Knockout of Shcbp1 sensitizes immunotherapy by regulating α-SMA positive cancer-associated fibroblasts.

The crucial role of cancer-associated fibroblasts (CAFs) in promoting T-cell exclusion has a significant impact on tumor immune evasion and resistance to immunotherapy. Therefore, enhancing T-cell infiltration into solid tumors has emerged as a pivotal area of research. We achieved a conventional knockout of Shcbp1 (Shcbp1-/- ) through CRISPR/Cas9 gene editing and crossed these mice with spontaneous breast cancer MMTV-PyMT mice, resulting in PyMT Shcbp1-/- mice. The different CAF subtypes were detected by flow cytometry analysis (FCA). We evaluated collagen and CAFs levels using Sirius red staining, immunohistochemistry (IHC), and immunofluorescence (IF). Primary tumor cells and CAFs were isolated from both PyMT Shcbp1+/+ and PyMT Shcbp1-/- mice. We analyzed CAFs' proliferation, invasion, migration, apoptosis, and cell cycle. Transwell coculture experiments were performed with primary tumor cells and CAFs to evaluate the role of CAFs in increasing the sensitivity of tumor cells to Erdafitinib. Tumors from PyMT Shcbp1+/+ and PyMT Shcbp1-/- mice were orthotopically transplanted to assess the therapeutic effect of the Erdafitinib and PD-1 combination. CAFs and T-cell infiltration in these tumors were assessed using FCA and IF. Knockout of Shcbp1 leads to a significant reduction in tumor burden, promotes longer survival, and decreases CAFs in MMTV-PyMT. Moreover, knockout of Shcbp1 enhances the sensitivity of Erdafitinib, leading to effective inhibition of CAFs' proliferation and invasion, as well as the induction of apoptosis. Additionally, it results in cell cycle arrest at the G2/M phase in vitro. Meanwhile, Shcbp1-/- CAFs change the sensitivity of Shcbp1-/- tumor cells to Erdafitinib compared to Shcbp1+/+ CAFs. Importantly, knockout of Shcbp1 boosts the effectiveness of Erdafitinib in combination with immune checkpoint blockade therapy by augmenting T-cell infiltration through CAFs regulation in vivo. Our findings demonstrate that knockout of Shcbp1 holds significant potential in enhancing the therapeutic response of Erdafitinib combined with PD-1 antibody treatment, offering promising prospects for future breast cancer therapies.

SHCBP1 Overexpression Aggravates Pancreatitis by Triggering the Loss of Primary Cilia.

Primary cilia are microtubule-based organelles that mediate various biological processes. Pancreatic cells are typically ciliated; however, the role of primary cilia in acute pancreatitis (AP) is largely unknown. Here, we report that the loss of primary cilia, mediated by SHCBP1 (SHC1 binding protein), exerted a provocative effect on AP. Primary cilia are extensively lost in inflamed pancreatic cells in vitro and in mouse tissues with AP in vivo. Abrogation of primary cilia aggravated lipopolysaccharide (LPS)-induced inflammation in pancreatic cells. Mechanistically, AP induced the overexpression of SHCBP1 mitotic factor, which is localized to the base of primary cilia. SHCBP1 deficiency relieved LPS- and cerulein-induced pancreatitis by preventing the loss of primary cilia in vitro and in vivo. Collectively, we reveal that inflammation-induced loss of primary cilia aggravates AP. Furthermore, abrogating SHCBP1 to prevent primary cilia loss is an efficient strategy to combat AP.

Therapeutic potential of SHCBP1 inhibitor AZD5582 in pancreatic cancer treatment.

Pancreatic cancer (PC) is a highly aggressive and deadly malignancy with limited treatment options and poor prognosis. Identifying new therapeutic targets and developing effective strategies for PC treatment is of utmost importance. Here, we revealed that SHCBP1 is significantly overexpressed in PC and negatively correlated with patient prognosis. Knockout of SHCBP1 inhibits the proliferation and migration of PC cells in vitro, and suppresses the tumor growth in vivo. In addition, we identified AZD5582 as a novel inhibitor of SHCBP1, which efficiently restrains the growth of PC in cell lines, organoids, and patient-derived xenografts. Mechanistically, we found that AZD5582 induced the apoptosis of PC cells by inhibiting the activity of PI3K/AKT signaling and preventing the degradation of TP53. Collectively, our study highlights SHCBP1 as a potential therapeutic target and its inhibitor AZD5582 as a viable agent for PC treatment strategies.

