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Aims: The development of rapamycin (RAP) and resveratrol (RSV) coloaded liposomes (RAP-RSV-LIP) for breast cancer therapy. Materials & methods: Liposomes were prepared using a high-pressure homogenization technique and evaluated according to their physicochemical characteristics, cellular uptake and cytotoxicity against tumoral and normal cells. Results & conclusion: The RAP-RSV-LIP showed negative surface charge, size around 100 nm, low polydispersity and high encapsulation efficiency for RAP and RSV (58.87 and 63.22%, respectively). RAP-RSV-LIP showed great stability over 60 days and a prolonged drug release profile. In vitro studies indicated that RAP-RSV-LIP were internalized in an estrogen receptor-positive human breast cancer cell line (MCF-7, 34.2%) and improved cytotoxicity when compared with free drugs. Therefore RAP-RSV-LIP showed great antitumoral potential against breast cancer cells.

Glioblastoma (GBM) is the highest-grade form of glioma, as well as one of the most aggressive types of cancer, exhibiting rapid cellular growth and highly invasive behavior. Despite significant advances in diagnosis and therapy in recent decades, the outcomes for high-grade gliomas (WHO grades III-IV) remain unfavorable, with a median overall survival time of 15–18 months. The concept of cancer stem cells (CSCs) has emerged and provided new insight into GBM resistance and management. CSCs can self-renew and initiate tumor growth and are also responsible for tumor cell heterogeneity and the induction of systemic immunosuppression. The idea that GBM resistance could be dependent on innate differences in the sensitivity of clonogenic glial stem cells (GSCs) to chemotherapeutic drugs/radiation prompted the scientific community to rethink the understanding of GBM growth and therapies directed at eliminating these cells or modulating their stemness. This review aims to describe major intrinsic and extrinsic mechanisms that mediate chemoradioresistant GSCs and therapies based on antineoplastic agents from natural sources, derivatives, and synthetics used alone or in synergistic combination with conventional treatment. We will also address ongoing clinical trials focused on these promising targets. Although the development of effective therapy for GBM remains a major challenge in molecular oncology, GSC knowledge can offer new directions for a promising future.

Background Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. Methods The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. Results The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. Conclusions In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers

Recently, many studies have reported the anticancer properties of flavonoid luteolin against a variety of tumors, but there is still a lack in the description of its mechanism of action. In attempt to better contribute to the literature, we evaluated the antiproliferative activity of luteolin extracted by Fridericia platyphylla in a panel of tumor cell lines representative of six different tissues. Luteolin presented antiproliferative activity for all the assessed tumor cell lines, being glioblastoma the most sensitive one. This compound was able to inhibit U-251 cells migration and tumorigenesis. Besides, luteolin leads U-251 tumor cells to apoptosis death by depolarisation of the mitochondrial membrane, ERK proteins phosphorylation, cleavage of PARP and Caspase 9, further inducing DNA damage by H2AX phosphorylation, which had not yet been described for glioblastomas. Altogether, our results reaffirm luteolin as a potential therapeutic drug.

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC., This work was supported by Barretos Cancer Hospital and the Public Ministry of Labor Campinas (Research, Prevention, and Education of Occupational Cancer) in Campinas, Brazil, CAPESDFATD (88887.137283/2017-00). INFG is the recipient of a FAPESP Ph.D. fellowship (2017/22305-9).

Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)-induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNA-dependent protein kinase (PKR) is a stress-sensitive pro-apoptotic kinase. Our previous study indicated that PKR-associated protein X, a stress-sensitive activator of PKR, is upregulated in the early stage of STZ-induced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZ-induced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2-α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, p-eIF2-α expression and cellular localization followed the trend of PKR, suggesting that this pro-apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.

Cervical cancer is the fourth most common cancer in women. Although cure rates are high for early stage disease, clinical outcomes for advanced, metastatic, or recurrent disease remain poor. To change this panorama, a deeper understanding of cervical cancer biology and novel study models are needed. Immortalized human cancer cell lines such as HeLa constitute crucial scientific tools, but there are few other cervical cancer cell lines available, limiting our understanding of a disease known for its molecular heterogeneity. This study aimed to establish novel cervical cancer cell lines derived from Brazilian patients. We successfully established one (HCB-514) out of 35 cervical tumors biopsied. We confirmed the phenotype of HCB-514 by verifying its' epithelial and tumor origin through cytokeratins, EpCAM and p16 staining. It was also HPV-16 positive. Whole-exome sequencing (WES) showed relevant somatic mutations in several genes including BRCA2, TGFBR1 and IRX2. A copy number variation (CNV) analysis by nanostring and WES revealed amplification of genes mainly related to kinases proteins involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and validated by western blot analysis. Furthermore, the HCB-514 cell line was sensitive to cisplatin. In summary, this novel Brazilian cervical cancer cell line exhibits relevant key molecular features and constitutes a new biological model for pre-clinical studies., Barretos Cancer Hospital Research Support Department (NAP) for sample collection, Barretos Cancer Hospital Biobank for sample processing, Dr. Flávia de Paula and Gabriela Fernandes for technical support of STRs and BRCA2 Sanger validation, respectively, and Dr. Laura Musselwhite (Duke University) for revising the manuscript. This study was supported by grants from the FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII- BIOPLAT - Process number 01.13.0469.00) and Barretos Cancer Hospital. PhD scholarship from FINEP (Grant numbers 384088/2014-7 and 380434/2015-6) and Barretos Cancer Hospital to MNR.

