The endonuclease FEN1 mediates activation of STAT3 and facilitates proliferation and metastasis in breast cancer.

BACKGROUND: The metastasis accounts for most deaths from breast cancer (BRCA). Understanding the molecular mechanisms of BRCA metastasis is urgently demanded. Flap Endonuclease 1 (FEN1), a pivotal factor in DNA metabolic pathways, contributes to tumor growth and drug resistance, however, little is known about the role of FEN1 in BRCA metastasis. METHODS AND RESULTS: In this study, FEN1 expression and its clinical correlation in BRCA were investigated using bioinformatics, showing being upregulated in BRCA samples and significant relationships with tumor stage, node metastasis, and prognosis. Immunohistochemistry (IHC) staining of local BRCA cohort indicated that the ratio of high FEN1 expression in metastatic BRCA tissues rose over that in non-metastatic tissues. The assays of loss-of-function and gain-of-function showed that FEN1 enhanced BRCA cell proliferation, migration, invasion, xenograft growth as well as lung metastasis. It was further found that FEN1 promoted the aggressive behaviors of BRCA cells via Signal Transducer and Activator of Transcription 3 (STAT3) activation. Specifically, the STAT3 inhibitor Stattic thwarted the FEN1-induced enhancement of migration and invasion, while the activator IL-6 rescued the decreased migration and invasion caused by FEN1 knockdown. Additionally, overexpression of FEN1 rescued the inhibitory effect of nuclear factor-κB (NF-κB) inhibitor BAY117082 on phosphorylated STAT3. Simultaneously, the knockdown of FEN1 attenuated the phosphorylation of STAT3 promoted by the NF-κB activator tumor necrosis factor α (TNF-α). CONCLUSIONS: These results indicate a novel mechanism that NF-κB-driven FEN1 contributes to promoting BRCA growth and metastasis by STAT3 activation.

Identification of macrophage-related genes correlated with prognosis and immunotherapy efficacy in non-small cell lung cancer.

Background: Malignant tumours, particularly non-small cell lung cancer (NSCLC), pose a significant threat to human health due to their prevalence and lethality. Treatment methods for NSCLC vary greatly among individuals, making it crucial to identify predictive markers. Moreover, during tumour initiation and progression, tumour cells can release signaling molecules to induce polarization of macrophages towards a more tumour friendly M2 phenotype, which can promote tumour growth, metastasis, and drug resistance. Methods: We employed a comprehensive approach, combining bulk RNA-seq and single-cell sequencing analysis. Results: In our study, we used bulk RNA-seq and single-cell sequencing methods to analyze differential cells in NSCLC and adjacent tissues, searching for relevant marker genes that can predict prognosis and drug efficacy. We scrutinized biological phenomena such as macrophage-related gene methylation, copy number variation, and alternative splicing. Additionally, we utilized a co-culture technique of immune and tumour cells to explore the role of these genes in macrophage polarization. Our findings revealed distinct differences in macrophages between cancerous and adjacent tissues. We identified ANP32A, CCL20, ERAP2, MYD88, TMEM126B, TUBB6, and ZNF655 as macrophage-related genes that correlate with NSCLC patient prognosis and immunotherapy efficacy. Notably, ERAP2, TUBB6, CCL20, and TMEM126B can induce macrophage M0 to M2 polarization, promoting tumour proliferation. Conclusion: These findings significantly contribute to our understanding of the NSCLC tumour immune microenvironment. They pave the way for further research into the potential of these genes as targets for regulating tumour occurrence and development.

Sanguinarine induces apoptosis in osteosarcoma by attenuating the binding of STAT3 to the single-stranded DNA-binding protein 1 (SSBP1) promoter region.

BACKGROUND AND PURPOSE: Osteosarcoma, a primary malignant bone tumour prevalent among adolescents and young adults, remains a considerable challenge despite protracted progress made in enhancing patient survival rates over the last 40 years. Consequently, the development of novel therapeutic approaches for osteosarcoma is imperative. Sanguinarine (SNG), a compound with demonstrated potent anticancer properties against various malignancies, presents a promising avenue for exploration. Nevertheless, the intricate molecular mechanisms underpinning SNG's actions in osteosarcoma remain elusive, necessitating further elucidation. EXPERIMENTAL APPROACH: Single-stranded DNA-binding protein 1 (SSBP1) was screened out by differential proteomic analysis. Apoptosis, cell cycle, reactive oxygen species (ROS) and mitochondrial changes were assessed via flow cytometry. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) were used to determine protein and gene levels. The antitumour mechanism of SNG was explored at a molecular level using chromatin immunoprecipitation (ChIP) and dual luciferase reporter plasmids. KEY RESULTS: Our investigation revealed that SNG exerted an up-regulated effect on SSBP1, disrupting mitochondrial function and inducing apoptosis. In-depth analysis uncovered a mechanism whereby SNG hindered the JAK/signal transducer and activator of transcription 3 (STAT3) signalling pathway, relieved the inhibitory effect of STAT3 on SSBP1 transcription, and inhibited the downstream PI3K/Akt/mTOR signalling axis, ultimately activating apoptosis. CONCLUSIONS AND IMPLICATIONS: The study delved further into elucidating the anticancer mechanism of SNG in osteosarcoma. Notably, we unravelled the previously undisclosed apoptotic potential of SSBP1 in osteosarcoma cells. This finding holds substantial promise in advancing the development of novel anticancer drugs and identification of therapeutic targets.