PICH Activates Cyclin A1 Transcription to Drive S-Phase Progression and Chemoresistance in Gastric Cancer.

The chemotherapeutic agent 5-fluorouracil (5-FU) remains the backbone of postoperative adjuvant treatment for gastric cancer. However, fewer than half of patients with gastric cancer benefit from 5-FU-based chemotherapies owing to chemoresistance and limited clinical biomarkers. Here, we identified the SNF2 protein Polo-like kinase 1-interacting checkpoint helicase (PICH) as a predictor of 5-FU chemosensitivity and characterized a transcriptional function of PICH distinct from its role in chromosome separation. PICH formed a transcriptional complex with RNA polymerase II (Pol II) and ATF4 at the CCNA1 promoter in an ATPase-dependent manner. Binding of the PICH complex promoted cyclin A1 transcription and accelerated S-phase progression. Overexpressed PICH impaired 5-FU chemosensitivity in human organoids and patient-derived xenografts. Furthermore, elevated PICH expression was negatively correlated with survival in postoperative patients receiving 5-FU chemotherapy. Together, these findings reveal an ATPase-dependent transcriptional function of PICH that promotes cyclin A1 transcription to drive 5-FU chemoresistance, providing a potential predictive biomarker of 5-FU chemosensitivity for postoperative patients with gastric cancer and prompting further investigation into the transcriptional activity of PICH. SIGNIFICANCE: PICH binds Pol II and ATF4 in an ATPase-dependent manner to form a transcriptional complex that promotes cyclin A1 expression, accelerates S-phase progression, and impairs 5-FU chemosensitivity in gastric cancer.

Role of heparanase in sepsis­related acute kidney injury (Review).

Sepsis-related acute kidney injury (S-AKI) is a common and significant complication of sepsis in critically ill patients, which can often only be treated with antibiotics and medications that reduce S-AKI symptoms. The precise mechanism underlying the onset of S-AKI is still unclear, thus hindering the development of new strategies for its treatment. Therefore, it is necessary to explore the pathogenesis of S-AKI to identify biomarkers and therapeutic targets for its early diagnosis and treatment. Heparanase (HPA), the only known enzyme that cleaves the side chain of heparan sulfate, has been widely studied in relation to tumor metabolism, procoagulant activity, angiogenesis, inflammation and sepsis. It has been reported that HPA plays an important role in the progression of S-AKI. The aim of the present review was to provide an overview of the function of HPA in S-AKI and to summarize its underlying molecular mechanisms, including mediating inflammatory response, immune response, autophagy and exosome biogenesis. It is anticipated that emerging discoveries about HPA in S-AKI will support HPA as a potential biomarker and therapeutic target to combat S-AKI.

Targeting UBE2T Potentiates Gemcitabine Efficacy in Pancreatic Cancer by Regulating Pyrimidine Metabolism and Replication Stress.

BACKGROUND & AIMS: Although small patient subsets benefit from current targeted strategies or immunotherapy, gemcitabine remains the first-line drug for pancreatic cancer (PC) treatment. However, gemcitabine resistance is widespread and compromises long-term survival. Here, we identified ubiquitin-conjugating enzyme E2T (UBE2T) as a potential therapeutic target to combat gemcitabine resistance in PC. METHODS: Proteomics and metabolomics were combined to examine the effect of UBE2T on pyrimidine metabolism remodeling. Spontaneous PC mice (LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre; KPC) with Ube2t-conditional knockout, organoids, and large-scale clinical samples were used to determine the effect of UBE2T on gemcitabine efficacy. Organoids, patient-derived xenografts (PDX), and KPC mice were used to examine the efficacy of the combination of a UBE2T inhibitor and gemcitabine. RESULTS: Spontaneous PC mice with Ube2t deletion had a marked survival advantage after gemcitabine treatment, and UBE2T levels were positively correlated with gemcitabine resistance in clinical patients. Mechanistically, UBE2T catalyzes ring finger protein 1 (RING1)-mediated ubiquitination of p53 and relieves the transcriptional repression of ribonucleotide reductase subunits M1 and M2, resulting in unrestrained pyrimidine biosynthesis and alleviation of replication stress. Additionally, high-throughput compound library screening using organoids identified pentagalloylglucose (PGG) as a potent UBE2T inhibitor and gemcitabine sensitizer. The combination of gemcitabine and PGG diminished tumor growth in PDX models and prolonged long-term survival in spontaneous PC mice. CONCLUSIONS: Collectively, UBE2T-mediated p53 degradation confers PC gemcitabine resistance by promoting pyrimidine biosynthesis and alleviating replication stress. This study offers an opportunity to improve PC survival by targeting UBE2T and develop a promising gemcitabine sensitizer in clinical translation setting.

