Karina Lani Silva

Variation of MDR Proteins Expression and Activity Levels According to Clinical Status and Evolution of CML Patients

The involvement of the multidrug resistance (MDR) mediated by ABC transporter proteins P‐glycoprotein (Pgp) and multidrug resistance‐associated protein‐1 (MRP1) overexpressions in patients with chronic myeloid leukemia (CML) are not completely understood. Pgp and MRP1 expressions and activity were analyzed in samples from 158 patients with chronic myeloid leukemia (CML). Using flow cytometry, Pgp expression was more frequently observed in early chronic (P = 0.00) and in advanced (P = 0.02) CML phases when it was compared to MRP1 expression. Variation of MDR expression and activity were observed during the CML evolution in patients previously treated with interferon and imatinib. In the K562‐Lucena cell line, Pgp positive, imatinib caused an enhancing in Pgp expression at protein and mRNA levels, whereas in the Pgp negative cell line, this drug was capable of decreasing MDR1/Pgp mRNA levels. Our result emphasizes the importance of understanding the different aspects of MDR status in patients with CML when they are under investigation in determining imatinib resistance.

FOXM1 targets XIAP and Survivin to modulate breast cancer survival and chemoresistance

Drug resistance is a major hurdle for successful treatment of breast cancer, the leading cause of deaths in women throughout the world. The FOXM1 transcription factor is a potent oncogene that transcriptionally regulates a wide range of target genes involved in DNA repair, metastasis, cell invasion, and migration. However, little is known about the role of FOXM1 in cell survival and the gene targets involved. Here, we show that FOXM1-overexpressing breast cancer cells display an apoptosis-resistant phenotype, which associates with the upregulation of expression of XIAP and Survivin antiapoptotic genes. Conversely, FOXM1 knockdown results in XIAP and Survivin downregulation as well as decreased binding of FOXM1 to the promoter regions of XIAP and Survivin. Consistently, FOXM1, XIAP, and Survivin expression levels were higher in taxane and anthracycline-resistant cell lines when compared to their sensitive counterparts and could not be downregulated in response to drug treatment. In agreement with our in vitro findings, we found that FOXM1 expression is significantly associated with Survivin and XIAP expression in samples from patients with IIIa stage breast invasive ductal carcinoma. Importantly, patients co-expressing FOXM1, Survivin, and nuclear XIAP had significantly worst overall survival, further confirming the physiological relevance of the regulation of Survivin and XIAP by FOXM1. Together, these findings suggest that the overexpression of FOXM1, XIAP, and Survivin contributes to the development of drug-resistance and is associated with poor clinical outcome in breast cancer patients.

XIAP and P-glycoprotein co-expression is related to imatinib resistance in chronic myeloid leukemia cells.

P-glycoprotein (Pgp) and XIAP co-expression has been discussed in the process of the acquisition of multidrug resistance (MDR) in cancer. Here, we evaluated XIAP and Pgp expression in chronic myeloid leukemia (CML) samples, showing a positive correlation between them. Furthermore, we evaluated the effects of imatinib in XIAP and Pgp expression using CML cell lines K562 (Pgp(-)) and K562-Lucena (Pgp(+)). Imatinib increased XIAP and Pgp expression in K562-Lucena cells, while in K562 cells a downregulation of these proteins was observed, suggesting that imatinib induces an increment of MDR phenotype of CML cells that previously exhibit high levels of Pgp/XIAP co-expression.

Apoptotic effect of fludarabine is independent of expression of IAPs in B-cell chronic lymphocytic leukemia.

