RESUMO
Osteopontin (OPN) is upregulated in several types of tumor and has been associated with chemoresistance. However, the contribution of OPN splicing isoforms (OPNSIs) to chemoresistance requires further investigation. The present study aimed to evaluate the expression patterns of each tested OPNSI in cisplatin (CDDP)resistant ovarian carcinoma cell lines, focusing on the role of the OPNc isoform (OPNc) in drug resistance. ACRP ovarian cancer cells resistant to CDDP, as well as their parental cell line A2780, were used. Analyses of the transcriptional expression of OPNSIs, epithelialmesenchymal transition (EMT) markers and EMTrelated cytokines were performed using reverse transcriptionquantitative PCR. OPNc was silenced in ACRP cells using antiOPNc DNA oligomers and stably overexpressed by transfecting A2780 cells with a mammalian expression vector containing the full length OPNc cDNA. Functional assays were performed to determine cell proliferation, viability and colony formation. The results demonstrated that among the three tested OPNSIs, OPNc was the most upregulated transcript in the ACRP cells compared with the parental A2780 cells. In addition, the expression levels of Pglycoprotein multidrug transporter were upregulated in CDDPresistant ACRP cells compared with those in A2780 cells. OPNc knockdown sensitized ACRP cells to CDDP treatment and downregulated Pgp expression levels compared with those in the negative control group. Additionally, silencing of OPNc impaired cell proliferative and colony formation abilities, as well as reversed the expression levels of EMT markers and EMTrelated cytokines compared with those in the negative control cells. Notably, although stable OPNc overexpression resulted in increased A2780 cell proliferation, it notably increased CDDP sensitivity compared with that in the cells transfected with a control vector. These results suggested that OPNc silencing may represent a putative approach to sensitize resistant ovarian cancer cells to chemotherapeutic agents.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Osteopontina/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Processamento Alternativo , Linhagem Celular Tumoral , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/uso terapêutico , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Osteopontina/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Among osteopontin splice variants (OPN-SV), the expression profile of osteopontin-4 (OPN4) and osteopontin-5 (OPN5) has not been addressed in distinct cancer types. We herein aimed to investigate their expression in several cancer cell lines, besides comparing it in relation to the three previously described OPN-SV: OPNa, OPNb and OPNc. Total RNA from cancer cell lines, including prostate (PC3 and DU145), ovarian (A2780), breast (MCF-7 and MDA-MB-231), colorectal (Caco-2, HT-29 and HCT-116), thyroid (TT, TPC1 and 8505c) and lung (A549 and NCI-H460) was extracted, followed by cDNA synthesis. OPN-SV transcript analysis by RT-PCR or RT-qPCR were performed using OPN-SV specific oligonucleotides and gapdh and actin transcripts were used as housekeeping controls. OPN4 and OPN5 transcripts displayed co-expression in most tested cell lines. OPN4 was found expressed in similar or higher levels in relation to OPN5. Moreover, in most tested cell lines, OPN4 is also expressed in similar levels to OPNa or OPNb. The expression of OPN5 is also generally variable in relation to the other OPN-SV, but expressed in similar or higher levels in relation to OPNc, depending on each tested cell line. OPN4 and OPN5 seem to be co-expressed in several tumor types and OPN4 is one of the most overexpressed OPN-SV in distinct tumor cell lines. Once both OPN4 and OPN5 are differentially expressed and also evidence tumor-specific expression patterns, we hypothesize that similarly to the other OPN-SV, they also possibly contribute to key aspects of tumor progression, what should be further functionally investigated in distinct tumor models.
Assuntos
Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Osteopontina/biossíntese , Células A549 , Células CACO-2 , Células HCT116 , Células HT29 , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Osteopontina/genética , Células PC-3 , Isoformas de Proteínas/biossínteseRESUMO
Murici (Byrsonima crassifolia (L.) Kunth and B. verbascifolia (L.) DC.) and tapereba (Spondias mombin) are Amazonian fruits that contain bioactive compounds. Biochemical and molecular characterization of these fruits can reveal their potential use in preventing diseases, including cancer. The extracts were characterized regarding the presence and profile of carotenoids by high-performance liquid chromatography (HPLC), total phenolic content by the Folin-Ciocalteu assay, and antioxidant activity by antioxidant value 2,2-diphenyl-1-picrylhydrazyl (DPPH) content analysis, 22,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) content analysis, Ferric-Reducing Ability of Plasma (FRAP), and Oxygen Radical Absorbance Capacity (ORAC) analysis. The extracts of tapereba and murici studied were important sources of total carotenoids and lutein, respectively. The extracts were then tested for their effect on the viability of the A2780 ovarian cancer (OC) cell line and its cisplatin (CDDP)-resistant derived cell line, called ACRP, by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Their influence on cell cycle and apoptosis were analyzed by using flow cytometry. Murici and tapereba cell extracts exhibited a strong bioactivity by inhibiting A2780 and ACRP cell viability by 76.37% and 78.37%, respectively, besides modulating the cell cycle and inducing apoptotic cell death. Our results open new perspectives for the development of innovative therapeutic strategies using these Amazon fruit extracts to sensitize ovarian cancer cells to current chemotherapeutic options.
Assuntos
Anacardiaceae/química , Apoptose/efeitos dos fármacos , Frutas/química , Inibidores do Crescimento/farmacologia , Malpighiaceae/química , Neoplasias Ovarianas/fisiopatologia , Extratos Vegetais/farmacologia , Brasil , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Inibidores do Crescimento/química , Humanos , Extratos Vegetais/químicaRESUMO
Drug resistance represents a major issue in treating breast cancer, despite the identification of novel therapeutic strategies, biomarkers, and subgroups. We have previously identified the LQB-223, 11a-N-Tosyl-5-deoxi-pterocarpan, as a promising compound in sensitizing doxorubicin-resistant breast cancer cells, with little toxicity to non-neoplastic cells. Here, we investigated the mechanisms underlying LQB-223 antitumor effects in 2D and 3D models of breast cancer. MCF-7 and MDA-MB-231 cells had migration and motility profile assessed by wound-healing and phagokinetic track motility assays, respectively. Cytotoxicity in 3D conformation was evaluated by measuring spheroid size and performing acid phosphatase and gelatin migration assays. Protein expression was analyzed by immunoblotting. Our results show that LQB-223, but not doxorubicin treatment, suppressed the migratory and motility capacity of breast cancer cells. In 3D conformation, LQB-223 remarkably decreased cell viability, as well as reduced 3D culture size and migration. Mechanistically, LQB-223-mediated anticancer effects involved decreased proteins levels of XIAP, c-IAP1, and Mcl-1 chemoresistance-related proteins, but not survivin. Survivin knockdown partially potentiated LQB-223-induced cytotoxicity. Additionally, cell treatment with LQB-223 resulted in changes in the mRNA levels of epithelial-mesenchymal transition markers, suggesting that it might modulate cell plasticity. Our data demonstrate that LQB-223 impairs 3D culture growth and migration in 2D and 3D models of breast cancer exhibiting different phenotypes.