Activation of Stimulation of Interferon Genes (STING) Signal and Cancer Immunotherapy.

Stimulator of interferon gene (STING), an intracellular receptor in the endoplasmic reticulum, could induce the production of cytokines such as type I interferon (IFN) by activating the cGAS-STING signal pathway. In recent years, activation of STING has shown great potential to enhance anti-tumor immunity and reshape the tumor microenvironment, which is expected to be used in tumor immunotherapy. A number of STING agonists have demonstrated promising biological activity and showed excellent synergistic anti-tumor effects in combination with other cancer therapies in preclinical studies and some clinical trials. The combination of STING agonists and ICI also showed a potent effect in improving anti-tumor immunity. In this review, we introduce the cGAS-STING signaling pathway and its effect in tumor immunity and discuss the recent strategies of activation of the STING signaling pathway and its research progress in tumor immunotherapy.

Piperlongumine inhibits the progression of osteosarcoma by downregulating the SOCS3/JAK2/STAT3 pathway via miR-30d-5p.

AIMS: The present study evaluated the functions of Piperlongumine (PL) in osteosarcoma (OS) cell growth and metastasis both in vitro and in vivo. MAIN METHODS: MTT assay was conducted to test the cytotoxic effects of PL on the human osteoblasts line HFOB1.19 and the human normal chondrocyte line C28/I2T. FITC-Annexin V and propidium iodide (PI) were used to examine cell apoptosis. The migration, invasion and relative epithelial-mesenchymal transition were examined by Transwell assay and Western blotting. Reverse transcription-quantitative PCR (RT-qPCR) was performed to analyze the cytokine signaling 3 (SOCS3) mRNA expression. TargetScan database was used to predict the target of SOCS3. The binding association between miR-30d-5p and SOCS3 in U2OS and MG63 cells was evaluated by the dual-luciferase reporter assay. A xenograft model was constructed to evaluate the effect of PL on OS cell growth in vivo. KEY FINDINGS: The results revealed that PL inhibited the growth, migration, invasion, epithelial-mesenchymal transition, and promoted the apoptosis of OS cells dose-dependently. In addition, PL upregulated the protein levels of suppressor of SOCS3, while it inactivated the JAK2/STAT3 pathway, which was accompanied by a decreased level of microRNA (miR)-30d-5p. Furthermore, SOCS3was confirmed as a novel target of miR-30d-5p. Overexpression of miR-30d-5p not only led to decreased expression of SOCS3, but also dampened the antitumor effect of PL on OS. SIGNIFICANCE: The present data demonstrated that PL inhibited the progression of OS via downregulation of the SOCS3-mediated JAK2/STAT3 pathway by inhibiting miR-30d-5p.

Tea polyphenol EGCG inhibited colorectal-cancer-cell proliferation and migration via downregulation of STAT3.

BACKGROUND: Green tea is a popular beverage worldwide and epigallocatechin-3-gallate (EGCG) is the most bioactive polyphenol in green tea. Our study aims to investigate the anti-proliferation and anti-migration effects of EGCG against colorectal-cancer SW480, SW620, and LS411N cells, and elucidate the underlying mechanism. METHODS: The in vitro anti-proliferation and anti-migration effects of EGCG against colon-cancer cells were evaluated using MTT, scratch-wound-healing, and transwell-migration assays. The effects of EGCG on apoptosis were assessed by Annexin V-FITC/PI double staining and JC-1 staining. Besides, Western blotting was employed to detect the protein-expression level and elucidate the underlying pathways. Real-time qPCR and dual-luciferase reporter assay were adopted to determine the mRNA level and promoter activity. RESULTS: Our results demonstrated that treatment with EGCG resulted in significant inhibition of cell proliferation by the induction of apoptosis. EGCG also inhibited SW480 cell migration in a dose-dependent manner as assessed by wound-healing and transwell-migration assays. Western blot confirmed that EGCG induced apoptosis by the activation of Caspase-3 and PARP. In addition, both STAT3 and phosphorylated STAT3 (p-STAT3) were downregulated significantly by EGCG in three selected colorectal-cancer cell lines. EGCG treatment also resulted in a significant decrease in Bcl-2, MCL-1, and Vimentin, and an increase in E-cadherin. When STAT3 was inhibited, EGCG showed no obvious effect on cell proliferation and migration. Further investigation by luciferase-reporter-activity assay showed that EGCG suppressed the promoter activity of STAT3 and downregulated the transcription of STAT3. CONCLUSION: Our study presents evidence on the anti-proliferation and anti-migration effects of EGCG against colorectal-cancer SW480, SW620, and LS411N cells by downregulating the expression of STAT3 and suggests that EGCG could be an effective and natural supplement for colon-cancer treatment.

