Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer.

Pancreatic cancer is highly lethal, of which 90% is pancreatic ductal adenocarcinoma (PDAC), with a 5-year survival rate of less than 12%, lacking effective treatment options and late diagnosis. Furthermore, the tumors show an intense resistance to cytotoxic chemotherapies. As autophagy is elevated in PDAC, targeting the autophagic pathway is regarded as a promising strategy for cancer treatment. Immunofluorescence and transmission electron microscopy were utilized to assess the autophagic flux. Label-free quantitative phosphoproteomics was used to figure out critically altered tyrosine phosphorylation of the proteins. Tumor-bearing mice were used to validate that SH2 TrM-(Arg)9 restrained the growth of tumor cells. SH2 TrM-(Arg)9 inhibited collagen-induced autophagy via blocking the DDR1/PYK2/ERK signaling cascades. SH2 TrM-(Arg)9 improved the sensitivity of PANC-1/GEM cells to gemcitabine (GEM). Inhibition of autophagy by SH2 TrM-(Arg)9 may synergized with chemotherapy and robusted tumor suppression in pancreatic cancer xenografts. SH2 TrM-(Arg)9 could enter into PDAC cells and blockade autophagy through inhibiting DDR1/PYK2/ERK signaling and may be a new treatment strategy for targeted therapy of PDAC.

Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic disease with high mortality. Currently, pirfenidone and nintedanib are the only approved drugs for IPF by the U.S. Food and Drug Administration (FDA), but their efficacy is limited. The activation of multiple phosphotyrosine (pY) mediated signaling pathways underlying the pathological mechanism of IPF has been explored. A Src homology-2 (SH2) superbinder, which contains mutations of three amino acids (AAs) of natural SH2 domain has been shown to be able to block phosphotyrosine (pY) pathway. Therefore, we aimed to introduce SH2 superbinder into the treatment of IPF. Methods: We analyzed the database of IPF patients and examined pY levels in lung tissues from IPF patients. In primary lung fibroblasts obtained from IPF patient as well as bleomycin (BLM) treated mice, the cell proliferation, migration and differentiation associated with pY were investigated and the anti-fibrotic effect of SH2 superbinder was also tested. In vivo, we further verified the safety and effectiveness of SH2 superbinder in multiple BLM mice models. We also compared the anti-fibrotic effect and side-effect of SH2 superbinder and nintedanib in vivo. Results: The data showed that the cytokines and growth factors pathways which directly correlated to pY levels were significantly enriched in IPF. High pY levels were found to induce abnormal proliferation, migration and differentiation of lung fibroblasts. SH2 superbinder blocked pY-mediated signaling pathways and suppress pulmonary fibrosis by targeting high pY levels in fibroblasts. SH2 superbinder had better therapeutic effect and less side-effect compare to nintedanib in vivo. Conclusions: SH2 superbinder had significant anti-fibrotic effects both in vitro and in vivo, which could be used as a promising therapy for IPF.

Aptamer-SH2 superbinder-based targeted therapy for pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) exhibits the poorest prognosis of all solid tumors with a 5-year survival rate of less than 10% and a median survival of 6 months after diagnosis. Numerous targeted agents have been developed and evaluated to improve the survival benefit in patients with PDAC. Unfortunately, most agents have been proven futile mainly owing to the dense stroma and the sophisticated signaling pathways of PDAC. Here, we show the potent effectiveness of Aptamer-SH2 superbinder-(Arg)9 conjugate on the treatment of PDAC. In this conjugate, DNA aptamer selected against PDAC cell line confers the function of specifically recognizing and binding to the PDAC cells and activated pancreatic stellate cells (PSCs) in stroma; cell penetrating peptide (Arg)9 facilitates the intracellular delivery of fused proteins; SH2 superbinder conducts the drastic blockade of multiple phosphotyrosines (pY)-based signaling pathways in tumor cells. METHODS: PDAC-associated pY were reanalyzed by bioinformatics screen. XQ-2d and SH2 superbinder-(Arg)9 were crosslinked with BMH to form XQ-2d-SH2 CM-(Arg)9 conjugate. Immunofluorescence was utilized to assess the potency of the conjugate entering cells. MTT and wound healing assays were performed to evaluate the proliferation or migration of PANC-1 and BxPC-3 cells, respectively. Western blot and Pulldown assays revealed that conjugate influenced several pY-based signaling pathways. Tumor-bearing mice were used to validate XQ-2d-SH2 CM-(Arg)9, which restrained the growth and metastasis of cancer cells. RESULTS: XQ-2d-His-SH2 CM-(Arg)9 conjugate restrained proliferation, invasion, and metastasis of PDAC cells with potent efficacy via blocking the activity of several pY-related signaling cascades. XQ-2d-His-SH2 CM-(Arg)9 could eliminate the dense stroma of PDAC and then arrive at tumor tissues. CONCLUSIONS: XQ-2d-SH2 CM-(Arg)9 conjugate may efficiently destroy the pancreatic stroma and show potent antitumor efficacy with minimal toxic effect by regulating tumor cell proliferation and metastasis in vitro and in vivo, which makes it to be a promising targeted therapy of PDAC.

