Bioactive silica nanoparticles target autophagy, NF-κB, and MAPK pathways to inhibit osteoclastogenesis.

Spherical 50 nm silica-based nanoparticles (SiNPs) promote healthy bone homeostasis and maintenance by supporting bone forming osteoblast lineage cells while simultaneously inhibiting the differentiation of bone resorbing osteoclasts. Previous work demonstrated that an intraperitoneal injection of SiNPs in healthy mice - both young and old - increased bone density and quality, suggesting the possibility that SiNPs represent a dual action therapeutic. However, the underlying mechanisms governing the osteoclast response to SiNPs have yet to be fully explored and defined. Therefore, the goals of this study were to investigate the cellular and molecular mechanisms by which SiNPs inhibit osteoclastogenesis. SiNPs strongly inhibited RANKL-induced osteoclast differentiation within the first hours and concomitantly inhibited early transcriptional regulators such as Nfatc1. SiNPs simultaneously stimulated expression of autophagy related genes p62 and LC3ß dependent on ERK1/2 signaling pathway. Intriguingly, SiNPs were found to stimulate autophagosome formation while inhibiting the autophagic flux necessary for RANKL-stimulated osteoclast differentiation, resulting in the inhibition of both the canonical and non-canonical NF-κB signaling pathways and stabilizing TRAF3. These results suggest a model in which SiNPs inhibit osteoclastogenesis by inhibiting the autophagic machinery and RANKL-dependent functionality. This mechanism of action defines a novel therapeutic strategy for inhibiting osteoclastogenesis.

Role of autophagy-related protein expression in patients with rectal cancer treated with neoadjuvant chemoradiotherapy.

BACKGROUND: Autophagy, a cellular degradation process, has complex roles in tumourigenesis and resistance to cancer treatment in humans. The aim of this study was to explore the expression levels of autophagy-related proteins in patients with rectal cancer and evaluate their clinical role in the neoadjuvant chemoradiotherapy setting. METHODS: All specimens evaluated were obtained from 101 patients with colorectal cancer who had undergone neoadjuvant chemoradiotherapy and curative surgery. The primary outcomes measured were the expression levels of two autophagy-related proteins (microtubule-associated protein 1 light chain 3 beta (LC3ß) and beclin-1) by immunohistochemistry and their association with clinicopathological parameters and patient survival. RESULTS: Among the 101 patients, the frequency of high expression of beclin-1 was 31.7% (32/101) and that of LC3ß was 46.5% (47/101). A pathologic complete response was inversely associated with LC3ß expression (P = 0.003) and alterations in the expression of autophagy-related proteins (P = 0.046). In the multivariate analysis, however, autophagy-related protein expression did not show prognostic significance for relapse-free survival or overall survival. CONCLUSIONS: High expression of autophagy-related proteins shows a strong negative association with the efficacy of neoadjuvant chemoradiotherapy in patients with rectal cancer. Autophagy has clear implications as a therapeutic target with which to improve the efficacy of neoadjuvant chemoradiotherapy.

Remodelamento da matriz extracelular da medula óssea em desnutrição protéica: possível relação da via de AKT com a expressão de fibronectina e metaloproteinases de matriz / Extracellular matriz remodeling of the bone marrow in protein malnutrition: possible relationship of AKT pathway with expression of fibronectin and matriz metalloproteínases

A desnutrição proteica (DP) pode ocasionar alterações na matriz extracelular (MEC) de diferentes órgãos e tecidos, inclusive o hematopoético, com comprometimento funcional. Estudos do nosso laboratório demonstraram, em modelo murino de DP, aumento da expressão proteica de fibronectina (FN) no estroma medular ósseo in vivo, principalmente na região subendosteal (local de fixação da célula tronco progenitora hemopoética). Já in vitro, no estroma medular ósseo, observou-se tanto o aumento quanto a diminuição de FN e a presença de suas isoformas. Essas alterações de FN parecem estar envolvidas com a hipoplasia da medula óssea (MO) em camundongos desnutridos. As modificações quantitativas de FN podem ser devidas: (i) à ação das metaloproteinases de matriz (MMP) responsáveis pela degradação das proteínas da MEC; (ii) aos inibidores de metaloproteinases (TIMP) que regulam a degradação da MEC; (iii) às alterações transcricionais, reguladas pela via de AKT/mTOR, que controla os splicing alternativos na FN, resultando em isoformas dessa proteína; (iv) a processos pós-transcricionais modulados por LC3, que aumenta a tradução do RNAm de FN. Assim, o objetivo deste estudo foi elucidar os mecanismos que alteram o turnover de FN no estroma medular ósseo em modelo murino de DP. Utilizamos camundongos, C57BL/6J machos, adultos, separados em dois grupos: controle e desnutrido, alimentados, ad libitum, com ração contendo 12% e 2% de proteína, respectivamente. Após cinco semanas de indução à desnutrição os camundongos foram eutanasiados, e coletado o material biológico. Avaliamos: o estado nutricional, o hematológico, a histologia da MO femoral bem como a determinação imunohistoquímica da FN, MMP-2 e MMP-9, determinação da expressão de FN e suas isoformas em células totais da MO, o estabelecimento do estroma medular ósseo in vitro, por 28 e 35 dias de cultivo. A partir das culturas foram avaliadas a expressão de RNAm de FN e suas isoformas, MMP-2, MMP-9, TIMP-1, TIMP-2, AKT, mTOR e LC3α e ß, quantificação de MMP-2, MMP-9, TIMP-1, TIMP-2,TNFα, TGFß e IL-1ß e determinação de LC3ß e proteínas da via de AKT/mTOR. Não observamos alterações na expressão do RNAm de FN e suas isoformas ex vivo e in vitro, mas um aumento da deposição de FN na MO.Também não observamos modificações na imunolocalização de MMP-2 e MMP-9 na MO e na atividade dessas proteínas no sobrenadante de culturas de células estromais in vitro, mas houve aumento da expressão do RNAm de MMP-9 em 28 dias de cultivo. Não detectamos alterações na expressão de RNAm e na concentração de TIMP-1 e TIMP-2 no sobrenadante das culturas. Houve redução significativa de TNFα e TGFß no sobrenadante das culturas de 28 dias. Observamos aumento da expressão do RNAm de mTOR em culturas de 28 dias e LC3α e LC3ß em 35 dias de células estromais. Encontramos menor fosforilação de PI3K, AKT, PTEN, mTOR e mTOR total e aumento de LC3ß em culturas de 28 dias, mas redução de LC3ß em 35 dias. Em função dos dados inferimos que a DP conduz a alterações da FN que não estão relacionadas à ação de MMPs e TIMPs e sim a modificações de LC3ß e da via de AKT/mTOR

