Inhibition of PPP1R15A alleviates osteoporosis via suppressing RANKL-induced osteoclastogenesis.

Osteoporosis results from overactivation of osteoclasts. There are currently few drug options for treatment of this disease. Since the successful development of allosteric inhibitors, phosphatases have become attractive therapeutic targets. Protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), is a stress-responsive protein, which promotes the UPR (unfolded protein response) and restores protein homeostasis. In this study we investigated the role of PPP1R15A in osteoporosis and osteoclastogenesis. Ovariectomy (OVX)-induced osteoporosis mouse model was established, osteoporosis was evaluated in the left femurs using micro-CT. RANKL-stimulated osteoclastogenesis was used as in vitro models. We showed that PPP1R15A expression was markedly increased in BMMs derived from OVX mice and during RANKL-induced osteoclastogenesis in vitro. Knockdown of PPP1R15A or application of Sephin1 (a PPP1R15A allosteric inhibitor in a phase II clinical trial) significantly inhibited osteoclastogenesis in vitro. Sephin1 (0.78, 3.125 and 12.5 µM) dose-dependently mitigated the changes in NF-κB, MAPK, and c-FOS and the subsequent nuclear factor of activated T cells 1 (NFATc1) translocation in RANKL-stimulated BMMs. Both Sephin1 and PPP1R15A knockdown increased the phosphorylated form of eukaryotic initiation factor 2α (eIF2α); knockdown of eIF2α reduced the inhibitory effects of Sephin1 on NFATc1-luc transcription and osteoclast formation. Furthermore, Sephin1 or PPP1R15A knockdown suppressed osteoclastogenesis in CD14+ monocytes from osteoporosis patients. In OVX mice, injection of Sephin1 (4, 8 mg/kg, i.p.) every two days for 6 weeks significantly inhibited bone loss, and restored bone destruction and decreased TRAP-positive cells. This study has identified PPP1R15A as a novel target for osteoclast differentiation, and genetic inhibition or allosteric inhibitors of PPP1R15A, such as Sephin1, can be used to treat osteoporosis. This study revealed that PPP1R15A expression was increased in osteoporosis in both human and mice. Inhibition of PPP1R15A by specific knockdown or an allosteric inhibitor Sephin1 mitigated murine osteoclast formation in vitro and attenuated ovariectomy-induced osteoporosis in vivo. PPP1R15A inhibition also suppressed pathogenic osteoclastogenesis in CD14+ monocytes from osteoporosis patients. These results identify PPP1R15A as a novel regulator of osteoclastogenesis and a valuable therapeutic target for osteoporosis.

Spatially Resolved Metabolomics Combined with the 3D Tumor-Immune Cell Coculture Spheroid Highlights Metabolic Alterations during Antitumor Immune Response.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.

Involvement of Rictor/mTORC2/Akt/GLUT4 pathway in the regulation of energy metabolism in the gastric smooth muscle of diabetic rats.

Diabetes mellitus can be accompanied by a variety of complications. The purpose of the present study was to characterize the Rictor/mTOR complex 2 (mTORC2)/Akt/glucose transporter 4 (GLUT4) pathway and its effects on energy metabolism in the gastric smooth muscle of diabetic rats. Diabetes was induced in rats using streptozotocin and their phenotype was compared with untreated rats. The relationship between gastric motility and energy metabolism was analyzed by comparing the contraction and ATP metabolism of muscle strips. Western blotting was used to detect the expression of key proteins in the pathway. The diabetic rats demonstrated less frequent and less powerful gastric smooth muscle contractions. The concentrations of ADP, AMP, and ATP, and the energy charge in gastric smooth muscle changed in different periods of diabetes, and these changes were consistent with changes in mechanistic target of rapamycin (mTOR) protein content. The expression of the key intermediates in signal transduction in the Rictor/mTORC2/Akt/GLUT4 pathway also underwent significant changes. Rictor protein expression increased during the development of diabetes, but the activation of mTORC2 did not increase with the increase in Rictor expression. GLUT4 translocation is regulated by Akt and its expression change during the development of diabetes. These findings suggest that altered energy metabolism is present in gastric smooth muscle that is associated with changes in the Rictor/mTORC2/Akt/GLUT4 pathway. Rictor/mTORC2/Akt/GLUT4 pathway may be involved in the regulation of energy metabolism in the gastric smooth muscle of diabetic rats and the development of diabetic gastroparesis.