Cervical cancer is the third most common in Brazilian women. The chemotherapy used for the treatment of this disease can cause many side effects, then, to overcome this problem, new treatment options are necessary. Natural compounds represent one of the most promising sources for the development of new drugs. In this study, 13 different species of 6 families from the Brazilian Cerrado vegetation biome were screened against human cervical cancer cell lines (CCC). Some of these species were also evaluated in one normal keratinocyte cell line (HaCaT). The effect of crude extracts on cell viability was evaluated by a colorimetric method (MTS assay). Extracts from Annona crassiflora, Miconia albicans, Miconia chamissois, Stryphnodendron adstringens, Tapirira guianensis, Xylopia aromatica, and Achyrocline alata showed half-maximal inhibitory concentration (IC50) values &lt, 30 μg/mL for at least one CCC. A. crassiflora and S. adstringens extracts were selective for CCC. Mass spectrometry (Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (ESI FT-ICR MS)) of A. crassiflora identified fatty acids and flavonols as secondary compounds. One of the A. crassiflora fractions, 7C24 (from chloroform partition), increased H2AX phosphorylation (suggesting DNA damage), PARP cleavage, and cell cycle arrest in CCC. Kaempferol-3-O-rhamnoside and oleic acid were bioactive molecules identified in 7C24 fraction. These findings emphasize the importance of investigating bioactive molecules from natural sources for developing new anti-cancer drugs.

Introduction: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. Methods: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex, flow cytometry and transwell assays were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. Results: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. Conclusion: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

The following are available online at http://www.mdpi.com/2073-4409/9/2/485/s1, Figure S1: Validation of WNK2 overexpression by RT-PCR., Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1—baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line., This project was supported by the Barretos Cancer Hospital Internal Research Funds (PAIP) to Rui Manuel Reis and by the Public Ministry of Labor Campinas (Research, Prevention, and Education of Occupational Cancer Project), Campinas, Brazil. Ana Laura Vieira Alves is the recipient of a FAPESP master fellowship (2016/18907-0).

Made available in DSpace on 2020-12-10T19:54:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Barretos Cancer Hospital Public Ministry of Labor Campinas (Research, Prevention and Education of Occupational Cancer) National Institute for Health Research (NIHR) Biomedical Research Center at the Royal Marsden Institute of Cancer Research in London NIHR The 5'-methylthioadenosine phosphorylase (MTAP) gene is located in the chromosomal region 9p21. MTAP deletion is a frequent event in a wide variety of human cancers; however, its biological role in tumorigenesis remains unclear. The purpose of this study was to characterize the MTAP expression profile in a series of gliomas and to associate it with patients' clinicopathological features. Moreover, we sought to evaluate, through glioma gene-edited cell lines, the biological impact of MTAP in gliomas. MTAP expression was evaluated in 507 glioma patients by immunohistochemistry (IHC), and the expression levels were associated with patients' clinicopathological features. Furthermore, an in silico study was undertaken using genomic databases totalizing 350 samples. In glioma cell lines, MTAP was edited, and following MTAP overexpression and knockout (KO), a transcriptome analysis was performed by NanoString Pan-Cancer Pathways panel. Moreover, MTAP's role in glioma cell proliferation, migration, and invasion was evaluated. Homozygous deletion of 9p21 locus was associated with a reduction of MTAP mRNA expression in the TCGA (The Cancer Genome Atlas) - glioblastoma dataset (p < 0.01). In addition, the loss of MTAP expression was markedly high in high-grade gliomas (46.6% of cases) determined by IHC and Western blotting (40% of evaluated cell lines). Reduced MTAP expression was associated with a better prognostic in the adult glioblastoma dataset (p < 0.001). Nine genes associated with five pathways were differentially expressed in MTAP-knockout (KO) cells, with six upregulated and three downregulated in MTAP. Analysis of cell proliferation, migration, and invasion did not show any significant differences between MTAP gene-edited and control cells. Our results integrating data from patients as well as in silico and in vitro models provide evidence towards the lack of strong biological importance of MTAP in gliomas. Despite the frequent loss of MTAP, it seems not to have a clinical impact in survival and does not act as a canonic tumor suppressor gene in gliomas. Barretos Canc Hosp, Mol Oncol Res Ctr, BR-14784400 Barretos, SP, Brazil Barretos Canc Hosp, Dept Pathol, BR-14784400 Barretos, SP, Brazil Barretos Canc Hosp, Dept Neurosurg, BR-14784400 Barretos, SP, Brazil AC Camargo Canc Ctr, BR-01508010 Sao Paulo, SP, Brazil Inst Canc Res, London SW7 3RP, England Barretos Sch Hlth Sci, Dr Paulo Prata FACISB, BR-14785002 Barretos, SP, Brazil Univ Estadual Paulista Unesp, Botucatu Med Sch, Dept Pathol, BR-18618970 Botucatu, SP, Brazil Univ Minho, Sch Med, Life & Hlth Sci Res Inst ICVS, P-4710057 Braga, Portugal 3Bs PT Govt Associate Lab, P-4806909 Braga, Portugal Univ Estadual Paulista Unesp, Botucatu Med Sch, Dept Pathol, BR-18618970 Botucatu, SP, Brazil FAPESP: 2016/06833-2 FAPESP: 2016/18907-0 FAPESP: 2017/22305-9 CNPq: 100707/2014-9 CNPq: 116477/2014-8 CNPq: 1282245/2014-0 CNPq: 472447/2013-0

The identification of signaling pathways that are involved in gliomagenesis is crucial for targeted therapy design. In this study we assessed the biological and therapeutic effect of ingenol-3-dodecanoate (IngC) on glioma. IngC exhibited dose-time-dependent cytotoxic effects on large panel of glioma cell lines (adult, pediatric cancer cells, and primary cultures), as well as, effectively reduced colonies formation. Nevertheless, it was not been able to attenuate cell migration, invasion, and promote apoptotic effects when administered alone. IngC exposure promoted S-phase arrest associated with p21CIP/WAF1 overexpression and regulated a broad range of signaling effectors related to survival and cell cycle regulation. Moreover, IngC led glioma cells to autophagy by LC3B-II accumulation and exhibited increased cytotoxic sensitivity when combined to a specific autophagic inhibitor, bafilomycin A1. In comparison with temozolomide, IngC showed a mean increase of 106-fold in efficacy, with no synergistic effect when they were both combined. When compared with a known compound of the same class, namely ingenol-3-angelate (I3A, Picato®), IngC showed a mean 9.46-fold higher efficacy. Furthermore, IngC acted as a potent inhibitor of protein kinase C (PKC) activity, an emerging therapeutic target in glioma cells, showing differential actions against various PKC isotypes. These findings identify IngC as a promising lead compound for the development of new cancer therapy and they may guide the search for additional PKC inhibitors., This research received was funded by Amazonia Fitomedicamentos Ltda, and Barretos Cancer Hospital, all from Brazil.

