One of the main reasons for cancer's low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate... Show moreOne of the main reasons for cancer's low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate the immunosuppression, which favors tumor progression. Extracellular vesicles (EVs) have shown great potential for targeted therapies as, depending on their biological origin, they can present different therapeutic properties, such as enhanced accumulation in the target tissue or modulation of the immune system. In the current study, EVs were isolated from M1-macrophages (M1-EVs) pre-treated with hyaluronic acid (HA) and the beta-blocker carvedilol (CV). The resulting modulated-M1 EVs (MM1-EVs) were further loaded with doxorubicin (MM1-DOX) to assess their effect in a mouse model of metastatic tumor growth. The cell death and cell migration profile were evaluated in vitro in 4T1 cells. The polarization of the RAW 264.7 murine macrophage cell line was also analyzed to evaluate the effects on the TME. Tumors were investigated by qRT-PCR and immunohistochemistry. MM1-DOX reduced the primary tumor size and metastases. NF-kappa B was the major gene downregulated by MM1-DOX. Furthermore, MM1-DOX reduced the expression of M2-TAM (CD-163) in tumors, which resulted in increased apoptosis (FADD) as well as decreased expression of MMP-2 and TGF-beta. These results suggest a direct effect in tumors and an upregulation in the TME immunomodulation, which corroborate with our in vitro data that showed increased apoptosis, modulation of macrophage polarization, and reduced cell migration after treatment with M1-EVs combined with HA and CV. Our results indicate that the M1-EVs enhanced the antitumor effects of DOX, especially if combined with HA and CV in an animal model of metastatic cancer. Show less
Photodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the... Show morePhotodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the photosensitizers (PS) toward the malignant tumors, resulting in prolonged periods of skin photosensitivity. In this work, we present a simple and straightforward strategy to increase the tumor distribution, selectivity, and efficacy of lipophilic PS zinc phthalocyanine (ZnPc) in colon cancer by their stabilization in purified, naturally secreted extracellular vesicles (EVs). The PS ZnPc was incorporated in EVs (EV-ZnPc) by a direct incubation strategy that did not affect size distribution or surface charge. By using co-culture models simulating a tumor microenvironment, we determined the preferential uptake of EV-ZnPc toward colon cancer cells when compared with macrophages and dendritic cells. We observed that PDT promoted total tumor cell death in normal and immune cells, but showed selectivity against cancer cells in co-culture models. In vivo assays showed that after a single intravenous or intratumoral injection, EV-ZnPc were able to target the tumor cells and strongly reduce tumor growth over 15 days. These data expose opportunities to enhance the potential and efficacy of PDT using simple non-synthetic strategies that might facilitate translation into clinical practice. Show less
Lima, M.L.D.; Medeiros, C.A.C.X. de; Guerra, G.C.B.; Santos, R.; Bader, M.; Pirih, F.Q.; ... ; Araujo, A.A. de 2021
Background: The aim of this study was to evaluate the role of AT1 and AT2 receptors in a periodontal inflammation experimental model. Methods: Periodontal inflammation was induced by LPS... Show moreBackground: The aim of this study was to evaluate the role of AT1 and AT2 receptors in a periodontal inflammation experimental model. Methods: Periodontal inflammation was induced by LPS/Porphyromonas gingivalis. Maxillae, femur, and vertebra were scanned using Micro-CT. Maxillae were analyzed histopathologically, immunohistochemically, and by RT-PCR. Results: The vertebra showed decreased BMD in AT1 H compared with WT H (p < 0.05). The femur showed increased Tb.Sp for AT1 H and AT2 H, p < 0.01 and p < 0.05, respectively. The Tb.N was decreased in the vertebra (WT H-AT1 H: p < 0.05; WT H-AT2 H: p < 0.05) and in the femur (WT H-AT1 H: p < 0.01; WT H-AT2 H: p < 0.05). AT1 PD increased linear bone loss (p < 0.05) and decreased osteoblast cells (p < 0.05). RANKL immunostaining was intense for AT1 PD and WT PD (p < 0.001). OPG was intense in the WT H, WT PD, and AT2 PD when compared to AT1 PD (p < 0.001). AT1 PD showed weak immunostaining for osteocalcin compared with WT H, WT PD, and AT2 PD (p < 0.001). AT1 H showed significantly stronger immunostaining for osteonectin in fibroblasts compared to AT2 H (p < 0.01). Conclusion: AT1 receptor knockout changed bone density, the quality and number of bone trabeculae, decreased the number of osteoblast cells, and increased osteonectin in fibroblasts. Show less