Incidence of multiple myeloma in Kailuan cohort: A prospective community-based study in China.

BACKGROUND: Previous retrospective studies showed that the incidence and mortality rates for MM in China were lower than those in western countries. A large-scale prospective study on incidence and mortality rates of MM is still lacking. METHODS: Based on the prospective Kailuan Cohort study in China, we included all patients with MM in Kailuan Cohort from June 1, 2008 to December 31, 2016. Using the numbers of diagnosed cases and deaths during the study period as the numerators and the corresponding observed person-years as the denominators respectively, we calculated crude incidence and mortality rates. The 95% confidence intervals for crude incidence rate and mortality rate were estimated base on Poisson distribution. Rates were standardized by direct standardization according to the China population in 2000 and Segi' world standard population. RESULTS: A total of 22 members from Kailuan Cohort were first diagnosed with MM between 2008 and 2016. The calculated crude incidence rates were 2.8 (95% CI, 1.7-4.2) per 100,000 person-years for all participants. The standardized incidence rate was 0.9 per 100,000 person-years (95% CI, 0.5-2.1) when standardized by 2000 China population census data, and 1.0 per 100,000 person-years (95% CI, 0.6-1.8) when standardized by Segi's world standard population (WSP). The calculated crude mortality rates were 2.3 (95% CI, 1.4-3.6) per 100,000 person-years. The mortality standardized by 2000 China population census data was 0.7 per 100,000 person-years (95% CI, 0.3-1.9), and 0.9 per 100,000 population (95% CI, 0.5-1.7) when standardized by Segi's WSP. Both incidence and mortality for males were higher than that for females almost in all age groups. Both rates increased steadily with age. CONCLUSION: In this community-based prospective cohort study, we found that the incidence of MM in China was far lower than that in American and Europe.

Endocrine regulation of cancer stem cell compartments in breast tumors.

Cancer cells within breast tumors exist within a hierarchy in which only a small and rare subset of cells is able to regenerate growths with the heterogeneity of the original tumor. These highly malignant cancer cells, which behave like stem cells for new cancers and are called "cancer stem cells" or CSCs, have also been shown to possess increased resistance to therapeutics, and represent the root cause underlying therapy failures, persistence of residual disease, and relapse. As >90% of cancer deaths are due to refractory tumors, identification of critical molecular drivers of the CSC-state would reveal vulnerabilities that can be leveraged in designing therapeutics that eradicate advanced disease and improve patient survival outcomes. An expanding and complex body of work has now described the exquisite susceptibility of CSC pools to the regulatory influences of local and systemic hormones. Indeed, breast CSCs express a plethora of hormonal receptors, which funnel hormonal influences over every aspect of breast neoplasia - be it tumor onset, growth, survival, invasion, metastasis, or therapy resistance - via directly impacting CSC behavior. This article is intended to shed light on this active area of investigation by attempting to provide a systematic and comprehensive overview of the available evidence directly linking hormones to breast CSC biology.

The LINC01119-SOCS5 axis as a critical theranostic in triple-negative breast cancer.