Iron Overload-Induced Osteocyte Apoptosis Stimulates Osteoclast Differentiation Through Increasing Osteocytic RANKL Production In Vitro.

Iron overload is closely associated with osteoporosis, the potential cellular mechanism involved in decreased osteoblast differentiation and increased osteoclast formation. However, the effect of iron overload on the biological behavior in osteocytes has not been reported. This study aims to investigate the changes of osteocytic activity, apoptosis, and its regulation on osteoclastogenesis in response to iron overload. MLO-Y4 osteocyte-like cells and primary osteocytes from mice were processed with ferric ammonium citrate (FAC) and deferoxamine (DFO), the conditioned medium (CM) was harvested and co-cultured with Raw264.7 cells and bone marrow-derived macrophages (BMDMs) to induce them to differentiate into osteoclasts. Osteocyte apoptosis, osteoclast differentiation, osteocytic gene expression and protein secretion of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) was examined. Excessive iron has a toxic effect on MLO-Y4 osteocyte-like cells. Increased cell apoptosis in MLO-Y4 cells and primary osteocytes was induced by iron overload. The osteoclastic formation, differentiation-related gene expression, and osteoclastic bone-resorption capability were significantly increased after treated with the CM from iron overload-exposed osteocytes. Excessive iron exposure significantly promoted the gene expression and protein secretion of the RANKL in MLO-Y4 cells. Addition of RANKL-blocking antibody completely abolished the increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron. Moreover, the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone) was used to inhibit osteocyte apoptosis. The results showed osteocyte apoptosis induced by iron overload was reduced by QVD and accompanied by the decrease of soluble RANKL (sRANKL) in supernatant. The increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron was significantly decreased by QVD. These results indicated that iron overload-induced osteocyte apoptosis is required to regulate osteoclast differentiation by increasing osteocytic RANKL production. This study, for the first time, reveals the indirect effect of iron overload on osteoclast differentiation through regulating osteocytes.

Epothilone B Facilitates Peripheral Nerve Regeneration by Promoting Autophagy and Migration in Schwann Cells.

The search for drugs that can facilitate axonal regeneration and elongation following peripheral nerve injury has been an area of increasing interest in recent years. Epothilone B (EpoB) is an FDA-approved antineoplastic agent, which shows the capacity to induce α-tubulin polymerization and to improve the stability of microtubules. Recently, it has been increasingly recognized that EpoB has a regenerative effect in the central nervous system. However, the information currently available regarding the potential therapeutic effect of EpoB on peripheral nerve regeneration is limited. Here, we used a rat sciatic crush injury model system to determine that EpoB strikingly improved axonal regeneration and recovery of function. Also, EpoB (1 nM) did not result in significant apoptosis in Schwann cells (SCs) and showed little effect on their viability either. Interestingly, EpoB (1 nM) significantly enhanced migration in SCs, which was inhibited by autophagy inhibitors 3-methyladenine (3-MA). Since PI3K/Akt signaling has been implicated in regulating autophagy, we further examined the involvement of PI3K/Akt in the process of EpoB-induced SC migration. We found that EpoB (1 nM) significantly inhibited phosphorylation of PI3K and Akt in SCs. Further studies showed that both EpoB-enhanced migration and autophagy were increased/inhibited by inhibition/activation of PI3K/Akt signaling with LY294002 or IGF-1. In conclusion, EpoB can promote axonal regeneration following peripheral nerve injury by enhancing the migration of SCs, with this activity being controlled by PI3K/Akt signaling-mediated autophagy in SCs. This underscores the potential therapeutic value of EpoB in enhancing regeneration and functional recovery in cases of peripheral nerve injury.

Methoxytetrahydro-2H-pyran-2-yl)methyl benzoate inhibits spinal cord injury in the rat model via PPAR-γ/PI3K/p-Akt activation.