N-acetylcysteine decreases malignant characteristics of glioblastoma cells by inhibiting Notch2 signaling.

BACKGROUND: Glioblastomas multiforme (GBM) is the most devastating primary intracranial malignancy lacking effective clinical treatments. Notch2 has been established to be a prognostic marker and probably involved in GBM malignant progression. N-acetylcysteine (NAC), a precursor of intracellular glutathione (GSH), has been widely implicated in prevention and therapy of several cancers. However, the role of NAC in GBM remains unclear and the property of NAC independent of its antioxidation is largely unknown. METHODS: The mRNA and protein levels of Notch family and other related factors were detected by RT-PCR and western blot, respectively. In addition, intracellular reactive oxygen species (ROS) was measured by flow cytometry-based DCFH-DA. Moreover, cell viability was assessed by CCK8 and cell cycle was analyzed by flow cytometry-based PI staining. The level of apoptosis was checked by flow cytometry-based Annexin V/PI. Cell migration and invasion were evaluated by wound healing and transwell invasion assays. At last, U87 Xenograft model was established to confirm whether NAC could restrain the growth of tumor. RESULTS: Our data showed that NAC could decrease the protein level of Notch2. Meanwhile, NAC had a decreasing effect on the mRNA and protein levels of its downstream targets Hes1 and Hey1. These effects caused by NAC were independent of cellular GSH and ROS levels. The mechanism of NAC-mediated Notch2 reduction was elucidated by promoting Notch2 degradation through Itch-dependent lysosome pathway. Furthermore, NAC could prevent proliferation, migration, and invasion and might induce apoptosis in GBM cells via targeting Notch2. Significantly, NAC could suppress the growth of tumor in vivo. CONCLUSIONS: NAC could facilitate Notch2 degradation through lysosomal pathway in an antioxidant-independent manner, thus attenuating Notch2 malignant signaling in GBM cells. The remarkable ability of NAC to inhibit cancer cell proliferation and tumor growth may implicate a novel application of NAC on GBM therapy.

All-trans-retinoid acid (ATRA) may have inhibited chondrogenesis of primary hind limb bud mesenchymal cells by downregulating Pitx1 expression.

Despite frequently well-established role of all-trans-retinoid acid (ATRA) in congenital limb deformities, its mechanism of action, thus far, is still ambiguous. Pitx1, which is expressed in the hindlimb bud mesenchyme, or its pathways may be etiologically responsible for the increased incidence of clubfoot. Here, we sought to investigate the mechanisms whereby Pitx1 regulated chondrogenesis of hindlimb bud mesenchymal cells in vitro. E12.5 embryonic rat hind limb bud mesenchymal cells were treated with ATRA at appropriate concentrations. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Hematoxylin-safranin-O-fast-green staining assays were used to observe cartilage nodules, and Pitx1 expression was examined by immunofluorescent microscopy. Real-time quantitative PCR and immunoblotting assays were applied to determine the mRNA expressions of Pitx1, Sox9 and type II collagen (Col2al), respectively. The results showed that ATRA inhibited the proliferation of hind limb bud cells dose-dependently. ATRA also induced a dose-dependent reduction in the number of cartilage nodules and the area of cartilage nodules compared with controls. Our real-time quantitative RT-PCR assays revealed that the mRNA expression of Pitx1, Sox9 and Col2al were significantly downregulated by ATRA. Furthermore, our immunofluorescent microscopy and Western blotting assays indicated that Pitx1 was mainly expressed in the cartilage nodules and the levels of Pitx1, Sox9 and Col2al were also downregulated by ATRA dose-dependently. The results indicated that ATRA may decrease chondrogenesis of hind limb bud mesenchymal cells by inhibiting cartilage-specific molecules, such as Sox9 and Col2al, via downregulating Pitx1 expression.

Lipopolysaccharide (LPS) promotes osteoclast differentiation and activation by enhancing the MAPK pathway and COX-2 expression in RAW264.7 cells.

