 Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences; 2 Institute of Neurobiology, Bulgarian Academy of Sciences, Acad. Georgi Bonchev St.; 3 Specialized Hospital for Active Treatment in Oncology, Plovdivsko Pole St. No.6, Sofia, Bulgaria; 4 Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. Georgi Bonchev St.; 5 Institute of Physical Chemistry “Ilie Murgulescu”, Splaiul Independentei St. No. 202, Bucharest 060021, Romania Background: The aim of our study was to evaluate the putative antitumor activity of newly synthesized Zn/Ag and Zn/Au complexes in vitro. Materials and Methods: The total of 16 complexes of Zn/Ag and Zn/Au with Schiff-base ligands (H2Salen, HSalampy, HSaldmen as well as Schiff bases obtained by condensation reactions involving 2,6-diformyl-p-cresol) were investigated. They were applied at a concentration range of 0.05– 100 mg/ml. A wide range of cell cultures were used as model systems: i) permanent cell lines from human (MCF-7, MDA-MB-231, A549, HT29, HepG2, HeLa, 8 MGBA, K562), rat (LSR-SF-SR) and chicken (LSCC-SFMc29) cancers, non-tumor human (Lep-3, MRC-5) cell lines; ii) primary cultures from human invasive ductal breast carcinomas; from Ehrlich ascites tumor in mouse, hepatoma of Zajdela in rat, Graffi myeloid tumor in hamster as well as bone-marrow cells and spleen lymphocytes of the same tumor-bearing animals; iii) sensitive human cell line A431 and its multidrug resistant clones expressing mdr1, mrp1 or abcg2 gene. Short- (4−72 h, with 2D cell cultures) and long- (16−18 days, with 3D colonies) term experiments were performed using methods with various molecular/cellular targets and mechanisms of action: MTT test, neutral red uptake cytotoxicity assay, crystal violet staining, trypan blue dye exclusion technique, double staining with acridine orange and propidium iodide, FACS, annexin V binding assay, TUNEL test, Comet assay and colony-forming method. Results: The compounds examined were proved to induce apoptosis and to decrease significantly viability and growth of the treated cancer cells in a time- and concentration-dependent manner. Zn/Au complexes were found to be the most active cytotoxic/cytostatic agents being more effective as compared to the ligands alone, corresponding Zn/Ag analogues and commercially available antitumor agents cis-platin, 5-fluorouracil and daunorubicin. Cell specific response was observed due to different origin of cells, histological type of cancers and tumor heterogeneity. Non-tumor cells, especially embryonic and bone-marrow cells, were found to be also sensitive to the cytotoxic/cytostatic action of the compounds that could be explained by their undifferentiated state, intensive proliferation, low expression of P-glycoprotein (in the case of bone marrow cells). Conclusions: The tested Zn/Au and Zn/Ag complexes with Schiff bases significantly decrease viability and proliferation of cultured human, rat, hamster, mouse and chicken cells obtained from cancers with different etiology, biology and behavior. Special attention deserves promising antitumor activity of Zn/Au complexes that could be administered with