Abstract Osteosarcoma is still associated with limited response to standard-of-care therapy and alarmingly elevated mortality rates, especially in low- and middle-income countries. Despite multiple efforts to repurpose β-blocker propranolol in oncology, its potential application in osteosarcoma management remains largely unexplored. Considering the unsatisfied clinical needs of this aggressive disease, we evaluated the antitumoral activity of propranolol using different in vitro and in vivo osteosarcoma preclinical models, alone or in addition to chemotherapy. Propranolol significantly impaired cellular growth in β2-adrenergic receptor-expressing MG-63 and U-2OS cells, and was capable of blocking growth-stimulating effects triggered by catecholamines. siRNA-mediated ADRB2 knockdown in MG-63 cells was associated with decreased cell survival and a significant attenuation of PPN anti-osteosarcoma activity. Direct cytostatic effects of propranolol were independent of apoptosis induction and were associated with reduced mitosis, G0/G1 cell cycle arrest and a significant down-regulation of cell cycle regulator Cyclin D1. Moreover, colony formation, 3D spheroid growth, cell chemotaxis and capillary-like tube formation were drastically impaired after propranolol treatment. Interestingly, anti-migratory activity of β-blocker was associated with altered actin cytoskeleton dynamics. In vivo, propranolol treatment (10 mg/kg/day i.p.) reduced the early angiogenic response triggered by MG-63 cells in nude mice. Synergistic effects were observed in vitro after combining propranolol with chemotherapeutic agent cisplatin. Sustained administration of propranolol (10 mg/kg/day i.p., five days a week), alone and especially in addition to low-dose metronomic cisplatin (2 mg/kg/day i.p., three times a week), markedly reduced xenograft progression. After histological analysis, propranolol and cisplatin combination resulted in low tumor mitotic index and increased tumor necrosis. β-blockade using propranolol seems to be an achievable and cost-effective therapeutic approach to modulate osteosarcoma aggressiveness. Further translational studies of propranolol repurposing in osteosarcoma are warranted.
Abstract Background: Osteosarcoma is still associated with poor response to therapy and alarmingly elevated mortality rates, especially in developing countries, highlighting the urgent need of novel therapeutic strategies. Given that β-adrenergic receptor (β-AR) signaling regulates many cellular processes involved in the initiation and progression of cancer, multiple efforts have been made to repurpose non-selective β-blocker propranolol in oncology. Considering the unsatisfied clinical needs of osteosarcoma, we evaluated the in vitro and in vivo antitumoral activity of propranolol as a monotherapy or in combination with metronomic chemotherapy, in β-AR-expressing human osteosarcoma cells. Methods: In vitro anti-osteosarcoma effects of propranolol were assessed on high-density cell growth, chemotaxis, colony-forming ability and three-dimensional spheroids. Cell cycle phase distribution and apoptosis were evaluated using flow cytometry analysis, and quantification of hypodiploid cell population and TUNEL labelling, respectively. Propranolol addition to chemotherapy was evaluated in vitro and in human osteosarcoma xenografts generated in athymic mice. Results: Propranolol significantly impaired MG-63 and U-2 OS cellular growth in a concentration range of 10-100 µM (IC 50 ≈ 45µM) and was capable of blocking in vitro protumoral effects triggered by catecholamines. Cytostatic activity of propranolol was associated to reduced mitosis and G 0 /G 1 cell cycle, but not to apoptosis induction. Moreover, β-blocker drastically reduced colony formation, spheroid progression and migration of osteosarcoma cells. Synergistic effects were observed in vitro after combining propranolol with chemotherapeutic agents methotrexate or cisplatin. In vivo , treatment with propranolol (10 mg/kg i.p.), alone and especially in addition to non-interrupted low-dose cisplatin (2 mg/kg i.p.), markedly abrogated xenograft progression, reducing tumor growth rates by 25 or 70%, respectively. After histological analysis, propranolol and cisplatin combined therapy resulted in low tumor mitotic index and increased tumor necrosis. Conclusions: Considering its robust antitumoral effects, β-AR antagonization using β-blocker propranolol seems to be an achievable and cost-effective therapeutic approach to modulate osteosarcoma aggressiveness, in addition to chemotherapy. Propranolol could be proposed as a co-adjuvant agent for the management of osteosarcoma, especially in low- and middle-income countries where access to treatment resources are limited. Further preclinical and clinical studies of propranolol repurposing in osteosarcoma are warranted.