The development of triple-negative breast cancer (TNBC) is critically regulated by certain tumor-microenvironment-associated cells called mesenchymal stem/stromal cells (MSCs), which we and others have shown promote TNBC progression by activating pro-malignant signaling in neighboring cancer cells. Characterization of these cascades would better our understanding of TNBC biology and bring about therapeutics that eliminate the morbidity and mortality associated with advanced disease. Here, we focused on the emerging class of RNAs called long non-coding RNAs or lncRNAs and utilized a MSC-supported TNBC progression model to identify specific family members of functional relevance to TNBC pathogenesis. Indeed, although some have been described to play functional roles in TNBC, activities of lncRNAs as mediators of tumor-microenvironment-driven TNBC development remain to be fully explored. We report that MSCs stimulate robust expression of LINC01119 in TNBC cells, which in turn induces suppressor of cytokine signaling 5 (SOCS5), leading to accelerated cancer cell growth and tumorigenesis. We show that LINC01119 and SOCS5 exhibit tight correlation across multiple breast cancer gene sets and that they are highly enriched in TNBC patient cohorts. Importantly, we present evidence that the LINC01119-SOCS5 axis represents a powerful prognostic indicator of adverse outcomes in TNBC patients, and demonstrate that its repression severely impairs cancer cell growth. Altogether, our findings identify LINC01119 as a major driver of TNBC development and delineate critical non-coding RNA theranostics of potential translational utility in the management of advanced TNBC, a class of tumors in most need of effective and targeted therapy.

In vivo library screening identifies the metabolic enzyme aldolase A as a promoter of metastatic lung colonization.

Elucidations of the factors that promote the growth of disseminated tumor cells (DTCs) into life-threatening lesions stand to provide much needed prognostic and therapeutic targets of translational utility for patients with metastatic cancer. To identify such regulators, we conducted gain-of-function cDNA library screening to discover genes that foster prostate cancer cell colonization of mouse lungs as an experimental model. Our efforts identified the metabolic enzyme aldolase A (ALDOA) as a driver of cancer cell motility, anchorage-independent growth, and metastatic colonization, and as a prognosticator of adverse patient outcome across many malignancies, including prostate, breast, pancreatic, and liver cancers. Metabolomics coupled with biochemical and functional analyses revealed that ALDOA triggered the activation of adenosine-5'-monophosphate (AMP)-activated protein kinase (AMPK), which we demonstrate played essential promalignant activities in ALDOA-expressing cells. Collectively, these findings unveiled vivo approaches to identify metastatic colonization regulators and uncovered previously undescribed roles for ALDOA-AMPK pathway in tumor progression.

The treatment option of progressive disease in breast cancer during neoadjuvant chemotherapy: a single-center experience.

Patients' responses to breast cancer neoadjuvant chemotherapy (NACT) differ because of heterogeneous tumor characteristics. Reports about NACT progression are sporadic. Here we enrolled 1187 patients who received NACT in our cancer center between January 1, 2007, and December 31, 2016. We analyzed the characteristics and treatments of patients with progressive disease (PD) or non-PD or pathological complete response (pCR). In total, 45 (3.8%) patients had PD. PD patients were associated with a significantly worse disease-free survival (DFS) (hazard ratio (HR) = 3.77; 95% CI, 1.77 to 8.00; P =.001) and overall survival (OS) (HR = 3.85; 95% CI, 1.77 to 8.35; P =.001). For the PD patients, 28 (62.2%) patients received mastectomy immediately after PD, and 17 (37.8%) changed to chemotherapy. DFS and OS exhibited no significant differences between these two salvage therapies. After a change to second chemotherapy, 58.8% (10/17) patients had PD or SD. With the exception of tumor size, pretreatment T stage, and histology type, no other significant differences were noted between PD and pCR patients. Our results demonstrated that PD patients were associated with a significantly worse prognosis. Based on these results, we suggest to give the addition of trastuzumab to HER-2 positive patients instead of changing the chemotherapy regimen and proceeding to surgery instead of further chemotherapy once patients have PD during NACT. Given that some similar characteristics exist between PD and pCR patients, more studies to identify novel molecular markers to predict disease response to NACT should be performed.

Impact on survival of estrogen receptor, progesterone receptor and Ki-67 expression discordance pre- and post-neoadjuvant chemotherapy in breast cancer.