Plant-based compounds are an option to explore and perhaps overcome the limitations of current antitumor treatments. Annona coriacea Mart. is a plant with a broad spectrum of biological activities, but its antitumor activity is still unclear. The purpose of our study was to determine the effects of A. coriacea fractions on a panel of cervical cancer cell lines and a normal keratinocyte cell line. The antitumor effect was investigated in vitro by viability assays, cell cycle, apoptosis, migration, and invasion assays. Intracellular signaling was assessed by Western blot, and major compounds were identified by mass spectrometry. All fractions exhibited a cytotoxic effect on cisplatin-resistant cell lines, SiHa and HeLa. C3 and C5 were significantly more cytotoxic and selective than cisplatin in SiHa and Hela cells. However, in CaSki, a cisplatin-sensitive cell line, the compounds did not demonstrate higher cytotoxicity when compared with cisplatin. Alkaloids and acetogenins were the main compounds identified in the fractions. These fractions also markedly decreased cell proliferation with p21 increase and cell cycle arrest in G2/M. These effects were accompanied by an increase of H2AX phosphorylation levels and DNA damage index. In addition, fractions C3 and C5 promoted p62 accumulation and decrease of LC3II, as well as acid vesicle levels, indicating the inhibition of autophagic flow. These findings suggest that A. coriacea fractions may become effective antineoplastic drugs and highlight the autophagy inhibition properties of these fractions in sensitizing cervical cancer cells to treatment., e FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/ 2013—FP XII-BIOPLAT), Barretos Cancer Hospital, CAPES, CNPq, FAPEMIG, UFSJ. RMR is a recipient of CNPq Productivity Grant

Glioblastoma (GBM) is the most frequent and aggressive type of brain tumor. There are limited therapeutic options for GBM so that new and effective agents are urgently needed. Euphol is a tetracyclic triterpene alcohol, and it is the main constituent of the sap of the medicinal plant Euphorbia tirucalli. We previously identified anti-cancer activity in euphol based on the cytotoxicity screening of 73 human cancer cells. We now expand the toxicological screening of the inhibitory effect and bioactivity of euphol using two additional glioma primary cultures. Euphol exposure showed similar cytotoxicity against primary glioma cultures compared to commercial glioma cells. Euphol has concentration-dependent cytotoxic effects on cancer cell lines, with more than a five-fold difference in the IC50 values in some cell lines. Euphol treatment had a higher selective cytotoxicity index (0.64-3.36) than temozolomide (0.11-1.13) and reduced both proliferation and cell motility. However, no effect was found on cell cycle distribution, invasion and colony formation. Importantly, the expression of the autophagy-associated protein LC3-II and acidic vesicular organelle formation were markedly increased, with Bafilomycin A1 potentiating cytotoxicity. Finally, euphol also exhibited antitumoral and antiangiogenic activity in vivo, using the chicken chorioallantoic membrane assay, with synergistic temozolomide interactions in most cell lines. In conclusion, euphol exerted in vitro and in vivo cytotoxicity against glioma cells, through several cancer pathways, including the activation of autophagy-associated cell death. These findings provide experimental support for further development of euphol as a novel therapeutic agent for GBM, either alone or in combination chemotherapy., The work was supported by the Amazonia Fitomedicamentos (FITO05/2012) Ltda. and Barretos Cancer Hospital, all from Brazil.

Anti-VEGF therapy with Bevacizumab is approved for glioblastoma treatment, however, it is known that tumors acquired resistance and eventually became even more aggressive and infiltrative after treatment. In the present study we aimed to unravel the potential cellular mechanisms of resistance to Bevacizumab in glioblastoma in vitro models. Using a panel of glioblastoma cell lines we found that Bevacizumab is able to block the secreted VEGF by the tumor cells and be internalized to the cytoplasm, inducing cytotoxicity in vitro. We further found that Bevacizumab increases the expression of hypoxic (HIF-1α and CAIX) and glycolytic markers (GLUT1 and MCT1), leading to higher glucose uptake and lactate production. Furthermore, we showed that part of the consumed glucose by the tumor cells can be stored as glycogen, hampering cell dead following Bevacizumab treatment. Importantly, we found that this change on the glycolytic metabolism occurs independently of hypoxia and before mitochondrial impairment or autophagy induction. Finally, the combination of Bevacizumab with glucose uptake inhibitors decreased in vivo tumor growth and angiogenesis and shift the expression of glycolytic proteins. In conclusion, we reported that Bevacizumab is able to increase the glucose metabolism on cancer cells by abrogating autocrine VEGF in vitro. Define the effects of anti-angiogenic drugs at the cellular level can allow us to discover ways to revert acquired resistance to this therapeutic approaches in the future, This study was partially developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and through the Competitiveness Factors Operational Programme (COMPETE), by Portuguese funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038, and by Brazilian MCTI/CNPq No 73/2013. VMG was recipient from a PhD fellowship (SFRH/BD/51997/2012) from Fundação para a Ciência e Tecnologia (FCT), Portugal. FC was recipient of a master FAPESP fellowship (n° 2014/03684-0) and “BEPE - Bolsa Estágio de Pesquisa no Exterior” (n° 2015/02691-6). OM is recipient of a post-doc fellowship (SFRH/BPD/108351/2015) from FCT, Portugal, info:eu-repo/semantics/publishedVersion

Breast cancer (BC) is a leading cause of cancer-associated mortality in females worldwide. MicroRNAs (miRNAs or miRs), a type of non-coding RNA, have been reported to be important in the regulation of BC onset and progression. Several studies have implicated the role of miR-183 and miR-494 in different types of cancer. However, the biological functions of these miRNAs in BC remain largely unknown. In the present study, the expression of both miRNAs was assessed in the MDA-MB-231 and MDA-MB-468 BC cell lines. It was hypothesized that miR-183 and miR-494 serve an important role in regulating the expression of key genes associated with the metastatic phenotype of BC cells. To further understand their role, the expression of these miRNAs was restored in selected BC cell lines. Functional assays revealed that overexpression of miR-183 or miR-494 modulated the proliferation and migration of MDA-MB-231 and MDA-MB-468 cells in vitro. Additionally, retinoblastoma 1 (RB1) was identified to be a downstream target of both miRNAs by in silico analysis. Western blotting revealed that upregulation of miR-183 was associated with downregulation of RB1 protein in MDA-MB-231 cells. In conclusion, the present results support the hypothesis that miR-183 and miR-494 serve a pivotal role in BC metastasis, and that miR-183 may act as an oncogene by targeting RB1 protein in MDA-MB-231 cells.