Bone degradation is a serious complication of chronic inflammatory diseases such as septic arthritis, osteomyelitis and infected orthopedic implant failure. At present, effective therapeutic treatments for lipopolysaccharide (LPS)-induced bone destruction are limited to antibiotics and surgical repair in chronic inflammatory diseases. The present study aimed to evaluate the mechanism of LPS on osteoclast differentiation and activation. RAW264.7 cells were non-induced, or induced by the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and then treated with LPS. Following treatment, the number of osteoclasts and cell viability were measured. The expression of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), cathepsin K (CK), carbonic anhydrase II (CAII) and cyclooxygenase-2 (COX-2) was determined by RT-PCR. Protein levels of RANK, tumor necrosis factor receptor-associated factor 6 (TRAF6), COX-2 and mitogen-activated protein kinases (MAPK) were measured using western blotting assays. LPS promoted osteoclast differentiation of RAW264.7 cells and differentiated osteoclasts. LPS significantly increased mRNA expression of osteoclast-related genes in RAW264.7 cells. Differentiated osteoclasts were treated with LPS (100 ng/ml) and the results showed a significantly increased mRNA expression of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2. Furthermore, LPS at 100 ng/ml significantly promoted the MAPK pathway including increasing the phosphorylation of c-Jun N-terminal kinases (JNK) and the phosphorylation of the extracellular signal-regulated kinase (ERK1/2). In conclusion, LPS promoted osteoclast differentiation and activation by enhancing RANK signaling and COX-2 expression. LPS also promoted osteoclast differentiation via activation of the JNK and ERK1/2 cell proliferation pathways.

Canonical Wnt signaling is required for Panax notoginseng saponin-mediated attenuation of the RANKL/OPG ratio in bone marrow stromal cells during osteogenic differentiation.

Panax notoginseng saponins (PNS) are known to regulate the osteogenic differentiation of bone marrow stromal cells (BMSCs). In the present study, we investigated whether PNS could promote the osteogenic differentiation of BMSCs through modulating the Wnt/ß-catenin signaling pathways, which are implicated in BMSCs osteogenesis. We found that PNS enhanced the mRNA expression of OPG, ß-catenin, and cyclin D1 while decreased the mRNA expression of RANKL and PPARγ2. The actions of PNS on BMSCs were reversed (or partially) by DKK-1, a classical inhibitor of Wnt/ß-catenin signaling. These results suggest that PNS stimulating bone formation by promoting the proliferation and osteogenic differentiation of BMSCs, and could also protect the skeletal system by decreasing bone resorption through reduction of RANKL/OPG expression via Wnt/ß-catenin signaling pathways.

Quercetin triggers apoptosis of lipopolysaccharide (LPS)-induced osteoclasts and inhibits bone resorption in RAW264.7 cells.

AIMS: Quercetin, a flavonoid present in vegetables, has anti-inflammatory properties and potential inhibitory effects on bone resorption. Up to date, the effect of quercetin on lipopolysaccharide (LPS)-induced osteoclastogenesis has not yet been reported. In the current study, we evaluated the effect of quercetin on LPS-induced osteoclast apoptosis and bone resorption. METHODS: RAW264.7 cells were non-treated, treated with LPS alone, or treated with both LPS and quercetin. After treatment, the number of osteoclasts, cell viability, bone resorption and osteoclast apoptosis were measured. The expressions of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP9) and cathepsin K (CK) were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of receptor activator of nuclear factor-ĸB (RANK), tumor necrosis factor receptor-associated factor 6 (TRAF6), cyclooxygenase-2 (COX-2), Bax, Bcl-2 and mitogenactivated protein kinases (MAPKs) were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with MAPK inhibitors. RESULTS: LPS directly promoted osteoclast differentiation of RAW264.7 cells and upregulated the protein expression of RANK, TRAF6 and COX-2; while quercetin significantly decreased the number of LPS-induced osteoclasts in a dose-dependent manner. None of the treatments increased cytotoxicity in RAW264.7 cells. Quercetin inhibited mRNA expressions of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2 in LPS-induced mature osteoclasts. Quercetin also induced apoptosis and inhibited bone resorptive activity in LPS-induced mature osteoclasts. Furthermore, quercetin promoted the apoptotic signaling pathway including increasing the phosphorylation of p38-MAPK, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), and Bax, while inhibited Bcl-2 expression. CONCLUSIONS: Quercetin could supress LPS-induced osteoclast bone resorption through blocking RANK signaling and inhibiting the expression of osteoclast-related genes. Quercetin also promoted LPS-induced osteoclast apoptosis via activation of the MAPK apoptotic signaling pathway. These findings suggest that quercetin could be of potential use as a therapeutic agent to treat bacteria-induced bone resorption.