Reveal the Mechanisms of Yi-Fei-Jian-Pi-Tang on Covid-19 through Network Pharmacology Approach.

Objectives: The Traditional Chinese Medicine (TCM) formula Yi-Fei-Jian-Pi-Tang (YFJPT) has been demonstrated effective against Corona Virus Disease 2019 (Covid-19). The aim of this article is to make a thorough inquiry about its active constituent as well as mechanisms against Covid-19 via TCM network pharmacology. Methods: All the ingredients of YFJPT are obtained from the pharmacology database of the TCM system. The genes which are associated with the targets are obtained by utilizing UniProt. The herb-target network is built up by utilizing Cytoscape. The target protein-protein interaction network is built by utilizing the STRING database and Cytoscape. The critical targets of YFJPT are explored by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Results: The outcomes show that YFJPT might has 33 therapeutic targets on Covid-19, namely, interleukin 2 (IL2), heme oxygenase 1 (HMOX1), interleukin 4 (IL4), interferon gamma (FNG), α nuclear factor of kappa light polypeptide gene enhancer in Bcells inhibitor, alpha (NFKBIA), nuclear factor-k-gene binding (NFKB), nitric oxide synthase 3 (NOS3), intercellular adhesion molecule 1 (ICAM1), hypoxia inducible factor 1 subunit alpha (HIF1A), mitogen-activated protein kinase 3 (MAPK3), epidermal growth factor receptor (EGFR), interleukin 10 (IL10), jun proto-oncogene (JUN), C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 8 (CXCL8), tumor protein p53 (TP53), interleukin 1 beta (IL1B), AKT serine/threonine kinase 1 (AKT1), tumor necrosis factor (TNF), interleukin 6 (IL6), erb-b2 receptor tyrosine kinase 2 (ERBB2), RELA proto-oncogene (RELA), NF-κB subunit, caspase 8 (CASP8), peroxisome proliferator activated receptor alpha (PPARA), TIMP metallopeptidase inhibitor 1 (TIMP1), transforming growth factor beta 1 (TGFB1), interleukin 1 alpha (IL1A), signal transducer and activator of transcription 1 (STAT1), mitogen-activated protein kinase 8 (MAPK8), myeloperoxidase (MPO), matrix metallopeptidase 3 (MMP3), matrix metallopeptidase 1 (MMP1), and NFE2 like bZIP transcription factor 2 (NFE2L2). The gene enrichment analysis prompts that YFJPT most likely contributes to patients related to Covid-19 by regulating the pathways of cancers. Conclusions: That will lay a foundation for the clinical rational application and further experimental research of YFJPT.

Therapeutic strategies targeting uPAR potentiate anti-PD-1 efficacy in diffuse-type gastric cancer.

The diffuse-type gastric cancer (DGC) is a subtype of gastric cancer (GC) associated with low HER2 positivity rate and insensitivity to chemotherapy and immune checkpoint inhibitors. Here, we identify urokinase-type plasminogen activator receptor (uPAR) as a potential therapeutic target for DGC. We have developed a novel anti-uPAR monoclonal antibody, which targets the domains II and III of uPAR and blocks the binding of urokinase-type plasminogen activator to uPAR. We show that the combination of anti-uPAR and anti-Programmed cell death protein 1 (PD-1) remarkably inhibits tumor growth and prolongs survival via multiple mechanisms, using cell line-derived xenograft and patient-derived xenograft mouse models. Furthermore, uPAR chimeric antigen receptor-expressing T cells based on the novel anti-uPAR effectively kill DGC patient-derived organoids and exhibit impressive survival benefit in the established mouse models, especially when combined with PD-1 blockade therapy. Our study provides a new possibility of DGC treatment by targeting uPAR in a unique manner.