Abstract The p38 pathway can be activated by three different MAP kinase kinases: MKK3, MKK4 and MKK6, each one activated by different stimuli, like DNA damage, oxidative stress, proinflammatory cytokines or growth factors. In consequence, the responses mediated by this kinase are also significantly diverse. Its participation in such different processes makes it reasonable for the role of p38 in cancer to be a topic of current debate. Several authors reported a pro-tumorigenic role of p38, through the stimulation of tumor progression, epidermal to mesenchymal transition and migration and chemotherapy resistance, as a consequence of the transduction of pro-survival signals. Due to the above mentioned, the combination of p38 inhibitors and chemotherapy is frequently proposed as an interesting clinical approach for cancer treatment. In contrast, several reports underline the importance of p38 suppression of tumor initiation, metastasis inhibition through dormancy maintenance, and also a decisive role in the action mechanism of antitumor treatments under evaluation. Specifically, an important amount of evidence point p38 as a key apoptosis mediator in cancer cells, as a response to treatment with anticancer drugs included in different clinical and preclinical protocols. Previously we studied the effect of p38 inhibition in a mouse mammary carcinoma model named F3II. We described that in vitro inhibition of p38 provokes an increased in cell adhesion, cell spreading, proliferation in 3D cultures, ERK phosphorylation and Integrin α5 expression. In vivo inhibition also resulted in shortened latency times and increased tumor growth rates. In this work, we studied the effect of p38 inhibition in the development of recurrences after tumor surgery. When SB203580 inhibitor was administered after surgery, mice developed larger recurrences. Apoptotic cells in histological samples were detected by TUNEL analysis, showing that SB203580 treatment decreased the number of apoptotic cells in tumor recurrences. In addition, mitotic cell count in recurrence tissue showed that SB203580 treatment increased the mitotic index in the tumors. In conclusion, in this work we demonstrated that in F3II cell line, p38 inhibition promotes tumor recurrence development after surgery, mainly blocking the apoptotic response, and promoting cell duplication. Results presented in this work position p38 as a tumor suppressor kinase. The evidence presented here highlight the possible risk of the administration of a p38 inhibitor to oncological patients that may present newly disseminated cells, or a residual tumor mass that remains after treatment. From our perspective, candidates to be treated with new p38 inhibitors should be carefully selected according to the tumor type and the stage of the disease, since the response might be extremely variable between the different conditions. Citation Format: Carla S. Capobianco, Maria F. Gottardo, Johanna E. Sidabra, Julio A. Aguirre Ghiso, Daniel F. Alonso, Hernan G. Farina. p38 kinase acts as a negative regulator of recurrence after surgery in a mammary carcinoma model, through apoptosis induction and proliferation inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5983.
Abstract Breast cancer is the first cause of death from female cancer. After the treatments against the primary tumor, a few cells can prevail in a state of quiescence for a long period of time, becoming minimal residual disease. There is a clear need for alternative techniques to enable an early detection of the disease for a better understanding of it and the generation of therapies capable of acting with a smaller tumor burden. Although many models have been developed for the detection of macrometastasis, there is a limitation of in vivo models that allow the identification of disseminated cells, micrometastasis, or analyze another steps of the metastasic cascade, as embolization or cell arrest. The aim of this work is the generation of in vivo models to enable the detection of disseminated tumor cells (DTCs) We began our study by evaluating the presence of disseminated tumor cells in three in vivo models and quantification by FACS. To this end, we generated the F3II murine triple negative mammary carcinoma line expressing the fluorescent protein GFP (F3II GFP); the F3II line was used as control. In s.c heterotopic model, we observed the presence of positive cells in tumors and livers of mice inoculated with F3II GFP (Student's t test) 30 days post inoculation. In the s.c orthotopic model, we detected positive cells in tumors of mice inoculated with F3II GFP (Student's t test). In both models, we did not observe significant differences in the presence of lung DTCs. In experimental metastasis models, we detected DTCs 15 days post inoculation in lungs of mice injected i.v with F3II GFP (Mann Whitney test).We carried out the search for cells disseminated at 24 h in order to obtain an in vivo model that evaluates the establishment capacity of these cells, and we observed that the percentage of GFP positive cells was higher in those mice inoculated iv with F3II GFP (Student´s t test). In addition, the presence of DTCs was detected by fluorescence microscopy in blood smears of these mice, suggesting that at 24 h there are fluorescent tumor cells in both lung and blood. In conclusion, although the established models allowed us to detect DTCs, the experimental metastasis model was more efficient in the detection and quantification of disseminated tumor cells. These models allows the detection and quantification of DTCs in the lung and blood circulation within 24 hours and 15 days after inoculation and could be used to study the mechanisms of metastasic dissemination involved in these cells and to test the capacity of various anti-tumor drugs to the early stage of metastasis Citation Format: Johanna Elena Sidabra, Carla Sabrina Capobianco, Maria Florencia Gottardo, Daniel Fernando Alonso, Hernan Gabriel Farina. Development of new alternative in vivo models to study early metastasic dissemination in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5118.