PURPOSE: To investigate whether estrogen receptor (ER), progesterone receptor (PR) and Ki-67 expression discordance before and after neoadjuvant chemotherapy (NAC) correlates with prognosis and treatment of breast cancer patients. METHODS: The study cohort included 482 breast cancer patients at the Zhejiang Cancer Hospital from January 1, 2008, to December 31, 2018. Core needle biopsies and excised tissue biopsies pre- and post-NAC were obtained. Immunohistochemistry was used to determine ER, PR and Ki-67 status. The relationship between biomarker discordance before and after NAC and clinicopathological features was compared retrospectively. RESULTS: ER (n = 482), PR (n = 482) and Ki-67 (n = 448) expression was assessed in the same lesion pre- and post-NAC. Discordance in the three markers pre- and post-NAC was observed in 50 (10.4%), 82 (17.0%) and 373 (77.4%) cases, respectively. Positive-to-negative PR expression changes were the most common type of discordance observed. The risk of death in patients with a PR positive-to-negative conversion was 6.58 times greater than for patients with stable PR expression. The risk of death in patients with increased Ki-67 expression following NAC treatment was 2.05 times greater than for patients with stable Ki-67 expression. CONCLUSION: Breast cancer patients showed changes in ER, PR and/or Ki-67 status throughout NAC, and these changes possibly influenced disease-free survival and overall survival. A switch to negative hormone receptor expression with increased Ki-67 expression following NAC could be indicators of a worse prognosis. Biomarker expression investigations following NAC may potentially improve patient management and survival.

Associations between folate metabolism enzyme polymorphisms and breast cancer: A meta-analysis.

We performed this meta-analysis to explore associations between folate metabolism enzyme polymorphisms and breast cancer (BC) in a larger pooled population. Systematic literature research was performed to identify eligible studies for pooled analyses. Totally 92 genetic association studies were included for analyses. The pooled analyses revealed significant findings for MTRR rs1801394 polymorphism in South Asians, for MTR rs1805087 polymorphism in Caucasians and East Asians, and for MTHFR rs1801133 polymorphism in East Asians. In conclusion, the present meta-analysis indicated that MTRR rs1801394, MTR rs1805087, and MTHFR rs1801133 polymorphisms could be used to identify individuals at high risk of developing BC.

Expression of SHP-1 and SOCS6 in patients with acute leukemia and their clinical implication.

BACKGROUND: To investigate the expression and clinical relevance of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and suppressor of cytokine signaling 6 (SOCS6) in acute leukemia (AL). PATIENTS AND METHODS: The enrolled AL patients were divided into three groups (newly diagnosed, relapsed, and complete remission [CR]). Healthy donors were also included as a control group in this study. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to measure messenger RNA (mRNA) expression of SHP-1 and SOCS6. Statistical analysis was conducted to analyze the correlation between mRNA levels of SHP-1 and SOCS6 with patient outcomes. RESULTS: mRNA expression of SHP-1 was significantly lower in AL patients than that in healthy donors. The newly diagnosed or relapsed AL patients had lower mRNA levels of SHP-1 than the patients in CR. In contrast, SOCS6 mRNA expression was significantly higher in newly diagnosed or relapsed patients than that in patients in CR as well as healthy donors. However, mRNA levels of both SHP-1 and SOCS6 were positively correlated with the patient remission. The chemotherapy-induced remission rate was higher in patients with detectable SHP-1 or SOCS6 expression than in patients with undetectable SHP-1 or SOCS6 expression. Furthermore, the AL patients with detectable SHP-1 mRNA expression had lower incidence rate of invasive fungal infection. CONCLUSION: The results suggest that expression patterns of SHP-1 and SOCS6 differ in AL patients. Despite the difference, expression of SHP-1 and SOCS6 is associated with favorable outcomes, suggesting an anticancer property of these two genes in AL.

Breast carcinoma associated with prolactinoma: A case report.