Cervical cancer is the third most commonly diagnosed tumor type and the fourth cause of cancer-related death in females. Therapeutic options for cervical cancer patients remain very limited. Annona crassiflora Mart. is used in traditional medicine as antimicrobial and antineoplastic agent. However, little is known about its antitumoral properties. In this study the antineoplastic effect of crude extract and derived partitions from A. crassiflora Mart in cervical cancer cell lines was evaluated. The crude extract significantly alters cell viability of cervical cancer cell lines as well as proliferation and migration, and induces cell death in SiHa cells. Yet, the combination of the crude extract with cisplatin leads to antagonistic effect. Importantly, the hexane partition derived from the crude extract presented cytotoxic effect both in vitro and in vivo, and initiates cell responses, such as DNA damage (H2AX activity), apoptosis via intrinsic pathway (cleavage of caspase-9, caspase-3, poly (ADP-ribose) polymerase (PARP) and mitochondrial membrane depolarization) and decreased p21 expression by ubiquitin proteasome pathway. Concluding, this work shows that hexane partition triggers several biological responses such as DNA damage and apoptosis, by intrinsic pathways, and was also able to promote a direct decrease in tumor perimeter in vivo providing a basis for further investigation on its antineoplastic activity on cervical cancer., This study was supported by grants from the FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 -FPXII-BIOPLAT), CAPES, FAPEMIG, UFSJ and Barretos Cancer Hospital, all from Brazil., info:eu-repo/semantics/acceptedVersion

Glioblastoma (GBM) is the most frequent and highest&ndash, grade brain tumor in adults. The prognosis is still poor despite the use of combined therapy involving maximal surgical resection, radiotherapy, and chemotherapy. The development of more efficient drugs without noticeable side effects is urgent. Coronarin D is a diterpene obtained from the rhizome extract of Hedychium coronarium, classified as a labdane with several biological activities, principally anticancer potential. The aim of the present study was to determine the anti&ndash, cancer properties of Coronarin D in the glioblastoma cell line and further elucidate the underlying molecular mechanisms. Coronarin D potently suppressed cell viability in glioblastoma U&ndash, 251 cell line, and also induced G1 arrest by reducing p21 protein and histone H2AX phosphorylation, leading to DNA damage and apoptosis. Further studies showed that Coronarin D increased the production of reactive oxygen species, lead to mitochondrial membrane potential depolarization, and subsequently activated caspases and ERK phosphorylation, major mechanisms involved in apoptosis. To our knowledge, this is the first analysis referring to this compound on the glioma cell line. These findings highlight the antiproliferative activity of Coronarin D against glioblastoma cell line U&ndash, 251 and provide a basis for further investigation on its antineoplastic activity on brain cancer.

Accepted manuscript, Crotoxin (CTX), a heterodimeric phospholipase present in venom of snakes of the genus Crotalus, has demonstrated a broad spectrum of pharmacological properties, such as antimicrobial, hemostatic, and antitumoral. However, the precise mechanism of its cytotoxicity and antitumoral properties remains to be determined. Therefore, in the present study, we isolated crotoxin (F1 CTX) through two steps DEAE-Sepharose and Heparin-Sepharose FF chromatography. The C-terminal sequence of the A- and B-chain protein fragment was determined by LC-MS/MS mass spectrometry, which showed 100% identity to crotoxin structure. In order to investigate its cytotoxic effects, we demonstrated that the F1 CTX fraction at 0-30 μg/mL concentrations for 72 h presented a heterogeneous response profile on nine human cancer-derived cell lines from four tumor types (pancreatic, esophagus, cervical cancer, and glioma). The glioma (GAMG and HCB151) and pancreatic (PSN-1 and PANC-1) cancer cells showed a higher sensitivity with IC50 of, FAPESP, the Hermínio Ometto University Center, the Hospital de Cancer de Barretos and FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT). We acknowledge the Mass Spectrometry Laboratory of the Brazilian Biosciences National Laboratory, CNPEM, Campinas, Brazil, for providing support on mass spectrometry analysis, info:eu-repo/semantics/publishedVersion

The latex from Euphorbia tirucalli is used in Brazil as a folk medicine for several diseases, including cancer. Recently, we showed a cytotoxic activity of E. tirucalli euphol in a wide range of cancer cell lines. Moreover, we showed that euphol inhibits proliferation, motility and colony formation in pancreatic cancer cells, induces autophagy and sensitizes glioblastoma cells to temozolomide cytotoxicity. Herein, we report in vitro activity of three semi-synthetic ingenol compounds derived from E. tirucalli, IngA (ingenol-3-trans-cinnamate), IngB (ingenol-3-hexanoate) and IngC (ingenol-3-dodecanoate), against a large panel of human cancer cell lines. Antineoplastic effects of the three semi-synthetic compounds were assessed using MTS assays on 70 cancer cell lines from a wide array of solid tumors. Additionally, their antitumor potential was compared with known compounds of the same class, namely ingenol-3-angelate (Picato®) and ingenol 3,20-dibenzoate and in combination with standard chemotherapeutic agents. We observed that IngA, B, and C exhibited dose-dependent cytotoxic effects. Amongst the semi-synthetic compounds, IngC displayed the best activity across the tumor cell lines. In comparison with ingenol-3-angelate and ingenol 3,20-dibenzoate, IngC showed a mean of 6.6 and 3.6-fold higher efficacy, respectively, against esophageal cancer cell lines. Besides, IngC sensitized esophageal cancer cells to paclitaxel treatment. In conclusion, the semi-synthetic ingenol compounds, in particular, IngC, demonstrated a potent antitumor activity on all cancer cell lines evaluated. Although the underlying mechanisms of action of IngC are not elucidated, our results provide insights for further studies suggesting IngC as a putative therapy for cancer treatment., Grants from Amazonia Fitomedicamentos Ltda, and Barretos Cancer Hospital, all from Brazil, supported this study.

