Elevated extracellular calcium ions accelerate the proliferation and migration of HepG2 cells and decrease cisplatin sensitivity.

Hepatoblastoma is the most frequent liver malignancy in children. HepG2 has been discovered as a hepatoblastoma-derived cell line and tends to form clumps in culture. Intriguingly, we observed that the addition of calcium ions reduced cell clumping and disassociated HepG2 cells. The calcium signal is in connection with a series of processes critical in the tumorigenesis. Here, we demonstrated that extracellular calcium ions induced morphological changes and enhanced the epithelial-mesenchymal transition in HepG2 cells. Mechanistically, calcium ions promoted HepG2 proliferation and migration by up-regulating the phosphorylation levels of focal adhesion kinase (FAK), protein kinase B, and p38 mitogen-activated protein kinase. The inhibitor of FAK or Ca 2+/calmodulin-dependent kinase Ⅱ (CaMKⅡ) reversed the Ca 2+-induced effects on HepG2 cells, including cell proliferation and migration, epithelial-mesenchymal transition protein expression levels, and phosphorylation levels of FAK and protein kinase B. Moreover, calcium ions decreased HepG2 cells' sensitivity to cisplatin. Furthermore, we found that the expression levels of FAK and CaMKⅡ were increased in hepatoblastoma. The group with high expression levels of FAK and CaMKⅡ exhibited significantly lower ImmunoScore as well as CD8 + T and NK cells. The expression of CaMKⅡ was positively correlated with that of PDCD1 and LAG3. Correspondingly, the expression of FAK was negatively correlated with that of TNFSF9, TNFRSF4, and TNFRSF18. Collectively, extracellular calcium accelerates HepG2 cell proliferation and migration via FAK and CaMKⅡ and enhances cisplatin resistance. FAK and CaMKⅡ shape immune cell infiltration and responses in tumor microenvironments, thereby serving as potential targets for hepatoblastoma.

Cu2+-Pyropheophorbide a-Cystine Conjugate: Synergistic Photodynamic/Chemodynamic Therapy and Glutathione Depletion Improves the Antitumor Efficacy and Downregulates the Hypoxia-Inducing Factor.

Cancer immune escape, metastasis, recurrence, and multidrug resistance are all associated with hypoxia in the tumor microenvironment (TME). We synthesized a CuPPaCC conjugate for reactive oxygen species (ROS)-mediated cancer therapy. CuPPaCC continuously produced cytotoxic ROS and oxygen through a photo-chemocycloreaction, alleviated hypoxia, and inhibited the expression of a hypoxia-inducing factor (HIF-1α). CuPPaCC was synthesized from pyromania phyllophyllic acid a (PPa), cystine (CC), and copper ions, and its structure was characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The ability of CuPPaCC to produce ROS and oxygen after photodynamic therapy (PDT) in vitro and in vivo was investigated. The ability of CuPPaCC to consume glutathione was investigated. CuPPaCC toxicity (light and dark) in CT26 cells was analyzed by MTT and live/dead cell staining. The anticancer effect of CuPPaCC in vivo was investigated in CT26 Balb/c mice. When stimulated by the TME, CuPPaCC released Cu2+ and PPaCC, and the singlet oxygen yield increased from 34 to 56.5%. The dual ROS-generating mechanism via a Fenton-like reaction/photoreaction and dual glutathione depletion via Cu2+/CC multiplied the antitumor efficacy of CuPPaCC. The photo-chemocycloreaction continued to produce oxygen and maintained high ROS levels even after PDT, significantly alleviating hypoxia in the TME and downregulating the expression of HIF-1α. CuPPaCC thus showed excellent antitumor activity in vitro and in vivo. These results showed that the strategy could be effective in improving the antitumor efficacy of CuPPaCC and could be used as a synergistic regimen for cancer therapy.

Cu2+-pyropheophorbide-a-cystine conjugate-mediated multifunctional mesoporous silica nanoparticles for photo-chemodynamic therapy/GSH depletion combined with immunotherapy cancer.