ARHGAP11A Promotes the Malignant Progression of Gastric Cancer by Regulating the Stability of Actin Filaments through TPM1.

The mechanism underlying the poor prognosis of gastric cancer, including its high degree of malignancy, invasion, and metastasis, is extremely complicated. Rho GTPases are involved in the occurrence and development of a variety of malignant tumors. ARHGAP11A, in the Rho GTPase activating protein family, is highly expressed in gastric cancer, but its function and mechanism have not yet been explored. In this study, the effect of ARHGAP11A on the occurrence and development of gastric cancer and the mechanism related to this effect were studied. The expression of ARHGAP11A was increased in gastric cancer cells and tissues, and high ARHGAP11A expression in tissues was related to the degree of tumor differentiation and poor prognosis. Moreover, ARHGAP11A knockout significantly inhibited cell proliferation, cell migration, and invasion in vitro and significantly inhibited the tumorigenic ability of gastric cancer cells in nude mice in vivo. Further studies revealed that ARHGAP11A promotes the malignant progression of gastric cancer cells by interacting with TPM1 to affect cell migration and invasion and the stability of actin filaments. These results suggest that ARHGAP11A plays an important role in gastric cancer and may become a useful prognostic biomarker and therapeutic target for gastric cancer patients.

Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumor cell mitosis and impairs trastuzumab sensitivity to gastric cancer.

Trastuzumab is the backbone of HER2-directed gastric cancer therapy, but poor patient response due to insufficient cell sensitivity and drug resistance remains a clinical challenge. Here, we report that HER2 is involved in cell mitotic promotion for tumorigenesis by hyperactivating a crucial HER2-SHCBP1-PLK1 axis that drives trastuzumab sensitivity and is targeted therapeutically. SHCBP1 is an Shc1-binding protein but is detached from scaffold protein Shc1 following HER2 activation. Released SHCBP1 responds to HER2 cascade by translocating into the nucleus following Ser273 phosphorylation, and then contributing to cell mitosis regulation through binding with PLK1 to promote the phosphorylation of the mitotic interactor MISP. Meanwhile, Shc1 is recruited to HER2 for MAPK or PI3K pathways activation. Also, clinical evidence shows that increased SHCBP1 prognosticates a poor response of patients to trastuzumab therapy. Theaflavine-3, 3'-digallate (TFBG) is identified as an inhibitor of the SHCBP1-PLK1 interaction, which is a potential trastuzumab sensitizing agent and, in combination with trastuzumab, is highly efficacious in suppressing HER2-positive gastric cancer growth. These findings suggest an aberrant mitotic HER2-SHCBP1-PLK1 axis underlies trastuzumab sensitivity and offer a new strategy to combat gastric cancer.

A novel UBE2T inhibitor suppresses Wnt/ß-catenin signaling hyperactivation and gastric cancer progression by blocking RACK1 ubiquitination.

Dysregulation of the Wnt/ß-catenin signaling pathway is critically involved in gastric cancer (GC) progression. However, current Wnt pathway inhibitors being studied in preclinical or clinical settings for other cancers such as colorectal and pancreatic cancers are either too cytotoxic or insufficiently efficacious for GC. Thus, we screened new potent targets from ß-catenin destruction complex associated with GC progression from clinical samples, and found that scaffolding protein RACK1 deficiency plays a significant role in GC progression, but not APC, AXIN, and GSK3ß. Then, we identified its upstream regulator UBE2T which promotes GC progression via hyperactivating the Wnt/ß-catenin signaling pathway through the ubiquitination and degradation of RACK1 at the lysine K172, K225, and K257 residues independent of an E3 ligase. Indeed, UBE2T protein level is negatively associated with prognosis in GC patients, suggesting that UBE2T is a promising target for GC therapy. Furthermore, we identified a novel UBE2T inhibitor, M435-1279, and suggested that M435-1279 acts inhibit the Wnt/ß-catenin signaling pathway hyperactivation through blocking UBE2T-mediated degradation of RACK1, resulting in suppression of GC progression with lower cytotoxicity in the meantime. Overall, we found that increased UBE2T levels promote GC progression via the ubiquitination of RACK1 and identified a novel potent inhibitor providing a balance between growth inhibition and cytotoxicity as well, which offer a new opportunity for the specific GC patients with aberrant Wnt/ß-catenin signaling.