Neuroendocrine tumors (NETs) comprise a heterogeneous group of malignancies that express neuropeptides as synaptophysin, chromogranin A and NSE, among others. Vasopressin (AVP) is a neuropeptide with an endocrine, paracrine and autocrine effect in normal and pathological tissues. AVP receptors are present in human lung, breast, pancreatic, colorectal and gastrointestinal tumors. While AVP V1 receptors are associated with stimulation of cellular proliferation, AVP V2 receptor (V2r) is related to antiproliferative effects. dDAVP is a synthetic analog of AVP that acts as a selective agonist for the V2r, which shows antitumor properties in breast and colorectal cancer models. Recently, we developed a derivative of dDAVP named [V4Q5]dDAVP, which presents higher antitumor effects in a breast cancer model compared to the parental compound. The goal of present work was to explore the antitumor properties of the V2r agonist dDAVP and its novel analog [V4Q5]dDAVP on aggressive human lung (NCI-H82) and prostate cancer (PC-3) cell lines with neuroendocrine (NE) characteristics. We study the presence of specific NE markers (CgA and NSE) and V2r expression in NCI-H82 and PC-3. Both cell lines express high levels of NE markers NSE and CgA but then incubation with dDAVP diminished expression levels of both markers. DDAVP and [V4Q5]dDAVP significantly reduced proliferation, doubling time and migration in both tumor cell cultures. [V4Q5]dDAVP analog showed a higher cytostatic effect than dDAVP, on cellular proliferation in the NCI-H82 cell line. Silencing of V2r using siRNA significantly attenuated the inhibitory effects of [V4Q5]dDAVP on NCI-H82 cell proliferation. We preliminary explored the in vivo effect of dDAVP and [V4Q5]dDAVP on NCI-H82 SCLC xenografts. Treated tumors (0.3 ugkg-1 thrice a week) grew slower rate in comparison to vehicle-treated animals. In this work we demonstrated that the specific agonists of V2r, dDAVP and [V4Q5]dDAVP displays antitumor capacity on different human models of lung and prostate cancers with NE features, showing their potential therapeutic benefits in the treatment of these aggressive tumors.
Abstract Breast cancer is the first cause of death from female cancer. After treatments against the primary tumor, a few cells can prevail in a dormant state for a long period, becoming minimal residual disease. These cells can resume growth and establish as metastasis, which are responsible for 90% of deaths from cancer. Patients with breast cancer diagnosed at early stage with small tumours have a 25-30% chance of recurrence after 10-15 years after treatment. These new growth often entails chemo and radio resistance and there is not response to conventional treatment. For all these reasons, there is a clear need for the development of novel therapies against these cells in a dormant state. The main limitation on the study of these dormant cells is the lack of relevant in vitro an in vivo models. In this work, we isolated and characterized two novel cell lines F3II-TP and F3II-NM. F3II-TP represents tumours with high proliferation rate whereas F3II-NM represents a dormant cell phenotype. The comparison between these cell lines is expected to yield new molecular targets for the design of new drugs that point dormant cells. The study began with the isolation and establishment of new tumour cell lines. After inoculation of the F3II murine mammary carcinoma cell line, we established cell populations in vitro, one from the primary tumour and another from a spontaneous metastatic nodule, F3II TP and F3II NM cell lines respectively. First, we analysed the expression of EMT markers such as E-cadherin, N-cadherin, Vimentin, Pan-Cytoqueratin and β-catenin by immunofluorescence. In in vitro assays, F3II NM presented major doubling time, major adhesion capacity and lower clonogenic potential. In addition, the expression of the dormancy related genes such as NR2F1 and BHLHE41 was analysed by qPCR, and we found that F3II-NM showed higher levels of transcript than F3II-TP. In addition, F3II NM showed lower chemo sensibility to cisplatin and in an orthotopic model of BALB/c mice, presented a longer latency time and lower tumour growth rate in comparison to F3II-TP. Taking all these together, F3II NM present a dormant phenotype and F3II TP a proliferative one. Finally, analysis and comparison of the cell lines using LFQ-Mass spectrometry revealed 256 differentially expressed proteins between the lines studied. Overexpressed genes of survival paths were identified in F3II-NM, such as DDX42, COX-2, ATGB4 involved in the inhibition of apoptosis, promotion of tumour recurrence via SOX-2 and therapy resistance, respectively. To sum up, we established a new interesting dormant tumour cell model, F3II NM that could help to a better understanding of the behaviour and survival of dormant cells. Moreover, we identified interesting protein targets that encourage the possibility of design new drugs that point and eliminate dormant cells Citation Format: Johanna E. Sidabra, Carla S. Capobianco, Maria F. Gottardo, Daniel F. Alonso, Hernan G. Farina. Isolation and characterization of novel murine mammary cell line with dormant phenotype and identification of new potential targets by mass spectrometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2649.
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