Despite the efficiency of fludarabine in the induction of clinical responses in B-cell chronic lymphocytic leukemia (B-CLL) patients, resistance to this drug has been documented. The present study tested whether resistance to fludarabine is related to the expression of inhibitor of apoptosis proteins (IAPs) family members. We analyzed the expression of c-IAP1, c-IAP2 and XIAP, by immunocytochemistry, in 30 blood samples from B-CLL patients and correlated protein expression to fludarabine-induced apoptosis estimated by an annexin-V assay. Expression of c-IAP1, c-IAP2 and XIAP were found predominantly in the cytoplasm, and a wide range of staining intensities was observed among distinct samples. No correlation was found between the levels of IAPs expression and prognostic factors such as age, gender, lymphocyte doubling time, white blood cell count or previous treatment. The expression of IAPs also failed to predict the sensitivity to fludarabine-induced apoptosis. Alternative pathways of cell death may explain the independence of fludarabine-induced apoptosis from the high expression of IAPs.

Modulation of reactive oxygen species by antioxidants in chronic myeloid leukemia cells enhances imatinib sensitivity through survivin downregulation.

Survivin, a member of the inhibitor of apoptosis protein family and a target for new drugs, is modulated by reactive oxygen species in several types of neoplasms including leukemias. The aim of this study is to find mechanisms to enhance sensitivity to imatinib in imatinib-responsive cells. In this study, we demonstrated through fluorescein isothiocyanate-labeled annexin V for apoptotic cells detection and western blotting that by inhibiting catalase activity, imatinib apoptosis induction was significantly enhanced (P<0.05) through diminishing survivin expression in K562 cells. These findings might be of clinical relevance and might help improve the chemotherapeutic use of imatinib mesylate.

Survivin overexpression correlates with an apoptosis-resistant phenotype in chronic myeloid leukemia cells.

Survivin is a member of the inhibitor of apoptosis protein family (IAP) that acts in both inhibition of apoptosis and regulation of the cell cycle. Despite the fact that survivin is overexpressed in almost all human malignancies, its expression is undetectable in most normal adult tissues, which is what makes it a potential target for anticancer interventions. The aim of this work was to investigate whether survivin is involved in resistance to idarubicin (ida), a drug commonly used in leukemia treatment. Cytotoxic assays using MTT showed that 1 µM of ida could inhibit 50% of cell viability in K562, a chronic myeloid leukemia cell line. Western blotting analysis revealed that survivin expression was increased in the cell line after treatment with ida 0.5 and 1 µM concentrations, protecting cells from ida-induced apoptosis. However, the highest ida concentrations tested were able to inhibit survivin levels and induce apoptosis in K562 cells, as evaluated by morphology and caspase-3 and -9 activation. These results indicate that survivin expression is involved in ida resistance in K562 leukemic cells. Flow cytometry analysis of the cell cycle showed that ida induced G2/M arrest in these cells and there was a statistically significant positive correlation between survivin expression and the percentage of cells in G2/M phase. This work supports the idea that survivin may contribute to an apoptosis-resistant phenotype by inhibiting ida-induced apoptosis and preventing cells from progressing in the cell cycle.

CPT-11-induced cell death in leukemic cells is not affected by the MDR phenotype

CPT-11 is a topoisomerase I (Topo I) inhibitor which was initially described as active in multi-drug resistance (MDR) tumors. The MDR phenomenon is characterized by the overexpression of efflux pumps which are able to extrude a range of drugs non-related chemical or functionally. In this work, we treated leukemic cells with CPT-11 300 microM at 24h and compared its cytotoxicity with the activity of efflux pumps and with cell cycle phase. Our findings show that CPT-11 has a potent anti-tumor activity in leukemic cells regardless MDR phenotype and the cell cycle phase, suggesting new avenues to be explored in leukemia treatment.

Characterization and distribution of extracellular matrix components and receptors in GH3B6 prolactin cells.

GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50–70 % of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: α5β1 (FN receptor), α6β1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75 %) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

Abstract A51: Antineoplastic activity of novel synthetic compound pterocarpanquinone LQB-118 in lung cancer cells

Introduction and objectives: Lung cancer is a significant health concern in many countries as it is the leading cause of cancer-related mortality worldwide. Although significant advances have been made in the treatment of this disease, either traditional chemotherapy or target agents fail to provide long-term benefits for the majority of patients. Intrinsic or acquired drug resistance is a major cause of failure in the treatment of lung cancer and remains an unsolved pharmacological problem. Therefore, the development of new therapies is needed to improve overall survival of these patients. The pterocarpanquinone LQB-118, a molecular hybrid structurally related to lapachol has emerged as a possible cytotoxic molecule for cancer cells by our group. This compound showed to be cytotoxic against leukemic cells regardless their multidrug resistance profiles while low toxicity was observed against normal peripheral blood mononucleated cells (PBMC). LQB-118 has already been patented in Brazil at National Institute of Industry Propriety under the code 020080139198. Although, cytotoxic effect of LQB-118 has been demonstrated in some tumor cells, the pathway through which this compound induces cell death remains unknown in lung cancer cells. Hence, in the present work, we analyzed the cell death process induced by LQB-118 in non-small cell lung cancer (NSCLC) cell line A549. Materials and Methods: Cisplatin, is one of the most commonly used chemotherapeutic agent in the treatment of NSCLC and was used as comparision to LQB-118 effects. For this purpose we carried out a cell viability assay by MTT and a clonogenic survival assay by crystal violet. To study apoptosis induction by compounds the annexin-V/propidium iodide (PI) label, sub-G0 content/cell cycle profile and activation of caspase-3 were examined by FACS analysis. Western blot was performed to evaluate the capability of LQB-118 in changing the expression of some proteins involved in proliferative and antiapoptotic pathways signaling. Results: We found that LQB-118 and cisplatin reduced cell viability in approximately 50% of cells at the concentration of 5μM after 48h of exposure and inhibited drastically colony formation. This cell viability reduction caused by LQB-118 was accompanied by an increase in the percentage of annexin-V/PI staining cells (around 30%) similar as observed for cisplatin. Activation of caspase-3 (around 25%) at 48h was also observed after LQB-118 exposure. However, the percentage of caspase-3 positive cells was higher after cisplatin treatment (around 60%). The new compound was also capable of inducing an arrest in G2/M cell cycle phase, however it was more pronounced in cisplatin treated cells. Interestingly, LQB-118 decreased significantly the expression of XIAP and survivin proteins, members of inhibitor of apoptosis proteins, responsible for drug resistance in several tumors. The inhibition of XIAP and survivin expression by LQB-118 was stronger than that observed for cisplatin treatment at 24h of culture. Analysis of epidermal growth factor receptor (EGFR) expression and proteins involved in signaling pathways downstream EGFR such as extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt was also evaluated. No changes in EGFR expression or ERK1/2 phosphorilation were observed. Nevertheless, an inhibition of phosphorilated Akt was showed after LQB-118 treatment, suggesting that this compound acts inducing apoptosis through PI3Kinase pathway inhibition. Conclusion: Our results suggest LQB-118 as a potential drug for NSCLC treatment once their effects in A549 cell line were similar that observed for cisplatin, with the advantage that LQB-118 seems to be less toxic than cisplatin in PBMC cells. Further studies are necessary to elucidate the mechanisms of action of this compound. Financial support: INCT, FAPERJ, CNPq and Programa de Oncobiologia. Citation Format: Karina Lani Silva, Paloma Silva de Souza, Paulo R.R. Costa, Raquel Ciuvalschi Maia. Antineoplastic activity of novel synthetic compound pterocarpanquinone LQB-118 in lung cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A51.

Analysis of survivin, XIAP and FoxM1 as potential chemoresistance factors in breast cancer cells

Materials and methods Human breast carcinoma cell lines MCF7 (wild-type p53) and MDA-MB-231 (mutant p53) were exposed to dox and citotoxicity was assessed through the MTT assay. Apoptosis was detected through analysis of flow cytometry DNA content and caspases-3, -7 and -9 levels. Survivin, XIAP, p53 and FoxM1 levels were assessed by Western blotting and Survivin and XIAP mRNA levels, by Real Time PCR. Transfections with the Survivin-encoding plasmid and siRNA against Survivin and XIAP were performed using Lipofectamine reagent.