Photodynamic therapy (PDT) and chemodynamic therapy (CDT) can activate immunogenicity, so PDT and CDT combined immunotherapy is a promising treatment strategy. However, insufficient hydrogen peroxide activity, hypoxia, and overexpressed glutathione in the tumor microenvironment (TME) significantly impaired the ability to activate immunogenicity. Thus, in this paper, self-reinforcing conjugates Cu2+-Pyropheophorbide-a-Cysteine (CuPPaCC), combined synergetic NIR and pH triggered PDT/CDT with glutathione depletion ability was constructed. CuPPaCC was encapsulated in mesoporous silica, and spherical HSCuPPaCC nanoparticles were prepared by Hyaluronic acid (HA) on the silica surface by Schiff base modification. HSCuPPaCC has tumor-specific targeting via HA mediated. In acidic solution, the Schiff base of HSCuPPaCC is destroyed and CuPPaCC is released (>70%), with excellent pH response release function. The results of the MTT analysis showed that the PDT/CDT synergistic anti-tumor effect was significant. HSCuPPaCC was activated in TME, catalyzing the decomposition of hydrogen peroxide to generate hydroxyl radicals and oxygen, alleviating TME hypoxia, replenishing oxygen to PDT, and significantly down regulating hypoxia factor HIF-1α expression. HSCuPPaCC has an excellent dual ROS mechanism and a dual depleting GSH mechanism resulting in a surge in intracellular ROS levels to efficiently kill cancer cells, enhance the ability to induce immunogenicity, and make tumors more sensitive to checkpoint PD-L1 blockade therapy. With the CT26 mouse model, not only the primary tumor was eradicated, but also the distal tumor at the end of treatment was completely suppressed by HSCuPPaCC combined with anti-PD-L1 immune checkpoint blockade therapy.

A carbon dot-doped Cu-MOF-based smart nanoplatform for enhanced immune checkpoint blockade therapy and synergistic multimodal cancer therapy.

Immune checkpoint blockade (ICB) is a kind of promising anti-tumor immunotherapy that can block the negative immune regulatory pathways using a particular antibody. Weak immunogenicity in most patients is a key obstacle to ICB therapy. Photodynamic therapy (PDT) is a non-invasive treatment that can enhance the immunogenicity of the host and realize systemic anti-tumor immunotherapy; yet tumor microenvironment hypoxia and glutathione overexpression severely restrict the PDT effect. To overcome the above issues, we design a combination therapy based on PDT and ICB. We prepared red carbon dot (RCD)-doped Cu-metal-organic framework nanoparticles (Cu-MOF@RCD) as smart nano-reactors because their tumor microenvironment and near-infrared light responsive property can decompose tumor endogenous H2O2 through Fenton-like reactions. Cu-MOF@RCD also shows clear near-infrared photothermal therapy (PTT) effect and has an ability to deplete glutathione (DG), which together enhances decomposition of cellular H2O2 and amplifies reactive oxygen species (ROS) levels in cells, thus leading to enhanced PDT and chemodynamic therapy (CDT) effect. Moreover, programmed cell death-ligand 1 antibody (anti-PD-L1) is used together to enable combination therapy, as Cu-MOF@RCD can significantly enhance host immunogenicity. In summary, the combination of Cu-MOF@RCD with anti-PD-L1 antibody exerts a synergistic PDT/PTT/CDT/DG/ICB therapy and can be used to eradicate the primary tumors and inhibit the growth of untreated distant tumors and tumor metastasis.

Inhibitor of nuclear factor kappa B kinase subunit epsilon regulates murine acetaminophen toxicity via RIPK1/JNK.

Drug-induced liver injury (DILI) still poses a major clinical challenge and is a leading cause of acute liver failure. Inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE) is essential for inflammation and metabolic disorders. However, it is unclear how IKBKE regulates cellular damage in acetaminophen (APAP)-induced acute liver injury. Here, we found that the deficiency of IKBKE markedly aggravated APAP-induced acute liver injury by targeting RIPK1. We showed that APAP-treated IKBKE-deficient mice exhibited severer liver injury, worse mitochondrial integrity, and enhanced glutathione depletion than wild-type mice. IKBKE deficiency may directly upregulate the expression of total RIPK1 and the cleaved RIPK1, resulting in sustained JNK activation and increased translocation of RIPK1/JNK to mitochondria. Moreover, deficiency of IKBKE enhanced the expression of pro-inflammatory factors and inflammatory cell infiltration in the liver, especially neutrophils and monocytes. Inhibition of RIPK1 activity by necrostatin-1 significantly reduced APAP-induced liver damage. Thus, we have revealed a negative regulatory function of IKBKE, which acts as an RIPK1/JNK regulator to mediate APAP-induced hepatotoxicity. Targeting IKBKE/RIPK1 may serve as a potential therapeutic strategy for acute or chronic liver injury.

Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming.

Background: Chitinase-3-like protein 1 (CHI3L1) is overexpressed in various types of tumors, especially in glioma, and contributes to tumor progression. However, the definite role of CHI3L1 and involved pathway in glioma progression are not completely understood. Methods: CHI3L1 expression in human gliomas and its association with patient survival was determined using enzyme-linked immunosorbent assay, western blot, immunohistochemistry, and public databases. Single-cell RNA-seq was used to characterize the landscape of tumor and myeloid cells. Human proteome microarray assay was applied to identify the binding partners of CHI3L1. Protein-protein interactions were analyzed by co-immunoprecipitation and cellular co-localization. The roles of CHI3L1 in glioma proliferation and invasion were investigated in tumor cell lines by gain- and loss- of function, as well as in vivo animal experiments. Results: CHI3L1 was up-regulated in all disease stages of glioma, which was closely related with tumor survival, growth, and invasion. CHI3L1 was primarily expressed in glioma cells, followed by neutrophils. Moreover, glioma cells with high expression of CHI3L1 were significantly enriched in NF-κB pathway. Pseudo-time trajectory analysis revealed a gradual transition from CHI3L1low to CHI3L1high glioma cells, along with the NF-κB pathway gradually reversed from inhibition to activation. Intriguingly, CHI3L1 binds to actinin alpha 4 (ACTN4) and NFKB1, and enhances the NF-κB signaling pathway by promoting the NF-κB subunit nuclear translocation in glioma cells. Further, CHI3L1 were released into the tumor microenvironment (TME) and interacted with CD44 expressed on tumor-associated macrophages to activate AKT pathway, thereby contributing to M2 macrophage polarization. In addition, CHI3L1 positively correlated to the expression of immune checkpoints, such as CD274 (PD-L1) and HAVCR2 (LAG3), which then remodeled the TME to an immunosuppressive phenotype. Conclusion: Our research revealed that CHI3L1 facilitated NF-κB pathway activation within glioma cells and reprogramed the TME, thereby serving as a promising therapeutic target for glioma.

Perirenal adipose afferent nerves sustain pathological high blood pressure in rats.

Hypertension is a pathological condition of persistent high blood pressure (BP) of which the underlying neural mechanisms remain obscure. Here, we show that the afferent nerves in perirenal adipose tissue (PRAT) contribute to maintain pathological high BP, without affecting physiological BP. Bilateral PRAT ablation or denervation leads to a long-term reduction of high BP in spontaneous hypertensive rats (SHR), but has no effect on normal BP in control rats. Further, gain- and loss-of-function and neuron transcriptomics studies show that augmented activities and remodeling of L1-L2 dorsal root ganglia neurons are responsible for hypertension in SHR. Moreover, we went on to show that calcitonin gene-related peptide (CGRP) is a key endogenous suppressor of hypertension that is sequestered by pro-hypertensive PRAT in SHRs. Taken together, we identify PRAT afferent nerves as a pro-hypertensive node that sustains high BP via suppressing CGRP, thereby providing a therapeutic target to tackle primary hypertension.

Functional coupling of TRPM2 and extrasynaptic NMDARs exacerbates excitotoxicity in ischemic brain injury.