We report a 28-year-old woman who presented with a 6-year history of milk-like discharge from both of her nipples and was diagnosed with prolactinoma based on computed tomography (CT) findings and serum prolactin level. Further breast examination revealed a mass located in the upper outer region of the left breast. She underwent subtotal pituitary tumor resection. Thereafter, modified radical mastectomy was performed for left breast cancer. Twelve years after treatment, prolactinoma recurrence was detected, and bromocriptine therapy was administered. No recurrence of breast cancer was discovered. Based on this case report, we stress the importance of prolactin levels due to their possible biologic effects on breast cancer induction or growth.

Research progress on berberine with a special focus on its oral bioavailability.

The natural product berberine (BBR) has become a potential drug in the treatment of diabetes, hyperlipidemia, and cancer. However, the oral delivery of BBR is challenged by its poor bioavailability. It is necessary to improve the oral bioavailability of BBR before it can be used in many clinical applications. Understanding the pharmacokinetic characteristics of BBR will enable the development of suitable formulas that have improved oral bioavailability. The key considerations for BBR are how to enhance the drug absorption and to avoid the intestinal first-pass effect. This review summarizes the pharmacological activities of BBR and analyzes the factors that lead to its poor oral bioavailability. In particular, the therapeutic potential of BBR in new indications from the aspect of oral bioavailability is discussed. In conclusion, BBR is a promising drug candidate for metabolic disorders and cancer but faces considerable challenges due to its poor oral bioavailability.

Three types of dermal grafts in rats: the importance of mechanical property and structural design.

BACKGROUND: To determine how the mechanical property and micro structure affect tissue regeneration and angiogenesis, three types of scaffolds were studied. Acellular dermal matrices (ADM), produced from human skin by removing the epidermis and cells, has been widely used in wound healing because of its high mechanical strength. Collagen scaffolds (CS) incorporated with poly(glycolide-co-L-lactide) (PLGA) mesh forms a well-supported hybrid dermal equivalent poly(glycolide-co-L-lactide) mesh/collagen scaffolds (PMCS). We designed this scaffold to enhance the CS mechanical property. These three different dermal substitutes-ADM, CS and PMCSs are different in the tensile properties and microstructure. METHODS: Several basic physical characteristics of dermal substitutes were investigated in vitro. To characterize the angiogenesis and tissue regeneration, the materials were embedded subcutaneously in Sprague-Dawley (SD) rats. At weeks 1, 2, 4 and 8 post-surgery, the tissue specimens were harvested for histology, immunohistochemistry and real-time quantitative PCR (RT-qPCR). RESULTS: In vitro studies demonstrated ADM had a higher Young's modulus (6.94 MPa) rather than CS (0.19 MPa) and PMCS (3.33 MPa) groups in the wet state. Compared with ADMs and CSs, PMCSs with three-dimensional porous structures resembling skin and moderate mechanical properties can promote tissue ingrowth more quickly after implantation. In addition, the vascularization of the PMCS group is more obvious than that of the other two groups. The incorporation of a PLGA knitted mesh in CSs can improve the mechanical properties with little influence on the three-dimensional porous microstructure. After implantation, PMCSs can resist the contraction and promote cell infiltration, neotissue formation and blood vessel ingrowth, especially from the mesh side. Although ADM has high mechanical strength, its vascularization is poor because the pore size is too small. In conclusion, the mechanical properties of scaffolds are important for maintaining the three-dimensional microarchitecture of constructs used to induce tissue regeneration and vascularization. CONCLUSION: The results illustrated that tissue regeneration requires the proper pore size and an appropriate mechanical property like PMCS which could satisfy these conditions to sustain growth.

In vitro evaluation of Panax notoginseng Rg1 released from collagen/chitosan-gelatin microsphere scaffolds for angiogenesis.