Primary cilia act as microgravity sensors by depolymerizing microtubules to inhibit osteoblastic differentiation and mineralization.

Microgravity-induced bone deterioration is a major challenge in long-term spaceflights since the underlying mechanisms remain elusive. Previously, we reported that primary cilia of osteoblasts gradually disappeared in microgravity conditions, and cilia abrogation was necessary for the inhibition of osteogenesis induced by microgravity. However, the precise roles of primary cilia have not been fully elucidated. Here, we report that microgravity depolymerizes the microtubule network of rat calvarial osteoblasts (ROBs) reversibly but has no effect on the architecture of actin filaments. Preventing primary ciliogenesis by chloral hydrate or a small interfering RNA sequence (siRNA) targeting intraflagellar transport protein 88 (IFT88) effectively relieves microgravity-induced microtubule depolymerization, whereas the stabilization of microtubules using pharmacological approaches cannot prevent the disappearance of primary cilia in microgravity conditions. Furthermore, quantification of the number of microtubules emerging from the ciliary base body shows that microgravity significantly decreases the number of basal microtubules, which is dependent on the existence of primary cilia. Finally, microgravity-induced repression of the differentiation, maturation, and mineralization of ROBs is abrogated by the stabilization of cytoplasmic microtubules. Taken together, these data suggest that primary cilia-dependent depolymerization of microtubules is responsible for the inhibition of osteogenesis induced by microgravity. Our study provides a new perspective regarding the mechanism of microgravity-induced bone loss, supporting the previously established role of primary cilia as a sensor in bone metabolism.

Superior mesenteric artery first approach can improve the clinical outcomes of pancreaticoduodenectomy: A meta-analysis.

BACKGROUND AND AIM: Superior mesenteric artery (SMA) first approach was a new improvement for pancreaticoduodenectomy (PD), but there is no evidence whether this approach is advantageous to PD. This meta-analysis aimed to determine the effects of the superior mesenteric artery (SMA) first approach on outcomes of pancreaticoduodenectomy (PD). METHODS: Literature searches were conducted on PubMed, The Cochrane Library, EMBASE, Web of Science, Clinical Trials Registry and China Biology Medicine disc. We completed a meta-analysis of the SMA first approach in PD, assessing overall survival, R0 resection, blood loss, postoperative complications, operation time and postoperative stay. The odds ratios and weighted mean differences with 95% confidence intervals (CIs) were pooled. RESULTS: Eighteen studies comprising 1483 participants were included. Patients who received SMA-PD had significantly lower overall complication rate (OR 0.62, 95% CI 0.47 to 0.81, P = 0.001) and less blood loss (WMD -264.84, 95% CI -336.1 to -193.58, P < 0.001). The obviously increased R0 resection rate (OR 2.92, 95% CI 1.72 to 4.96, P < 0.001) and 3-year OS (OR 2.15, 95% CI 1.34 to 3.43, P = 0.001) were found in the SMA-PD group. CONCLUSION: The SMA-PD group had better clinical outcomes, particularly in long-term survival of pancreatic cancer patients; furthermore, the patients acquired superior clinical efficacy via the posterior approach in SMA-PD.

The Role of Shcbp1 in Signaling and Disease.

Src homolog and collagen homolog (Shc) proteins have been identified as adapter proteins associated with cell surface receptors and have been shown to play important roles in signaling and disease. Shcbp1 acts as a Shc SH2-domain binding protein 1 and is involved in the regulation of signaling pathways, such as FGF, NF-κB, MAPK/ERK, PI3K/AKT, TGF-ß1/Smad and ß -catenin signaling. Shcbp1 participates in T cell development, the regulation of downstream signal transduction pathways, and cytokinesis during mitosis and meiosis. In addition, Shcbp1 has been demonstrated to correlate with Burkitt-like lymphoma, breast cancer, lung cancer, gliomas, synovial sarcoma, human hepatocellular carcinoma and other diseases. Shcbp1 may play an important role in tumorigenesis and progression. Accordingly, recent studies are reviewed herein to discuss and interpret the role of Shcbp1 in normal cell proliferation and differentiation, tumorigenesis and progression, as well as its interactions with proteins.

Novel functions of the primary cilium in bone disease and cancer.