Excitotoxicity induced by NMDA receptor (NMDAR) activation is a major cause of neuronal death in ischemic stroke. However, past efforts of directly targeting NMDARs have unfortunately failed in clinical trials. Here, we reveal an unexpected mechanism underlying NMDAR-mediated neurotoxicity, which leads to the identification of a novel target and development of an effective therapeutic peptide for ischemic stroke. We show that NMDAR-induced excitotoxicity is enhanced by physical and functional coupling of NMDAR to an ion channel TRPM2 upon ischemic insults. TRPM2-NMDAR association promotes the surface expression of extrasynaptic NMDARs, leading to enhanced NMDAR activity and increased neuronal death. We identified a specific NMDAR-interacting motif on TRPM2 and designed a membrane-permeable peptide to uncouple the TRPM2-NMDAR interaction. This disrupting peptide protects neurons against ischemic injury in vitro and protects mice against ischemic stroke in vivo. These findings provide an unconventional strategy to mitigate excitotoxic neuronal death without directly targeting NMDARs.

CD5L deficiency attenuate acetaminophen-induced liver damage in mice via regulation of JNK and ERK signaling pathway.

CD5 molecule like (CD5L), a member of the scavenger receptor cysteine-rich domain superfamily, plays a critical role in immune homeostasis and inflammatory disease. Acetaminophen (APAP) is a safe and effective antipyretic analgesic. However, overdose may cause liver damage or even liver failure. APAP hepatotoxicity is characterized by extensive necrotic cell death and a sterile inflammatory response, in which the role of CD5L remains to be investigated. In this study, we found that the expression of CD5L was increased in the livers of mice after APAP overdose. Furthermore, CD5L deficiency reduced the increase of alanine transaminase (ALT) level, histopathologic lesion area, c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK) phosphorylation level, Transferase-Mediated dUTP Nick End-Labeling positive (TUNEL+) cells proportion, vascular endothelial cell permeability and release of inflammatory cytokines induced by excess APAP. Therefore, our findings reveal that CD5L may be a potential therapeutic target for prevention and treatment of APAP-induced liver injury.

CD36 deficiency ameliorates drug-induced acute liver injury in mice.

BACKGROUND: Acetaminophen (APAP) overdose causes hepatotoxicity and even acute liver failure. Recent studies indicate that sterile inflammation and innate immune cells may play important roles in damage-induced hepatocytes regeneration and liver repair. The scavenger receptor CD36 has its crucial functions in sterile inflammation. However, the roles of CD36 in APAP induced acute liver injury remain unclear and warrant further investigation. METHODS: WT C57BL/6 J and CD36-/- mice were intraperitoneally injected with APAP (300 mg/kg) after fasting for 16 h. Liver injury was evaluated by serum alanine aminotransferase (ALT) level and liver tissue hematoxylin and eosin (H&E) staining. Liver inflammatory factor expression was determined by real-time polymerase chain reaction (PCR). The protein adducts forming from the metabolite of APAP and the metabolism enzyme cytochrome P450 2E1 (CYP2E1) levels were measured by Western blot. Liver infiltrating macrophages and neutrophils were characterized by flow cytometry. RNA sequencing and Western blot were used to evaluate the effect of damage-associated molecular patterns (DAMP) molecule high mobility group B1 (HMGB1) on WT and CD36-/- macrophages. Moreover, PP2, a Src kinase inhibitor, blocking CD36 signaling, was applied in APAP model. RESULTS: The expression of CD36 was increased in the liver of mice after APAP treatment. Compared with WT mice, APAP treated CD36-/- mice show less liver injury. There was no significant difference in APAP protein adducts and CYP2E1 expression between these two strains. However, reduced pro-inflammatory factor mRNA expression and serum IL-1ß level were observed in APAP treated CD36-/- mice as well as infiltrating macrophages and neutrophils. Moreover, CD36 deficiency impaired the activation of c-Jun N-terminal kinase (JNK) caused by APAP. Interestingly, the lack of CD36 reduced the activation of extracellular regulated protein kinases (Erk) and v-akt murine thymoma viral oncogene homolog (Akt) induced by HMGB1. RNA transcription sequencing data indicated that HMGB1 has a different effect on WT and CD36-/- macrophages. Furthermore, treatment with PP2 attenuated APAP induced mouse liver injury. CONCLUSION: Our data demonstrated that CD36 deficiency ameliorated APAP-induced acute liver injury and inflammatory responses by decreasing JNK activation. CD36 might serve as a new target to reduce acute liver injury.

POU2AF1 promotes MSCs adipogenesis by inhibiting HDAC1 expression.