BACKGROUND: The emergence of skin substitutes provides a new approach for the treatment of wound repair and healing. The consistent and steady release of angiogenic factors is an important factor in the promotion of angiogenesis in skin substitutes, which usually lack, yet need, a vascular network. METHODS: In this study, ginsenoside Rg1, a natural compound isolated from Panax notoginseng (PNS), was incorporated into a collagen/chitosan-gelatin microsphere (CC-GMS) scaffold. The cumulative release kinetics were evaluated, and the effects of the released Rg1 on human umbilical vein endothelial cells (HUVECs) behavior, including proliferation, migration, tube formation, cell-cycle progression, cell apoptosis, and vascular endothelial growth factor (VEGF) secretion, were investigated. Additionally, HUVECs were cultured on the CC-GMS scaffold to test its biocompatibility. Standard Rg1 and VEGF were used as positive controls. RESULTS: The results indicated that the CC-GMS scaffold had good release kinetics. The Rg1 released from the CC-GMS scaffold did not lose its activity and had a significant effect on HUVEC proliferation. Both Rg1 and VEGF promoted HUVEC migration and tube formation. Rg1 did not induce HUVEC apoptosis but instead promoted HUVEC progression into the S and G2/M phases of the cell cycle. Rg1 significantly increased VEGF secretion compared with that in the control group. HUVEC culture on the CC-GMS scaffold indicated that this scaffold has good biocompatibility and that CC-GMS scaffolds containing different concentrations of Rg1 promote HUVEC attachment in a dose- and time-dependent manner. CONCLUSIONS: Rg1 may represent a new class of angiogenic agent that can be encapsulated in CC-GMS scaffolds to exert angiogenic effects in engineered tissue.

The progress of silver nanoparticles in the antibacterial mechanism, clinical application and cytotoxicity.

Nanotechnology is a highly promising field, with nanoparticles produced and utilized in a wide range of commercial products. Silver nanoparticles (AgNPs) has been widely used in clothing, electronics, bio-sensing, the food industry, paints, sunscreens, cosmetics and medical devices, all of which increase human exposure and thus the potential risk related to their short- and long-term toxicity. Many studies indicate that AgNPs are toxic to human health. Interestingly, the majority of these studies focus on the interaction of the nano-silver particle with single cells, indicating that AgNPs have the potential to induce the genes associated with cell cycle progression, DNA damage and mitochondrial associated apoptosis. AgNPs administered through any method were subsequently detected in blood and were found to cause deposition in several organs. There are very few studies in rats and mice involving the in vivo bio-distribution and toxicity, organ accumulation and degradation, and the possible adverse effects and toxicity in vivo are only slowly being recognized. In the present review, we summarize the current data associated with the increased medical usage of nano-silver and its related nano-materials, compare the mechanism of antibiosis and discuss the proper application of nano-silver particles.

Fabrication and characterization of poly(L-lactide-co-glycolide) knitted mesh-reinforced collagen-chitosan hybrid scaffolds for dermal tissue engineering.

Mechanical properties are essential considerations for the design of porous scaffolds in the field of tissue engineering. To develop a well-supported hybrid dermal substitute, poly(L-lactide-co-glycolide) (PLGA) yarns were knitted into a mesh with relative fixed loops, followed by incorporation into collagen-chitosan scaffolds (CCS) to obtain PLGA knitted mesh-reinforced CCS (PLGAm/CCS). The morphology and tensile strength in both the dry and wet state of PLGAm/CCS were investigated in vitro. To characterize the tissue response, specifically angiogenesis and tissue regeneration, PLGAm/CCS was embedded subcutaneously in Sprague-Dawley rats and compared with two control implants, i.e., PLGA mesh (PLGAm) and CCS. At weeks 1, 2, and 4 post surgery, tissue specimens were harvested for histology, immunohistochemistry, real-time quantitative PCR and Western blot analysis. These results demonstrated that the incorporation of PLGA knitted mesh into CCS can improve the mechanical strength with little influence on its mean pore size and porosity. After implantation, PLGAm/CCS can resist contraction and promote cell infiltration, neotissue formation, and blood vessel ingrowth, effectively. In conclusion, the mechanical strength of scaffolds can play a synergetic role in tissue regeneration and vascularization by maintaining its 3D microstructure. The ability of PLGAm/CCS to promote angiogenesis and induce in situ tissue formation demonstrates its strong potential in the field of skin tissue engineering.