An L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom was purified to homogeneity in a two-step procedure using molecular exclusion on Sephadex G-75, followed by Phenyl Sepharose FF chromatography. The molecular mass of the purified enzyme was 113 kDa, as determined by SDS-PAGE under reducing conditions. LAAOcdt showed amino acid homology to other L-amino acid oxidases isolated from different snake venoms. The comparative analysis of the internal peptide sequences of the NNPGILEYPVKPSEEGK fragments by LC-MS/MS spectrometry revealed 100% identity with C. durissus cumanensis LAAO. The purified protein catalyzed the oxidative deamination of L-amino acids, and the most specific substrates were L-Tyr and L-Phe. The enzyme presented optimum activity at pH 7.4 and at 44 °C. LAAOcdt also showed hemolytic activity (0.6-20 µg/µL) and induced both the formation plasma clots (5-100 µg/µL) and platelet aggregation (2.5 × 10(-3), 5.0 × 10(-3) and 10 × 10(-3) µg/mL), as well as bactericidal activity (2.5-10 µg/µL) against Staphylococcus aureus. Moreover, LAAOcdt exhibited cytotoxicity in distinct cancer cell lines, which presented a heterogeneous response profile. The mean IC50 value was 10.5 µg/mL. Glioma and pancreatic carcinoma cells were the most sensitive cell lines; they showed mean IC50 values of 7.2 µg/mL and 7.4 µg/mL, respectively. The exposure of the drug-sensitive cells to LAAOcdt for 24 h upregulated activated p-H2AX and efficiently decreased P42/P44 (ERK) activation in glioma cells (HCB151), which suggested an anti-proliferative effect. In addition, increased p21 expression was observed in SiHa cells, which showed a resistant phenotype. On the other hand, the flow cytometry and immunoblotting analyses showed that the enzyme did not induce cancer cell apoptosis. These results suggest that another cell death mechanism might contribute to the LAAOcdt-induced cytotoxicity. Taken together, this work may help to elucidate the function and structure of LAAOcdt by providing the basis for further investigations on its efficacy in cancer treatment., The current study was supported by grants from FAPESP (grant number: 2011/12267-6) and partially supported by FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT) and the Herminio Ometto University Center., info:eu-repo/semantics/publishedVersion

The incidence of malignant melanoma has increased greatly in recent decades presenting a high mortality rate despite intensive efforts in this area of research. Recent studies indicate that the chemokine receptor 4 (CXCR4) plays a critical role in cancer. Thus, it has been reported that CXCL12 binding to CXCR4 initiates various downstream signaling pathways that result in a plethora of responses involved in cell proliferation and metastasis. Recently, we demonstrated that CXCR4 silencing by RNA interference (RNAi) significantly reduced the number of pulmonary metastatic nodules. In the present study, we examined the effect of the intratumoral injection of CXCR4 short hairpin (shRNA) expressing plasmids on the growth of B16‑F10 melanoma in mice. In vitro transfection of these tumor cells with CXCR4 shRNA expressing plasmid (CXCR4 shRNA) significantly reduced the levels of CXCR4 mRNA (85%) and CXCR4 protein (70%) compared with the control. We showed that the tumor growth was significantly reduced (66%) in mice inoculated with transfected B16‑F10 melanoma cells when compared with the control group. We also found that the intratumoral injection of CXCR4 shRNA expressing plasmids results in a significant inhibition (70%) of B16‑F10 melanoma growth. This finding supports the hypothesis that a direct administration of RNAi‑based therapeutics into the target tumor is a promising approach for overcoming the hurdles of systemic delivery. The present study is the first demonstration that CXCR4 plays a critical role in B16-F10 melanoma growth. Currently there is great interest in the development of antagonists for therapeutic targeting CXCR4 expression. Considering our results and the fact that CXCR4 is highly conserved between human and mouse, this experimental model of cancer may be useful for the discovery of new CXCR4 antagonists with clinical implications.

A large number of classic antineoplastic agents are derived from plants. Euphorbia tirucalli L. (Euphorbiaceae) is a subtropical and tropical plant, used in Brazilian folk medicine against many diseases, including cancer, yet little is known about its true anticancer properties. The present study evaluated the antitumor effect of the tetracyclic triterpene alcohol, euphol, the main constituent of E. tirucalli in a panel of 73 human cancer lines from 15 tumor types. The biological effect of euphol in pancreatic cells was also assessed. The combination index was further used to explore euphol interactions with standard drugs. Euphol showed a cytotoxicity effect against several cancer cell lines (IC50 range, 1.41-38.89 µM), particularly in esophageal squamous cell (11.08 µM) and pancreatic carcinoma cells (6.84 µM), followed by prostate, melanoma, and colon cancer. Cytotoxicity effects were seen in all cancer cell lines, with more than half deemed highly sensitive. Euphol inhibited proliferation, motility and colony formation in pancreatic cancer cells. Importantly, euphol exhibited synergistic interactions with gemcitabine and paclitaxel in pancreatic and esophageal cell lines, respectively. To the best of our knowledge, this study constitutes the largest in vitro screening of euphol efficacy on cancer cell lines and revealed its in vitro anti-cancer properties, particularly in pancreatic and esophageal cell lines, suggesting that euphol, either as a single agent or in combination with conventional chemotherapy, is a potential anti-cancer drug., Amazônia Fitomedicamentos Ltda. (grant no. FITO 05/2012) and Barretos Cancer Hospital, all from Brazil, info:eu-repo/semantics/publishedVersion

Euphorbia umbellata (E. umbellata)belongs toEuphorbiaceaefamily, popularly known as Janauba, and contains in its latex a combination of phorbol esters with biological activities described to different cellular protein kinase C (PKC) isoforms. Here, we identified deoxi-phorbol esters present inE. umbellatalatex alcoholic extract able to increase HIV transcription and reactivate HIV from latency models. This activity was mediated by NF-kB activation followed by nuclear translocation and binding to HIV LTR promoter. In addition,E. umbellatelatex extract induced the production of pro inflammatory cytokines together with IL21 inin vitrohuman PBMC cultures. Our latex extract activates latent HIV in human PBMCs isolated from HIV positive patients as well as latent SIV in non-human primate primary CD4+T lymphocytes. These results strongly indicate that the phorbol esters present inE. umbellatalatex are promising candidate compounds for future clinical trials forshock and killtherapy to promote HIV cure and eradication.