Excessive production of visceral adipose is a major risk factor of many diseases. Inhibiting the adipogenesis of mesenchymal stem cells (MSCs) will be an efficient way to block adipose production. We illuminated POU class 2 homeobox associating factor 1 (POU2AF1) may promote MSCs adipogenesis by histone deacetylases 1 (HDAC1) signalling. Human retroperitoneal adipose-derived mesenchymal stem cells were isolated from overweight and control groups of patients. IncRNA microarray was used to identified gene expression levels. Adenovirus transduction and cellular small-interfering RNA transfection were used to achieve overexpression and interference of POU2AF1 or HDAC1. Adipogenesis was identified by Oil-red O staining, triglycende, cholesterol assay, real-time PCR and Western Blot. POU2AF1 expression was upregulated in retroperitoneal adipose tissue of overweight patients, and increased during adipogenesis. Overexpression of POU2AF1 promoted spontaneous adipogenesis without adipogenic treatment. Silencing of endogenous POU2AF1 in MSCs inhibited adipogenesis. Overexpression of POU2AF1 alleviated the translocation of HDAC1 to the nucleus. The mRNA level of HDAC1 was also reduced. Co-transfection of Ad-POU2AF1 and Ad-HDAC1 partially reversed the promotion effect of POU2AF1 overexpression in MSCs spontaneous adipogenic differentiation. POU2AF1 involves in the natural differentiation of human mesenchymal stem cells. Overexpression or silencing POU2AF1 could effectively induce or inhibit the adipogenesis by HDAC1 signaling.

Experimental Study on the Bone Morphogenetic Protein 1-Modified Bone Marrow Mesenchymal Stem Cell Sheets to Promote Mandibular Distraction Osteogenesis.

OBJECTIVE: The present study aims to increase the concentration of genetically modified bone marrow mesenchymal stem cells (BMSCs) in the distraction osteogenesis (DO) interstitial space and induce the conversion of BMSCs to osteoblasts to improve the osteogenic efficiency in DO and shorten the treatment period. METHODS: Bone morphogenetic protein 1 (BMP-1) and green fluorescent protein (GFP) gene-modified cell sheets of BMSCs were constructed by tissue engineering. Thirty-six New Zealand white rabbits were randomly divided into three groups: group A (the blank control group), group B (the GFP group) with the injection of GFP gene-modified BMSC sheets into the DO gap, and group C (the BMP-1 group) with the injection of BMP-1 gene-modified BMSC sheets into the DO gap. Rabbits in all three groups were distracted for 5 days at a distraction rate of 2.0 mm/d, once/day. After distraction, the above-mentioned cell sheet suspension was injected into the distraction gap to observe osteogenesis, which was observed by gross specimen observation, micro-computed tomography (Micro-CT) scanning, and histomorphology. RESULTS: The gross specimen observation showed that all animals had smooth and continuous bone cortex in the distraction region with relatively high hardness. The osteogenesis quality or hardness was ranked from the highest to the lowest, as Group C > Group B > Group A. Micro-CT and histomorphological observation revealed that group C had better maturation and bone volume of the new bone in the DO region at weeks 3 and 6 than groups B and A. CONCLUSION: BMP-1 gene-modified BMSC sheets could effectively promote the formation of new bone during rapid DO in the mandible, compensating for the poor osteogenesis caused by rapid distraction and providing a new approach to shorten the DO treatment period in clinical practice.

Chitinase-3 like-protein-1 function and its role in diseases.