Background and Purpose Inflammation associated with the tumour microenvironment (TME) is critical for cancer development, and immunotherapeutic strategies modulating the immune response in cancer... Show moreBackground and Purpose Inflammation associated with the tumour microenvironment (TME) is critical for cancer development, and immunotherapeutic strategies modulating the immune response in cancer have been crucial. In this study, a methotrexate-loaded (MTX) poly(lactic-co-glycolic acid)-based (PLGA) drug nanocarrier covered with polyethyleneimine (Pei) and hyaluronic acid (HA) was developed and combined with an PD-L1 antibody to investigate anti-cancer and immunomodulatory effects in breast cancer TME.Experimental Approach Naked or HA-coated PeiPLGA-MTX nanoparticles (NPs) were assessed on 4T1 breast cancer cells grown in culture and in a mouse model of orthotopic tumour growth. Tumours were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and cell migration were analysed in vitro in 4T1 cells. Polarization of murine macrophages (RAW cells) was also carried out.Key Results Naked or HA-coated PeiPLGA-MTX NPs used alone or combined with PD-L1 antibody modified the tumourigenic course by TME immunomodulation, leading to reduction of primary tumour size and metastases. STAT3 and NF-kappa B were the major genes downregulated by NPs. In tumor-associated macrophages (TAM) such regulation switched M2 phenotype (CD163) towards M1 (CD68) and reduced levels of IL-10, TGF-beta and CCL22. Moreover, malignant cells showed overexpression of FADD, APAF-1, caspase-3 and E-cadherin, and decreased expression of Bcl-2, MDR-1, survivin, vimentin, CXCR4 and PD-L1 after treatment with NPs.Conclusion and Implications NPs-mediated STAT3/NF-kappa B signalling axis suppression disrupted crosstalk between immune and malignant cells, reducing immunosuppression and critical pro-tumour events. These findings provide a promising therapeutic approach capable of guiding the immune TME to suppress the development of breast cancer. Show less
One of the challenges of nanotechnology is to improve the efficacy of treatments for diseases, in order to reduce morbidity and mortality rates. Following this line of study, we made a nanoparticle... Show moreOne of the challenges of nanotechnology is to improve the efficacy of treatments for diseases, in order to reduce morbidity and mortality rates. Following this line of study, we made a nanoparticle formulation with a small size, uniform surfaces, and a satisfactory encapsulation coefficient as a target for colorectal cancer cells. The results of binding and uptake prove that using the target system with folic acid works: Using this system, cytotoxicity and cell death are increased when compared to using free oxaliplatin. The data show that the system maximized the efficiency of oxaliplatin in modulating tumor progression, increasing apoptosis and decreasing resistance to the drug. Thus, for the first time, our findings suggest that PLGA-PEG-FA increases the antitumor effectiveness of oxaliplatin by functioning as a facilitator of drug delivery in colorectal cancer. Show less
Extracellular vesicles (EVs) are important mediators of intercellular communication that participate in many physiological/pathological processes. As such, EVs have unique properties related to... Show moreExtracellular vesicles (EVs) are important mediators of intercellular communication that participate in many physiological/pathological processes. As such, EVs have unique properties related to their origin, which can be exploited for drug delivery applications in cell regeneration, immunosuppression, inflammation, cancer treatment or cardioprotection. Moreover, their cell-like membrane organization facilitates uptake and accumulation in specific tissues and organs, which can be exploited to improve selectivity of cargo delivery. The combination of these properties with the inclusion of drugs or imaging agents can significantly improve therapeutic efficacy and selectivity, reduce the undesirable side effects of drugs or permit earlier diagnosis of diseases. In this review, we will describe the natural properties of EVs isolated from different cell sources and discuss strategies that can be applied to increase the efficacy of targeting drugs or other contents to specific locations. The potential risks associated with the use of EVs will also be addressed. Show less
Oliveira, A.L.C.D.L.; Santos-Silva, A.M. dos; Silva-Junior, A.A. da; Garcia, V.B.; Araujo, A.A. de; Geus-Oei, L.F. de; ... ; Araujo, R.F. de 2020
The inflammation has been identified as factor of tumor progression, which has increased the interest and use of molecules with anti-inflammatory and antioxidant activities in the cancer treatment.... Show moreThe inflammation has been identified as factor of tumor progression, which has increased the interest and use of molecules with anti-inflammatory and antioxidant activities in the cancer treatment. In this study, the antioxidant, anti-inflammatory, and antitumor potentials of carvedilol was explored in a different approach. The cholesterol (CHO) was investigated as facilitated agent in the action of carvedilol-loaded nanoparticles. Different formulations exhibited spherical and stable nanoparticle with mean diameter size < 250 nm. The cholesterol changed the copolymer-drug interactions and the encapsulation efficiency. The in vitro cancer study was performed using murine colorectal cancer cell line (CT-26) to observe the cell viability and apoptosis on MTS assay and flow cytometry, respectively. The experiments have demonstrated that cholesterol improved the performance of drug-loaded nanoparticles, which was much better than free drug. The in vivo inflammation peritonitis model revealed that carvedilol-loaded nanoparticles increased the level of glutathione and leukocyte migration mainly when the functionalized drug-loaded nanoparticles were tested, in a lower dose than the free drug. As hypothesized, the experimental data suggest that cholesterol-functionalized carvedilol-loaded PLGA nanoparticles can be a novel and promising approach in the inflammation-induced cancer therapy since showed anti-inflammatory, antioxidant, and antitumor effects.Graphical abstract Show less