Protein malnutrition (PM) can lead changes in extracellular matrix (ECM) from several organs and tissues, including hematopoietic, with functional impairments. Research from our laboratory demonstrated, in a murine model of protein malnutrition, increase in proteic expression of fibronectin (FN) in vivo bone marrow stroma, principally in subendosteal region (attachment site of hematopoietic stem/progenitor cell - HSPC). It was observed as both an increase and a decrease in the presence of FN and its isoforms in vitro bone marrow stroma. These FN changes seem to be related to bone marrow (BM) hypoplasia in malnourished mice. Quantitative FN changes may be due to: (i) action of matrix metalloproteinases (MMP) responsible for ECM proteins degradation; (ii) tissue inhibitors of metalloproteinases (TIMP) that regulate ECM degradation; (iii) transicional changes regulated by AKT/mTOR pathway, which controls alternative splicing in FN, resulting in isoforms from this protein; (iv) post-transcriptional processes modulated by LC3 that increases FN mRNA translation. Therefore, the aim of this study was to elucidade the mechanisms that changes the FN turnover in bone marrow stroma in a murine model of PM. C57BL/6J, adult and male mice were used and divided into two groups: control and malnourished, fed ad libitum with ration containing 12% and 2% of protein, respectively. After five weeks of induction malnutrition, mice were euthanized and the biological material was collected. We evaluated: nutritional and hematologic status, the femoral BM histology, immunohistochemistry determination of FN, MMP-2 and MMP-9, the FN and its isoforms expression determination in total BM cells, establishment of in vitro bone marrow stroma for 28 and 35 days of culture. From the cultures were evaluated FN mRNA expressions and its isoforms, MMP-2, MMP-9, TIMP-1, TIMP-2, AKT, mTOR, LC3α and ß, quantification of MMP-2, MMP-9, TIMP-1, TIMP-2,TNFα, TGFß and IL-1ß and determination of LC3ß and AKT/mTOR proteins. No changes were observed, ex vivo and in vitro, in the expression of FN mRNA and its isoforms, but there was a FN deposition increase in BM. We did not observe modifications in MMP-2 e MMP-9 immunolocalization in BM and in these proteins activity in the supernatant of in vitro stromal cell culture, but there was an increase in MMP-9 mRNA expression after 28 days of culture. We did not detect changes in mRNA and in TIMP-1 and TIMP-2 expressions in the supernatant of cultures. There was significant reduction of TNFα and TGFß in the cultures supernatant of 28 days. We observed an increase of mTOR RNAm in 28 days cultures and also LC3α and LC3ß in stromal cells with 35 days. We found lower phosphorylation of PI3K, AKT, PTEN, mTOR e total mTOR and an LC3ß increase in 28 days cultures, yet an LC3ß reduction in 35 days. According to the data we conclude that PM leads to FN changes that are not related to MMPs and TIMPs actions, but the LC3ß and AKT/mTOR pathway modifications

Methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate (HIMOXOL), a synthetic oleanolic acid derivative, induces both apoptosis and autophagy in MDA-MB-231 breast cancer cells.

HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate) is a synthetic derivative of oleanolic acid (OA). HIMOXOL revealed the highest cytotoxic effect among tested synthetic OA analogs. In this study we focused on elucidating the cytotoxic mechanism of HIMOXOL in MDA-MB-231 breast cancer cells. HIMOXOL reduced MDA-MB-231 cell viability with an IC50 value of 21.08±0.24µM. In contrast to OA, the tested compound induced cell death by activating apoptosis and the autophagy pathways. More specifically, we found that HIMOXOL was able to activate the extrinsic apoptotic pathway, which was proven by observation of caspase-8, caspase-3 and PARP-1 protein activation in Western blot analysis. An increase in the ratio of Bax/Bcl-2 protein levels was also detected. Moreover, HIMOXOL triggered microtubule-associated protein LC3-II expression and upregulated beclin 1. This observed compound activity was modulated by mitogen-activated protein kinases and NFκB/p53 signaling pathways. Together, these data suggest that HIMOXOL, a synthetic oleanolic acid derivative which activates dual cell death machineries, could be a potential and novel chemotherapeutic agent.