Non-enzymatic chitinase-3 like-protein-1 (CHI3L1) belongs to glycoside hydrolase family 18. It binds to chitin, heparin, and hyaluronic acid, and is regulated by extracellular matrix changes, cytokines, growth factors, drugs, and stress. CHI3L1 is synthesized and secreted by a multitude of cells including macrophages, neutrophils, synoviocytes, chondrocytes, fibroblast-like cells, smooth muscle cells, and tumor cells. It plays a major role in tissue injury, inflammation, tissue repair, and remodeling responses. CHI3L1 has been strongly associated with diseases including asthma, arthritis, sepsis, diabetes, liver fibrosis, and coronary artery disease. Moreover, following its initial identification in the culture supernatant of the MG63 osteosarcoma cell line, CHI3L1 has been shown to be overexpressed in a wealth of both human cancers and animal tumor models. To date, interleukin-13 receptor subunit alpha-2, transmembrane protein 219, galectin-3, chemo-attractant receptor-homologous 2, and CD44 have been identified as CHI3L1 receptors. CHI3L1 signaling plays a critical role in cancer cell growth, proliferation, invasion, metastasis, angiogenesis, activation of tumor-associated macrophages, and Th2 polarization of CD4+ T cells. Interestingly, CHI3L1-based targeted therapy has been increasingly applied to the treatment of tumors including glioma and colon cancer as well as rheumatoid arthritis. This review summarizes the potential roles and mechanisms of CHI3L1 in oncogenesis and disease pathogenesis, then posits investigational strategies for targeted therapies.

Celastrol attenuates arterial and valvular calcification via inhibiting BMP2/Smad1/5 signalling.

Vascular calcification is an important risk factor for the mortality and morbidity in chronic kidney disease (CKD). Unfortunately, until now there is no certain medication targeting vascular calcification in CKD. In this study, we explored the inhibitory effect of celastrol on high calcium-induced vascular calcification and the underlying molecular mechanisms. Cell proliferation assay showed that celastrol inhibited aortic valve interstitial cell (VIC) and vascular smooth muscle cell (VSMC) proliferation when its concentration was higher than 0.6 µmol/L. 0.8 µmol/L celastrol inhibited the expression of osteogenic genes and calcium deposition induced by high-calcium medium in both AVICs and VSMCs. In mouse vascular calcification model induced by adenine combined with vitamin D, alizarin red and immunostaining showed that celastrol inhibited pro-calcification gene expression and calcium deposition in aortic wall and aortic valve tissues. At the molecular level, celastrol inhibited the increase of BMP2, phosphorylated Smad1/5 (p-Smad1/5) and non-phosphorylated ß-catenin (n-p-ß-catenin) induced by high-calcium medium both in vitro and in vivo. Also, BMP2 overexpression reversed the anti-calcification effects of celastrol by recovering the decrease of p-Smad1/5 and n-p-ß-catenin. Furthermore, celastrol prevented the up-regulation of BMPRII and down-regulation of Smad6 induced by high calcium, and this protectory effect can be abolished by BMP2 overexpression. In conclusion, our data for the first time demonstrate that celastrol attenuates high calcium-induced arterial and valvular calcification by inhibiting BMP2/Smad1/5 signalling, which may provide a novel therapeutic strategy for arterial and valvular calcification in patients with CKD.

Lysine-222 succinylation reduces lysosomal degradation of lactate dehydrogenase a and is increased in gastric cancer.

BACKGROUND: Lysine succinylation is an emerging posttranslational modification that has garnered increased attention recently, but its role in gastric cancer (GC) remains underexplored. METHODS: Proteomic quantification of lysine succinylation was performed in human GC tissues and adjacent normal tissues by mass spectrometry. The mRNA and protein levels of lactate dehydrogenase A (LDHA) in GC and adjacent normal tissues were analyzed by qRT-PCR and western blot, respectively. The expression of K222-succinylated LDHA was measured in GC tissue microarray by the K222 succinylation-specific antibody. The interaction between LDHA and sequestosome 1 (SQSTM1) was measured by co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The binding of carnitine palmitoyltransferase 1A (CPT1A) to LDHA was determined by co-IP. The effect of K222-succinylated LDHA on tumor growth and metastasis was evaluated by in vitro and in vivo experiments. RESULTS: Altogether, 503 lysine succinylation sites in 303 proteins were identified. Lactate dehydrogenase A (LDHA), the key enzyme in Warburg effect, was found highly succinylated at K222 in GC. Intriguingly, this modification did not affect LDHA ubiquitination, but reduced the binding of ubiquitinated LDHA to SQSTM1, thereby decreasing its lysosomal degradation. We demonstrated that CPT1A functions as a lysine succinyltransferase that interacts with and succinylates LDHA. Moreover, high K222-succinylation of LDHA was associated with poor prognosis in patients with GC. Finally, overexpression of a succinylation-mimic mutant of LDHA promoted cell proliferation, invasion, and migration. CONCLUSIONS: Our data revealed a novel lysosomal pathway of LDHA degradation, which is mediated by the binding of K63-ubiquitinated LDHA to SQSTM1. Strikingly, CPT1A succinylates LDHA on K222, which thereby reduces the binding and inhibits the degradation of LDHA, as well as promotes GC invasion and proliferation. This study thus uncovers a new role of lysine succinylation and the mechanism underlying LDHA upregulation in GC.