Cisplatin induces apoptosis via upregulating Wrap53 in U-2OS osteosarcoma cells.

Wrap531α, a newly identified natural antisense transcript of p53, can regulate p53 expression upon DNA damage. We sought to investigate changes in Wrap53 and p53 levels in an osteosarcoma cell line (U-2OS) exposed to cisplatin and to study apoptosis before and after knockdown of Wrap53. Our RT-PCR analysis showed a dose- dependent 3 to 40-fold increase in Wrap53 mRNA transcript levels in U-2OS exposed to 5 to 20 µM cisplatin. An approximate 2-fold increase was also observed in transcript levels of p53 mRNA. Furthermore, transient knockdown of Wrap53 by siRNAs in U-2OS cells treated with 10 µM cisplatin reduced p53 mRNA transcript levels by up to 50% of those of controls. Immunoblotting analysis showed that in U-2OS cells treated with siRNA against exon 4 of the Wrap53 gene, the protein level of p53 was also markedly reduced. Our findings suggest that cisplatin upregulates the expression of p53 in osteosarcoma cells by upregulating the transcript levels of Wrap53. Finally, measurement of apoptotic cell death by flow cytometry showed that knockdown of Wrap53 reduced apotosis in U-2OS cells induced by cisplatin.

Panax notoginseng saponins promotes proliferation and osteogenic differentiation of rat bone marrow stromal cells.

AIM OF THE STUDY: Panax notoginseng saponins (PNS) is the main effective component of Panax notoginseng, have various pharmacologic activities such as antioxidant, anti-inflammatory, and estrogen-like bioactivities, have been shown to be an effective agent on anti-osteoporosis. Bone marrow stromal cells (BMSCs) play a crucial homeostatic role in skeletal modeling and remodeling due to their capability to differentiate into osteooblasts. Whether PNS has effect on osteogenic differentiation of BMSCs are unknown. This study was designed to investigate the effects of PNS on the proliferation and osteogenic differentiation of BMSCs in vitro. MATERIALS AND METHODS: When BMSCs cultivated in the basal medium or the osteogenic induction medium (OS with or without PNS), cell proliferation was analyzed using an MTT assay, the mineralization was assessed using Alizarin red S staining, the alkaline phosphatase activity was measured using a commercial kit, the mRNA level of osteogenic gene and PPARγ2 gene were determined using RT-PCR, the protein level of PPARγ2 was analyzed by Western blotting. RESULTS: BMSCs cultured in the basal medium with PNS caused a significant increase in proliferation. PNS treatment increased ALP activity, Alizarin red S staining and mRNA level of ALP, Cbfa 1, OC, and BSP, whereas decreased the mRNA level and protein expression of PPARγ2 during osteogenic induction. In addition, the effects of PNS treatment were dose-dependent relationship. CONCLUSION: PNS could stimulate BMSCs proliferation and promote their osteogenic differentiation by up-regulation expression of osteogenic marker gene and down-regulation expression of adipogenic marker gene in a dose-dependent manner. Thus, PNS may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs.

Panax notoginseng saponins potentiate osteogenesis of bone marrow stromal cells by modulating gap junction intercellular communication activities.

AIMS: The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. We investigated the effects of PNS on gap junction intercellular communication (GJIC) and osteogenesis-associated genes in rat bone marrow stromal cells (BMSCs). METHODS AND RESULTS: Our MTT assays demonstrated that PNS enhanced BMSC proliferation under basal medium culture in vitro. Alkaline phosphatase (ALP) assays and alizarin Red staining showed that PNS stimulated ALP activity and calcium deposition by BMSCs. Measurement of the traversing of Lucifer yellow through intercellular junctions revealed that PNS significantly stimulated GJIC activities. RT-PCR assays further showed that PNS augmented the increase in the mRNA levels of ALP, core-binding factor a1, and bone sialoprotein while decreasing the mRNA level of PPARγ2. PNS also reduced RANKL levels and increased osteoprotegerin levels. Gap junction inhibitor, 18a-glycyrrhetinic acid, could partially reverse the actions of PNS on BMSCs. CONCLUSIONS: Our findings indicate that PNS could promote osteogenesis of BMSCs by targeting osteogenesis-associated genes, which could be mediated by their actions on GJIC.