Background: Glioblastoma, the most frequent and malignant adult brain tumor, has been extensively studied. However, there is no effective treatment, and to overcome this challenging scenario, it is essential to improve preclinical biological models. This study aimed to molecularly characterize short-term glioblastoma primary cultures and to compare them with patient tumor profiles. Methods: Glioblastoma cell lines were established from Barretos Cancer Hospital patients diagnosed with glioblastoma. The cells were cultured with DMEM (+)10% FBS (+)1% PS and were molecularly characterized using array CGH (aCGH), next-generation and Sanger sequencing. Results: We established four short-term glioblastoma cultures and we found that the primary cells exhibited a diversity of chromosomal aberrations, with gain of chromosome 7 and loss of chromosomes 10, 13 and 17p being the most frequent alterations. Mutation profiling showed that hotspot TERT promoter mutations were present in 3/4 cases, followed by mutations in TP53 (2/4) and in the RB1, BRAF and PTEN (1/4) genes. A similar chromosomal and mutation pattern was observed in all short-term cultures and matched frozen tumors. Conclusions: Herein, short-term glioblastoma primary cultures were successfully characterized and had genetic make-ups that were similar to those of patient tumors, suggesting that short-term primary cultures are suitable in vitro models for studies of glioblastoma biology., Universal/CNPq (475358/2011-2-Reis RM), FAPESP (2012/19590-0-Reis RM) and the MCTI/CNPq No. 73/2013 (Reis RM) grants. Bidinotto LT was a recipient of the FAPESP fellowship (2011/08523-7 and 2012/08287-4), info:eu-repo/semantics/publishedVersion

Euphorbia umbellata (E. umbellata) belongs to Euphorbiaceae family, popularly known as Janauba, and its latex contains a combination of phorbol esters with biological activities described to different cellular protein kinase C (PKC) isoforms. Here, we identified deoxi-phorbol esters present in E. umbellata latex alcoholic extract that are able to increase HIV transcription and reactivate virus from latency models. This activity is probably mediated by NF-kB activation followed by nuclear translocation and binding to the HIV LTR promoter. In addition, E. umbellata latex extract induced the production of pro inflammatory cytokines in vitro in human PBMC cultures. This latex extract also activates latent virus in human PBMCs isolated from HIV positive patients as well as latent SIV in non-human primate primary CD4+ T lymphocytes. Together, these results indicate that the phorbol esters present in E. umbellata latex are promising candidate compounds for future clinical trials for shock and kill therapies to promote HIV cure and eradication., Research and experimental expenses were funded by the grant E26/2015064289 from FAPERJ, info:eu-repo/semantics/publishedVersion

The epidermal growth factor receptor (EGFR) is a member of the HER family of growth factors that activates several intracellular signaling pathways promoting proliferation and survival. EGFR over-expression is frequently associated with gene mutation or amplification, thereby constituting a major target for molecular therapies. Recently, a new generation of EGFR inhibitors has been developed with pan-HER properties and irreversible actions. AllitinibA (R) (AST1306) is an orally active, highly selective irreversible inhibitor of the HER family of receptor tyrosine kinases with promising efficacies. In the present study we aimed to investigate the cytotoxicity of allitinib in a large panel of human cancer-derived cell lines and to correlate its efficacy to the mutational status of the EGFR, KRAS, BRAF, PI3KCA and PTEN genes. In addition, we aimed to evaluate the functional role of KRAS mutations in the response to this new inhibitor. In total 76 different cancer-derived cell lines, representing 11 distinct histological types, were analyzed and classified into three groups: highly sensitive (HS), moderately sensitive (MS) and resistant (R). We found that 28 (36.8 %) cancer-derived cell lines exhibited a HS phenotype, 19 (25.0 %) a MS phenotype and 29 (38.1 %) a R phenotype. Allitinib showed a stronger cytotoxicity in head and neck, esophageal, melanoma and lung cancer-derived cell lines. We found that KRAS mutations were significantly associated with the R phenotype. To substantiate these results, an allitinib-sensitive lung cancer-derived cell line (H292) was transfected with plasmids carrying the two most common activating KRAS mutations (p.G12D and p.G12S). We found that both mutations reverted the allitinib-sensitive phenotype in these cells. The current study represents the largest in vitro assessment of allitinib cytotoxicity performed to date. Through this study, we identified cancer types that could potentially benefit from this drug. Additionally, our findings suggest that prevalent KRAS mutations constitute potential predictive biomarkers for allitinib response., This study was partially supported by FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT) and the Assistance Program and Incentive Research (PAIP), Barretos Cancer Hospital Sao Paulo, Brazil. A.L.C and R.M.R are recipients of a National Counsel of Technological and Scientific Development (CNPq) scholarship. M.N.R is recipient of a CNPq scholarship (380434/2015-6) and O.C.M is recipient of a Portuguese Foundation for Science and Technology (FCT) scholarship (SFRH/BPD/108351/2015)., info:eu-repo/semantics/publishedVersion