Overexpression of Bone Morphogenetic Protein-1 Promotes Osteogenesis of Bone Marrow Mesenchymal Stem Cells In Vitro.

BACKGROUND Osteogenesis of bone marrow mesenchymal stem cells (BMSCs) is an important research topic in the application of bone tissue engineering. Bone morphogenetic protein-1 (BMP-1) is important in bone formation and stability, but its effects on the osteogenesis of BMSCs are unclear. This study aimed to investigate the association of BMP-1 with the osteogenic capacity of BMSCs. MATERIAL AND METHODS Primary rabbit BMSCs were cultured and divided into a BMP-1-overexpressing group, a Green Fluorescent Protein-expressing (GFP) group, and a Control group. The transfection efficiency of BMP-1 was tested by Western blotting. Cell viabilities, alkaline phosphatase (ALP) activities, Ca2+ concentrations, and gross examinations of BMSC sheets were examined at different times. The osteogenic marker collagen I was assessed by immunohistochemical analysis. RESULTS The cell viability, ALP activity, and Ca2+ content of the BMP1-overexpressed group were significantly enhanced compared with the GFP group and Control group. Immunohistochemistry staining results showed that BMP-1 promoted the expression of type I collagen in BMSCs sheets. CONCLUSIONS Our results suggest that the overexpression of BMP-1 can promote the osteogenesis of BMSCs and provides an improved method of cell-based tissue engineering.

Cardioprotection of hydralazine against myocardial ischemia/reperfusion injury in rats.

This study aimed to investigate whether hydralazine could reduce cardiac ischemia/reperfusion (I/R) injury in rats. Anesthetized male Sprague-Dawley rats underwent myocardial I/R injury. Saline, hydralazine (HYD, 10-30 mg/kg) was administered intraperitoneally 10 min before reperfusion. After 30 min of ischemia and 24 h of reperfusion, the myocardial infarct size was determined using TTC staining. Heart function and oxidative stress were determined through biochemical assay and DHE staining. HE staining was used for histopathological evaluation. Additionally, the cardiomyocytes apoptosis and protein expression of PI3K-Akt-eNOS pathway marker were detected by TUNEL and Western blotting. The serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) and reactive oxygen species were significantly elevated in cardiac I/R group, but the superoxide dismutase (SOD) level was suppressed. However, intraperitoneal pretreatment with hydralazine at a dose of 10-30 mg/kg before cardiac I/R significantly limited the increase in CK-MB, LDH, oxidative stress, inflammatory factors, histological damage and apoptosis in the hearts. In addition, hydralazine also increased p-PI3K, p-AKT, p-eNOS expression and decreased Cleaved Caspase-3, Cleaved Caspase-9 expression in the hearts. Our results suggest that the cardioprotective effect of hydralazine against I/R injury might be a cooperation of the inhibition of oxidative stress, inflammatory response, apoptosis with the motivation of eNOS phosphorylation via activating the PI3K/AKT signal pathway.

CD47 Deficiency Attenuates Isoproterenol-Induced Cardiac Remodeling in Mice.