Spinal cord injury (SCI) is the most commonly seen trauma leading to disability in people worldwide. The purpose of current study was to determine the protective effect of methoxytetrahydro-2H-pyran-2-yl)methyl benzoate (HMPB) on SCI in rat model. TUNEL staining was used to examine apoptotic changes in spinal cord of SCI rats. The ELISA kits were employed to assess inflammatory processes and oxidative factors in the spinal cord tissues. Behavioral changes in SCI rats were assessed using Basso, Beattie, and Bresnahan (BBB) scoring system. Western blotting was used for assessment of proteins. The HMPB treatment of SCI rats reduced apoptotic cell number based on the concentration of dose administered. Treatment of SCI rats with HMPB enhanced BBB score and decreased accumulation of water content in SCI rats significantly. On treatment with HMPB the TNF-α and interleukin-6/1ß/18 levels were suppressed in SCI rats. Treatment with HMPB induced excessive release of SOD, CAT, and GSH molecules and decreased overproduction of MDA. The SCI induced upregulation of caspase-3/9 activity was completely alleviated by HMPB at 2 mg/kg dose. The HMPB treatment of SCI rats promoted peroxisome proliferator-activated receptor γ (PPAR-γ) expression, reduced cyclooxygenase (COX)-2 production and increased expression of p-Akt and phosphoinositide 3-kinase (p-PI3K). The study demonstrated that HMPB suppressed apoptosis, raised BBB score and inhibited inflammation in SCI rats. Moreover, activation of PI3K/Akt in the spinal cord tissues of SCI rats was promoted by HMPB. Therefore, HMPB has protective effect on SCI in the rat model.

Identification and Validation of a Potent Multi-miRNA Signature for Prediction of Prognosis of Osteosarcoma Patients.

BACKGROUND Osteosarcoma, the most common solid malignancy, has high incidence and mortality rates. We constructed a miRNA-based signature that can be used to assess the prognosis of osteosarcoma patients. MATERIAL AND METHODS The miRNA profile was derived from the Gene Expression Omnibus (GEO) website, with matched clinical records. The miRNA-based overall survival (OS)-predicting signature was established by LASSO Cox regression analysis. Receiver operating characteristic (ROC) curve and Kaplan-Meier (K-M) analyses were performed to examine the stability and discriminatory ability of the OS-predicting signatures. Pathway enrichment analyses were performed to uncover potential mechanisms. RESULTS Three miRNAs (miR-153, miR-212, and miR-591) independently related to the OS were extracted to build a risk score formula. The ROC curve and K-M analyses revealed good discrimination ability of the OS signature for osteosarcoma patients in both the training cohort (P=0.00015, AUC=0.962) and the validation cohort (P=0.0065, AUC=0.793). As shown in multivariate analysis, the classifier showed favorable predictive accuracy similar to the recurrence status to be an independent risk factor for osteosarcoma. Furthermore, the nomogram showed a synergistic effect by combining the clinicopathological features with our classifier. Also, the enrichment analyses of the target genes may contribute to improved treatment of osteosarcoma. CONCLUSIONS The 3-miRNA-based classifier serves as an effective prognosis-predicting signature for osteosarcoma patients.

EGCG Enhanced the Anti-tumor Effect of Doxorubicine in Bladder Cancer via NF-κB/MDM2/p53 Pathway.

Doxorubicin (DOX), the first-line chemotherapy for bladder cancer, usually induces side effects. We previously demonstrated that green tea polyphenol EGCG had potent anti-tumor effect in bladder cancer via down regulation of NF-κB. This study aimed to investigate the additive/synergistic effect EGCG and DOX against bladder cancer. Our results demonstrated that the combined use of DOX and EGCG inhibited T24 and SW780 cell proliferation. EGCG enhanced the apoptosis induction effect of DOX in both SW780 and T24 cells and resulted in significant differences. Besides, EGCG promoted the inhibitory effect of DOX against bladder cancer cell migration. In addition, the in vivo results demonstrated that DOX in combination with EGCG showed the most potent anti-tumor effects among DOX, EGCG and DOX+EGCG treatment groups. Further mechanistic studies determined that the combination of DOX and EGCG inhibited phosphorylated NF-κB and MDM2 expression, and up-regulated p53 expression in tumor, as assessed by western blot and immunohistochemistry. Western blot in SW780 cells also confirmed that the combined use of EGCG and DOX caused significant increase in p53, p21, and cleaved-PARP expression, and induced significant inhibition in phosphorylated NF-κB and MDM2. When NF-κB was inhibited, the expression of p53 and p-MDM2 were changed, and the combination of DOX and EGCG showed no obvious effect in transwell migration and cell viability. In conclusion, the novel application of chemotherapy DOX and EGCG demonstrated potent anti-tumor, anti-migration and anti-proliferation effects against bladder cancer. EGCG enhanced the anti-tumor effect of DOX in bladder cancer via NF-κB/MDM2/p53 pathway, suggesting the potential clinical application against bladder cancer patients.

EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway.

Epigallocatechin-3-gallate (EGCG), the bioactive polyphenol in green tea, has been demonstrated to have various biological activities. We previously found that EGCG inhibited SW780 tumor growth by down-regulation of NF-κB and MMP-9. This study demonstrated that EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway, without modulation of NF-κB. Our results showed that treatment of EGCG resulted in significant inhibition of cell proliferation by induction of apoptosis, without obvious toxicity to normal bladder SV-HUC-1 cells. EGCG also inhibited 5637 and T24 cell migration and invasion at 25-100 µM. Western blot confirmed that EGCG induced apoptosis in T24 and 5637cells by activation of caspases-3 and PARP. Besides, EGCG up-regulated PTEN and decreased the expression of phosphorylated PI3K, AKT in both T24 and 5637 cells. In addition, animal study demonstrated that EGCG (100 mg/kg, i.p. injected daily for 4 weeks) decreased the tumor weight in mice bearing T24 tumors by 51.2%, as compared with the untreated control. EGCG also decreased the expression of phosphorylated PI3K and AKT in tumor, indicating the important role of PI3K/AKT in EGCG inhibited tumor growth. When AKT was inhibited, EGCG showed no obvious effect in cell migration in T24 and 5637 cells. In conclusion, our study elucidated that EGCG was effective in inhibition of T24 and 5637 cell proliferation and migration, and presented evidence that EGCG inhibited cell proliferation and tumor growth by modulation of PI3K/AKT pathway.

Delayed release particles from vascular endothelial growth factor for repairing spinal cord ischemic injury of rats.

OBJECTIVE: To study the effect of delayed release particles from vascular endothelial growth factor (VEGF) on the reparation of ischemic injury of spinal cord in rats. METHODS: The spinal cord ischemia model of rats was established. The delayed release particles from VEGF were injected via the intubation of spinal subarachnoid space. The rehabilitation was observed by the assessment of unfold claw reflection, space between toes, spinal evoked potential (SEP) and motor evoked potential (MEP). RESULTS: VEGF prompted SEP and MEP appearance, improved the motor function of hind limbs. CONCLUSIONS: VEGF can promote the rehabilitation of spinal cord ischemic injury of rats.

Effects of salmon calcitonin on fracture healing in ovariectomized rats.

OBJECTIVE: To explore the effects of salmon calcitonin on the healing process of osteoporotic fractures in ovariectomized rats. METHODS: We performed this study in The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, China, during the period March 2002 to December 2004. We used 120 female adult Wistar rats in this experiment, among which 90 underwent ovariectomy (OVX) and the other 30 had sham-operation. All rats had their left tibias fractured 3 months later. The 90 OVX rats were randomly divided into 3 groups with 30 in each, while the 30 sham-operated rats served as control group. After the fracture the rats had subcutaneous injection of normal saline, salmon calcitonin and estrogen, respectively. X-ray film, histological examination, bone mineral density (BMD) measurement and biomechanics testing were carried out to evaluate the fracture healing. RESULTS: Compared with OVX rats treated with normal saline, the rats with salmon calcitonin had significantly higher BMD values in the left tibia, higher max torque, shear stress of the left tibia 8 weeks after fracture (p<0.05), and presented with stronger callus formation, shorter fracture healing time and faster normalization of microstructure of bone trabeculae. CONCLUSION: Salmon calcitonin can, not only increase BMD in osteoporotic bone, but also enhance the bone biomechanical properties and improve the process of fracture healing in fractured osteoporotic bone.

[Experimental study on polylactic glycolate acid microparticles with releasing-slowly vascular endothelial growth factor].

OBJECTIVE: To observe the degradation of the polylactic glycolate acid (PLGA) microparticles with releasing-slowly vascular endothelial growth factor (VEGF) synthesized by the method of emulsification-diffusion. METHODS: The method of emulsification-diffusion is to incorporate VEGF into microparticles composed of biodegradable PLGA. The controlled release of microparticles are acquired. The content of the VEGF released slowly from PLGA microparticles in vitro was detected with ELISA at different time. RESULTS: We synthesized 100 releasing-slowly VEGF PLGA microparticles with the size of 0.20-0.33 microm by 5 times. The contents were 62 +/- 11 ng/L, 89 +/- 14 ng/L, and 127 +/- 19 ng/L in the 1st, the 2nd and the 3rd months after degradation, respectively. CONCLUSION: The PLGA microparticles with releasing-slowly VEGF can be synthesized by the method of emulsification-diffusion.