Background: Glioblastomas are the most frequent and deadly brain tumors. Genomic alterations, as well as its high heterogeneity, are important features of these tumors. Despite the advances in the biology of these tumors, it is essential to develop models, such as primary tumor cell lines, that better mimic their genomic diversity and therapeutic behavior. We aimed to characterize the molecular profile of two (HCB2 and HCB151) established immortalized glioblastoma cell lines. Methods: Immortalized glioblastoma cell lines, HCB2 and HCB151, were established from Barretos Cancer Hospital patients diagnosed with glioblastoma. The cells were cultured with DMEM+10%BFS+1%PS and molecularly characterized, using arrayCGH, next-generation sequencing (Ion Torrent) and validated by Sanger sequencing, miRNA expression and CNV using the NanoString platform, and BD Lyoplate™ Human Cell Surface Marker Screening Panel using Accuri™ C6 flow cytometer. The tumorigenic capacity was further determined in vivo by the chicken chorioallantoic membrane (CAM) assay, and its response to temozolomide-based chemotherapy was done by MTS. Results: The aCGH analysis showed that the most frequent events was the gain of chromosome 7 and loss of chromosomes 10, 13, and 17p. HCB2 exhibited mutations in the TERT gene, while HCB151 showed mutations in the TP53, PTEN, LZTR1, and TERT genes. The immortalized cultures present an unique miRNA profile. The CAM assay showed that HCB2 and HCB151 were able to form vascularized tumors, with HCB151 more aggressive than HCB2. In addition, both HCB2 (IC50>1000 µM) and HCB151 (IC50=783 µM) showed resistance to TMZ when compared to commercial glioblastomas cell lines (23-441 µM). Conclusion: The present findings showed that the immortalized glioblastoma cell lines showed classic genomic features of glioblastomas and exhibited a similar biologic behavior, suggesting that these cell lines are a good model for glioblastoma preclinical studies. Citation Format: Adriana Cruvinel-Carloni, Renato José da Silva-Oliveira, Viviane Aline Oliveira-Silva, Marcela Nunes Rosa, Lucas Tadeu Bidinotto, Gustavo Noriz Berdinarelli, Raul Torrieri, Adriane Feijó Evangelista, Olga Catarina Martinho, Vera Mónica Gonçalves, Carlos Clara, Gisele Caravina de Almeida, Rui Manuel Reis. Omics profile of two immortalized Brazilian glioblastoma cell cultures [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A29.

Metastasis is a key factor that limits survival in the majority of patients with cancer. Thus, numerous efforts have been made to elucidate the molecular mechanisms involved in this phenomenon. B16‑F10 melanoma cells have been demonstrated to be highly metastatic to the lungs in mice. The aim of the current study was to investigate the role of CXC motif chemokine receptor 4 (CXCR4) in the metastatic potential of B16‑F10 melanoma cells in mice. In vitro transfection of B16‑F10 tumor cells with CXCR4 short hairpin RNA (shRNA) expressing plasmids (CXCR4 shRNA) significantly reduced the expression levels of CXCR4 mRNA (80%) and protein (68%), compared with the control. In addition, these results demonstrated that pulmonary metastasis was significantly inhibited (85%) in mice inoculated with CXCR4 shRNA‑transfected B16‑F10 melanoma cells. The polycation‑based nanoparticle (jetPEI) was used to investigate the effect of CXCR4 knockdown in vivo on the metastatic potential of B16‑F10 melanoma cells. The number of pulmonary metastatic nodules was significantly reduced (50%) in animals that received a retro‑orbital injection of jetPEI‑CXCR4‑1 shRNA. The current study demonstrated that CXCR4 serves a role in the metastatic potential of B16‑F10 melanoma cells. Currently there is a great interest in the development of antagonists for the therapeutic targeting of CXCR4 expression. Taking the results of the current study and the fact that CXCR4 is highly conserved between humans and mice into account, this experimental model of metastasis with B16‑F10 melanoma cells may aid in the discovery of CXCR4 antagonists with clinical implications.

It has been brought to our attention that there was an important omission of the word 'shRNA', which should have been featured in a couple of places in the above article. The authors incorrectly claimed that the RNA‑dependent protein kinase (PKR) acts as a tumor suppressor; in fact, it is the PKR shRNA that would fulfil the role of being a tumor suppressor. Therefore, the text in the Abstract on p. 2267, line 14, should have read "...these findings suggested that PKR shRNA acts as a tumor suppressor"; likewise, on p. 2272, towards the end of the final sentence of the second paragraph of the Discussion, the text should have read "The results show that PKR silencing inhibits tumor growth, suggesting that PKR shRNA acts as a tumor supressor...". The authors regret that this error was not corrected before the article went to print, and they thank the reader concerned for drawing the matter to our attention. [the original article was published in the Oncology Reports 32: 2267‑2273, 2014; DOI: 10.3892/or.2014.3410].

The RNA-dependent protein kinase (PKR) is a serine/threonine kinase that is involved in the regulation of important cell processes such as apoptosis, signal transduction, cell proliferation and differentiation. However, the role played by PKR in cancer remains controversial. RNA interference (RNAi) has currently become an important technique in understanding gene function. Previously, we showed that PKR shRNA downregulates PKR expression in B16-F10 melanoma cells and reduces the metastatic potential of these tumor cells. In the present study, we examined the effect of the intratumoral injection of PKR shRNA‑expressing plasmid on the growth of B16-F10 melanoma in mice. The results showed that this treatment significantly reduced tumor growth. Thus, these findings suggested that PKR acts as a tumor suppressor, a finding that is consistent with our previous study on the experimental model of metastasis. Moreover, the results suggested that this effect may be mediated by the transcription factor NF-κB. The present study confirmed the hypothesis that the direct administration of RNAi-based therapeutics in the target tumor is a promising approach for overcoming the obstacles of systemic delivery. The results also suggested that the intratumoral injection of PKR shRNA‑expressing vector is a novel therapeutic approach for human solid tumors such as cutaneous melanoma and breast cancer, since PKR is overexpressed in these tumors.

O presente trabalho utilizou a tecnologia do RNA de interferencia no modelo de melanoma murino com o objetivo de investigar o papel da proteina quinase dependente de RNA sobre o crescimento tumoral. Cerca de 7x10 5 celulas de melanoma B16-F10 foram transfectadas com shRNAs anti-PKR e Lipofectamina por 5 horas. O efeito do silenciamento foi analisado por PCR semi-quantitativo e Western blot apos 24 e 48 horas de transfeccao. Para os ensaios in vivo as celulas B16-F10 transfectadas com vetor psiSTRIKE PKR-2 shRNA foram inoculadas pela via subcutânea em camundongos C57BL/6 e apos 14 dias os camundongos foram sacrificados e o tumor removido e pesado. Nossos resultados demonstraram reducao significativa da expressao do RNAm da PKR e da proteina apos 48h de transfeccao. A analise do peso tumoral indicou uma reducao de 86% no crescimento tumoral indicando que esta quinase exerce um papel positivo na proliferacao celular.