The primary cilium, a sensory organelle that emanates from the cell surface of most mammalian cell types during growth arrest, has attracted the attention of many researchers over the past decade. Recently, a large number of new findings have assigned novel functions and roles to the primary cilium in signal transduction and related diseases, which has greatly augmented the importance of the cilium in human health and development. Here, we review emerging evidence supporting the primary cilium as a sensory organelle in signal transduction in microgravity, electromagnetic field sensing, chemosensation and tumorigenesis. We also present an overview of signal transduction crosstalk associated with the primary cilium in bone disease and cancer, including primary cilium-related Ca2+ signaling, parathyroid hormone signaling, cAMP signaling, BMP/Smad1/5/8 signaling and Wnt signaling. We anticipate that emerging discoveries about the function of the primary cilium will provide novel insight into the molecular mechanisms of stimulus sensation, signal transduction and pathogenesis.

Pulsed electromagnetic fields promote bone formation by activating the sAC-cAMP-PKA-CREB signaling pathway.

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly.

Icariin prevents bone loss by inhibiting bone resorption and stabilizing bone biological apatite in a hindlimb suspension rodent model.

Bone loss induced by microgravity is a substantial barrier to humans in long-term spaceflight. Recent studies have revealed that icariin (ICA) can attenuate osteoporosis in postmenopausal women and ovariectomized rats. However, whether ICA can protect against microgravity-induced bone loss remains unknown. In this study, the effects of ICA on a hindlimb suspension rodent model were investigated. Two-month-old female Wistar rats were hindlimb suspended and treated with ICA (25 mg·kg-1·d-1, i.g.) or a vehicle for 4 weeks (n = 6). The bone mass density of the hindlimbs was analyzed using dual-energy X-ray absorptiometry and micro-CT. mRNA expression of osteogenic genes in the tibia and the content of bone metabolism markers in serum were measured using qRT-PCR and ELISA, respectively. The bone mineral phase was analyzed using X-ray diffraction and atomic spectrometry. The results showed that ICA treatment significantly rescued the hindlimb suspension-induced reduction in bone mineral density, trabecular number and thickness, as well as the increases in trabecular separation and the structure model index. In addition, ICA treatment recovered the decreased bone-related gene expression, including alkaline phosphatase (ALP), bone glaprotein (BGP), and osteoprotegerin/receptor activator of the NF-κB ligand ratio (OPG/RANKL), in the tibia and the decreased bone resorption marker TRACP-5b levels in serum caused by simulated microgravity. Notably, ICA treatment restored the instability of bone biological apatite and the metabolic disorder of bone mineral elicited by simulated microgravity. These results demonstrate that ICA treatment plays osteoprotective roles in bone loss induced by simulated microgravity by inhibiting bone resorption and stabilizing bone biological apatite.

The flavonol glycoside icariin promotes bone formation in growing rats by activating the cAMP signaling pathway in primary cilia of osteoblasts.

Icariin, a prenylated flavonol glycoside isolated from the herb Epimedium, has been considered as a potential alternative therapy for osteoporosis. Previous research has shown that, unlike other flavonoids, icariin is unlikely to act via the estrogen receptor, but its exact mechanism of action is unknown. In this study, using rat calvarial osteoblast culture and rat bone growth models, we demonstrated that icariin promotes bone formation by activating the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway requiring functional primary cilia of osteoblasts. We found that icariin increases the peak bone mass attained by young rats and promotes the maturation and mineralization of rat calvarial osteoblasts. Icariin activated cAMP/PKA/CREB signaling of the osteoblasts by increasing intracellular cAMP levels and facilitating phosphorylation of both PKA and CREB. Blocking cAMP/PKA/CREB signaling with inhibitors of the cAMP-synthesizing adenylyl cyclase (AC) and PKA inhibitors significantly inhibited the osteogenic effect of icariin in the osteoblasts. Icariin-activated cAMP/PKA/CREB signaling was localized to primary cilia, as indicated by localization of soluble AC and phosphorylated PKA. Furthermore, blocking ciliogenesis via siRNA knockdown of a cilium assembly protein, IFT88, inhibited icariin-induced PKA and CREB phosphorylation and also abolished icariin's osteogenic effect. Finally, several of these outcomes were validated in icariin-treated rats. Together, these results provide new insights into icariin function and its mechanisms of action and strengthen existing ties between cAMP-mediated signaling and osteogenesis.

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells.

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3'-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.