Accumulating evidence indicates that ceramide (Cer) and palmitic acid (PA) possess the ability to modulate switching of macrophage phenotypes and possess anti-tumorigenic effects; however, the... Show moreAccumulating evidence indicates that ceramide (Cer) and palmitic acid (PA) possess the ability to modulate switching of macrophage phenotypes and possess anti-tumorigenic effects; however, the underlying molecular mechanisms are largely unknown. The aim of the present study was to investigate whether Cer and PA could induce switching of macrophage polarization from the tumorigenic M2- towards the pro-inflammatory M1-phenotype, and whether this consequently altered the potential of colorectal cancer cells to undergo epithelial-mesenchymal transition (EMT), a hallmark of tumor progression. Our study showed that Cer- and PA-treated macrophages increased expression of the macrophage 1 (M1)-marker CD68 and secretion of IL-12 and attenuated expression of the macrophage 2 (M2)-marker CD163 and IL-10 secretion. Moreover, Cer and PA abolished M2 macrophage-induced EMT and migration of colorectal cancer cells. At the molecular level, this coincided with inhibition of SNAI1 and vimentin expression and upregulation of E-cadherin. Furthermore, Cer and PA attenuated expression levels of IL-10 in colorectal cancer cells co-cultured with M2 macrophages and downregulated STAT3 and NF-kappa B expression. For the first time, our findings suggest the presence of an IL-10-STAT3-NF-kappa B signaling axis in colorectal cancer cells co-cultured with M2 macrophages, mimicking the tumor microenvironment. Importantly, PA and Cer were powerful inhibitors of this signaling axis and, consequently, EMT of colorectal cancer cells. These results contribute to our understanding of the immunological mechanisms that underlie the anti-tumorigenic effects of lipids for future combination with drugs in the therapy of colorectal carcinoma. Show less
Apoptosis signaling pathways, drug resistance, and metastasis are important targets to develop new cancer treatments. We developed cholesterol-coated Poly(d,l-Lactide-co-Glycolic Acid) (PLGA)... Show moreApoptosis signaling pathways, drug resistance, and metastasis are important targets to develop new cancer treatments. We developed cholesterol-coated Poly(d,l-Lactide-co-Glycolic Acid) (PLGA) nanoparticles for effective encapsulation and delivery of retinoic acid and oxaliplatin to analyze their antitumor activity in colorectal cancer. The cell viability and proliferation of tumoral cells lines (CT-26 and SW-480) decreased when compared to control in vitro after treatment with the nanoparticles. In addition, apoptosis of CT-26 cells increased. Importantly, cytoprotection of nontumor cells was detected. Expression of pro-apoptotic proteins was upregulated, while anti-apoptotic proteins were downregulated either in vitro or in vivo. In addition, drug resistance and metastasis factors were downregulated in vivo. Human colorectal tumors that highly expressed BCL-2 and Ki-67 had a greater tendency towards death within 60 months. Our results show that loading oxaliplatin combined with retinoic acid and cholesterol in a nanoparticle formulation enables determination of optimal antitumor activity and subsequent treatment efficacy. Show less
Vasconcelos, R.C.; Ferreira, C.; Araujo, E.M. de; Motta, F.; Bomio, M.; Araujo, R.F. de; ... ; Araujo, A.A. de 2020
The aim of this study was to characterize and evaluate zirconia/hydroxyapatite in a critical size calvarial defect model in rats. Zirconia/hydroxyapatite (80/20) scaffold was characterized by X-ray... Show moreThe aim of this study was to characterize and evaluate zirconia/hydroxyapatite in a critical size calvarial defect model in rats. Zirconia/hydroxyapatite (80/20) scaffold was characterized by X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM). Critical size (8 mm) calvarial defects were created in wistar rats (n=48) and divided into four groups (90 days): G0 Group: positive control; G1 Group: hydroxyapatite; G2 Group: Zirconia; G3 Group: Zirconia/hydroxyapatite (80/20). Calvaria were subjected to Micro CT, histological and immunohistochemical analyses (RANK, RANKL, OPG, osteocalcin and FGF-2). IL-1 beta, IL-10 and TNF-alpha levels were analyzed by Elisa Immunoassay. The XRD analysis confirmed the formation of a crystalline structure and SEM showed the presence of regions corresponding to Zirconia and Hydroxyapatite. The Micro CT showed increased bone volume (BV/TV) and bone mineral density (BMD) in the G3 group (P<0.05). In addition, discrete periosteal bone formation was found at the interface of the defect edge and the external surface of the scaffold in the G3 group, showing osteocytes inside and osteoblasts (P<0.05) with scarce mononuclear inflammatory cells (P<0.01) in the central region of the defect. The immunostaining was moderate for RANKL, Osteocalcin and FGF-2 in the G3 group (P<0.5), while it was intense for OPG (P<0.001). IL-1 beta levels were decreased and IL-10 levels increased (P<0.05). Zirconia/hydroxyapatite (80/20) scaffold repair in critical size calvarial defects increased bone density, osteoblast and osteoclast cell numbers, FGF-2, osteocalcin and OPG immunostaining and IL-10 levels. Show less