Durante a anidrobiose (um estado ametabólico muito estável), o nematóide Panagrolaimus superbus tolera vários tipos de estresses físicos. A fim de melhor compreender essa extremotolerância, P. superbus foi submetido a regimes de hiperaceleração (RHA) de até 400.000 x g. Surpreendentemente, foi observado que esse verme tolera a exposição à RHA tanto dessecado (i.e., em anidrobiose) como hidratado. Para verificar se esse fenômeno era específico para essa espécie ou algo observável em outros organismos, os mesmos procedimentos experimentais foram realizados no organismo modelo Caenorhabditis elegans. Intrigantemente, C. elegans também mostrou o mesmo perfil de sobrevivência. Ademais, o desenvolvimento, comportamento, morfologia e crescimento populacional desse nematóide também foram analisados após a exposição ao RHA, não sendo observada quaisquer mudanças nesses parâmetros em função da exposição à hipergravidade. Em seguida, foram realizadas buscas (tBLASTn) no genoma de C. elegans por homólogos de genes relacionados ao gravitropismo que são naturalmente encontrados em plantas. Essa busca resultou nos genes dnj- 16 (homólogo ao ARG1), ipla-1 (homólogo ao SGR2) e uma sequência não caracterizada (homóloga a TWD1). Especial atenção foi despendida ao gene dnj-16, uma vez que é o mais conservado entre eles. As análises de RT-qPCR revelaram que o dnj-16 é ligeiramente regulado para baixo durante o RHA, o que não era esperado caso ele possuísse função semelhante ao seu homólogo em plantas. A análise do estado metabólico desse nematoide durante o RHA lançou luz sobre os dados de RT-qPCR, mostrando que a queda na expressão de dnj-16 é provavelmente devida à centrifugação. Posteriormente, diversas análises in silico foram realizadas a fim de caracterizar o gene dnj-16 e a sua respectiva proteína. Inicialmente, a análise comparativa dos domínios DnaJ, transmembrana e coiled coil das proteínas dnj-16, ARG1, ARL1 e ARL2 apontou para uma grande semelhança não apenas na sequência como na estrutura dessas proteínas. Essa grande similaridade motivou análises para desvendar o papel e a origem do gene dnj-16. Três hipóteses ((i) homologia, (ii) convergência e (iii) transferência gênica horizontal (TGH)) foram consideradas na investigação desse intrigante gene. Os resultados obtidos nas análises in silico apontaram para uma TGH mediada por RNA, potencialmente ocorrida a 1325 m.a., como a hipótese mais plausível para explicar a origem de dnj-16 e algumas espécies parasitas do gênero Phytophthora como prováveis mediadores dessa transferência. Dessa forma, os dados apresentados nessa tese mostram pela primeira vez que C. elegans é tolerante a RHA ordens de magnitude mais altas do que se pensava serem compatíveis com a vida multicelular. Além disso, os dados sugerem que dnj-16 foi transferido horizontalmente de plantas para nematoides e que a ultracentrifugação leva a uma redução no metabolismo de C. elegans, o que ajudaria a explicar a sua sobrevivência sob tal condição extrema. Por fim, o conjunto de dados desse trabalho representa contribuições originais para a compreenção da biologia, da genética e da evolução de C. elegans. During anhydrobiosis (a very stable ametabolic state), the nematode Panagrolaimus superbus tolerates many types of physical stresses. In order to better understand this extremotolerance, P. superbus underwent hyperacceleration regimes (RHA) of up to 400,000 x g. Surprisingly, it was observed that this worm tolerated RHA exposure both dried (i.e., during anhydrobiosis) and hydrated. In order to verify if this phenomenon was specific for this species or something observable in other organisms, the same experimental procedures were performed in the model organism Caenorhabditis elegans. Intriguingly, C. elegans also showed the same survival profile. In addition, the development, behavior, morphology and population growth of this nematode were also analyzed after the exposure to RHA and no changes were observed in these parameters due to the hypergravity exposure. Thereafter, searches (tBLASTn) were performed on C. elegans genome by homologs of gravitropism related genes that are naturally found in plants. These searches resulted in the genes dnj-16 (homologous to ARG1), ipla-1 (homologous to SGR2) and an uncharacterized sequence (homologous to TWD1). Special attention was given to dnj-16 gene, since it is the most conserved among them. RT-qPCR analyzes revealed that dnj-16 is slightly down regulated during RHA, which was not expected if it had similar function to its homologue in plants. Analysis of the metabolic status of this nematode during RHA shed light on the RT-qPCR data, showing that the decrease in dnj-16 expression was probably due to centrifugation. Subsequently, several in silico analyzes were performed in order to characterize the dnj-16 gene and its respective protein. Initially, the comparative analyzis of the DnaJ, transmembrane and coiled coil domains of dnj-16, ARG1, ARL1 and ARL2 proteins pointed to a great similarity not only in the sequence as well as in the structure of these proteins. This great similarity motivated analyzes in order to uncover the real nature of the dnj-16 gene. Three hypotheses ((i) homology, (ii) convergence and (iii) horizontal gene transfer (HGT)) were considered in the investigation of this intriguing gene. The results obtained in the in silico analyzes indicated an RNA mediated TGH, potentially occurred 1325 my (millions of years), as the most plausible hypothesis to explain the origin of dnj-16 and some parasitic species of the Phytophthora genus as probable mediators of this transference. Therefore, data presented in this thesis show for the first time that C. elegans tolerates RHA of magnitude orders higher than it was thought to be compatible with multicellular life. In addition, the data suggest that dnj-16 was transferred horizontally from plants to nematodes and that ultracentrifugation leads to a reduction in C. elegans metabolism, which would help explain its survival under such extreme condition. Finally, the data set of this work represent original contributions to the understanding of the C. elegans\' biology, genetics and evolution.