In this study, we investigated whether CD47 deficiency attenuates isoproterenol- (ISO-) induced cardiac remodeling in mice. Cardiac remodeling was induced by intraperitoneal (i.p.) injection of ISO (60 mg·kg-1·d-1 in 100 µl of sterile normal saline) daily for 14 days and was confirmed by increased levels of lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB), increased heart weight to body weight (HW/BW) ratios, and visible cardiac fibrosis. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were found to be significantly higher in the ISO group than in the control group, while superoxide dismutase (SOD) levels were suppressed in the ISO group. However, CD47 knockout significantly limited ISO-induced increases in LDH, CK-MB, and HW/BW ratios, cardiac fibrosis, oxidative stress, and apoptosis in the heart. In addition, CD47 deficiency also increased p-AMPK and LAMP2 expression and decreased HDAC3, cleaved Caspase-3, cleaved Caspase-9, LC3II, and p62 expression in cardiac tissues. In conclusion, CD47 deficiency reduced i.p. ISO-induced cardiac remodeling probably by inhibiting the HDAC3 pathway, improving AMPK signaling and autophagy flux, and rescuing autophagic clearance.

Study on Connective Tissue Growth Factor Expressed in Patients with ST-Segment Elevation Myocardial Infarction.

BACKGROUND: This study measured the change in connective tissue growth factor levels after the onset of unstable angina and ST-segment elevation myocardial infarction, and studied its correlation with peak creatine kinase-MB (CK-MB) enzyme. It also discussed the significance of myocardial fibrosis after myocardial infarction. To detect the serum levels of connective tissue growth factor in patients with ST-segment elevation myocardial infarction and its relationship with the maximum level of CK-MB. METHODS: We selected 50 patients with ST-segment elevation myocardial infarction and 50 patients with unstable angina. Connective tissue growth factor levels were examined 24 h, 2 d, 7 d, and 14 d after the onset of ST-segment elevation myocardial infarction, and within 24 h for unstable angina, using enzyme-linked immunosorbent assay (ELISA). The maximum level of CK-MB was detected by immunosuppression. RESULTS: The serum level of connective tissue growth factor in the unstable angina patients was 10.34 ± 2.00 ng/mL, and the levels in the ST-segment elevation myocardial infarction patients were 16.76 ± 3.17 ng/mL at 24 h, 29.87 ± 4.90 ng/mL at 2 d, 45.02 ± 8.35 ng/mL at 7 d, and 31.61 ± 4.40 ng/mL at 14 d. Compared with the unstable angina patients, the connective tissue growth factor levels in the ST-segment elevation myocardial infarction patients were significantly higher since day 1 (p < 0.01). The maximum level of CK-MB was correlated with connective tissue growth factor levels at 7 d (r = 0.859, p = 0.000). CONCLUSIONS: Connective tissue growth factor was significantly expressed in the ST-segment elevation myocardial infarction patients, indicating that it might play an important role in myocardial fibrosis.

c-Myc Overexpression Promotes Oral Cancer Cell Proliferation and Migration by Enhancing Glutaminase and Glutamine Synthetase Activity.

BACKGROUND: This study aimed to investigate whether glutaminase (GLS) and glutamine synthetase (GS) are involved in c-Myc-mediated tumor development in oral cancer. METHODS: The correlation between the expressions of c-Myc, GLS, and GS in clinical samples and the clinicopathologic features of oral cancer were examined using immunohistochemistry and quantitative real-time polymerase chain reaction. After overexpressing the c-Myc gene and using an inhibitor of GLS or GS, functional experiments were performed to confirm the effects of c-Myc, GLS and GS on proliferation, cell cycle and migration in KB oral cancer cells. The expressions of E-cadherin and N-cadherin were determined by immunofluorescence assays in KB cells overexpressing c-Myc in the presence of GLS or GS inhibitors. RESULTS: The protein expression of GS was correlated with the Tumor, Lymph Node, and Metastasis (TNM) stage. In addition, c-Myc mRNA levels were positively correlated with GS mRNA levels. Overexpression of c-Myc increased the colonies derived from oral cancer cells and caused more cells to be in S phase compared with the mock-vehicle group. The migratory speed of KB cells was promoted by overexpression of c-Myc compared to the mock-vehicle group. However, these effects were effectively reversed in the presence of GLS or GS inhibitor. Furthermore, c-Myc could inhibit E-cadherin protein expression while promoting N-cadherin expression by enhancing the activity of GLS and GS. CONCLUSIONS: c-Myc overexpression promotes oral cancer cell proliferation and migration by enhancing GLS and GS activity. Our findings are beneficial for the identification of novel molecular targets for the prevention and treatment of oral cancer.