Cinnamaldehyde Suppressed EGF-Induced EMT Process and Inhibits Ovarian Cancer Progression Through PI3K/AKT Pathway.

Ovarian cancer is one of the most common gynecological malignancies in women worldwide with a poor survival rate. Cinnamaldehyde (CA), a bioactive substance isolated from cinnamon bark, is a natural drug and has shown that it can inhibit the progression of other tumors. However, the role of CA in ovarian cancer and its mechanism is poorly understood. In this study, wound healing assays, plate cloning, CCK-8, and transwell assays were used to determine cell proliferation and invasion. Western blot and flow cytometry were used to detect apoptosis levels. Western blot and immunofluorescence were used to detect changes in cellular EMT levels. The Western blot was used to detect levels of the PI3K/AKT signaling pathway. In vivo, we established a subcutaneous transplantation tumor model in nude mice to verify the role of CA in the progression and metastasis of ovarian cancer. Our data showed that in vitro CA was able to inhibit the cell viability of ovarian cancer. The results of scratch assay and transwell assay also showed that CA inhibited the proliferation and invasion ability of A2780 and SKOV3 cells. In addition, CA promoted apoptosis by increasing the expression of cleaved-PARP and cleaved-caspase 3 in ovarian cancer cells. Mechanistically, we found that CA inhibited the EGF-induced PI3K/AKT signaling pathway and reduced the phosphorylation levels of mTOR, PI3K, and AKT. The EGF-induced EMT process was also abolished by CA. The EMT process induced by AKT-specific activator SC79 was also suppressed by CA. Furthermore, in in vivo, CA significantly repressed the progression of ovarian cancer as well as liver metastasis. In all, our results suggest that CA inhibits ovarian cancer progression and metastasis in vivo and in vitro and inhibits EGF-induced EMT processes through the PI3K/AKT signaling pathway.

Association between changes in thioredoxin reductase and other peripheral blood biomarkers with response to PD-1 inhibitor-based combination immunotherapy in non-small cell lung cancer: a retrospective study.

Background: Immunotherapy deeply changed the treatment paradigm of advanced non-small cell lung cancer (NSCLC) in the past years. However, the objective response rate (ORR) after immunotherapy is about 20-30% of NSCLC patients. Therefore, identification of predictive biomarkers is crucial for selecting patients with NSCLC who would most benefit from programmed cell death receptor protein 1 (PD-1) inhibitor-based immunotherapy. Methods: We retrospectively collected medical records and thioredoxin reductase (TrxR) data from 90 patients with a NSCLC who received PD-1 inhibitor-based combination therapy. Serum biomarkers were also measured at 6- and 12-week post-treatment and compared with their baseline values. Associations between changes in serum biomarkers, clinical characteristics and treatment efficacy were evaluated using univariate tests. The patients who were still alive were followed up remotely by phone or email to assess survival. The association between serum biomarkers and TrxR with overall survival (OS) and progression-free survival (PFS) were assessed by univariate and multivariate Cox proportional hazard regression. Nomogram prediction models were constructed using factors associated with PFS and OS, respectively. Results: The median follow-up time among the 90 patients was 19.7 (range, 13.6 to 25.8) months. Median PFS and OS were 13.6 [95% confidence interval (CI): 13.5 to 13.7] and 19.7 (95% CI: 13.6 to 25.8) months, respectively. Patients with decreased carcinoembryonic antigen (CEA), albumin (Alb), and TrxR values at 6- and 12-week post-treatment compared to baseline had statistically significantly improved disease remission rates (P<0.05). Patients with decreased white blood cell (WBC), neutrophil-to-lymphocyte ratio (NLR), derived NLR (dNLR) at week 6, and decreased Alb, CEA, and lymphocyte-to-monocyte ratio (LMR) at week 12 had statistically significantly increased ORRs (P<0.05). According to the univariate and multivariate Cox regression analyses, we included adenocarcinoma, Eastern Cooperative Oncology Group performance status (ECOG PS), and CEA change at week 6 post-treatment as predictors for PFS, and adenocarcinoma, change in absolute lymphocyte count (ALC), and TrxR at week 6 as predictors for OS in the nomogram models. Each nomogram was also validated internally using a bootstrap method with 1,000 resamples. Conclusions: Change in TrxR at 6 weeks post-treatment in combination with other clinical and hematological biomarkers could be used as a predictor for treatment outcome and prognosis in NSCLC patients after PD-1 inhibitor-based combination immunotherapy.

PKG II secreted via the classical endoplasmic reticulum-Golgi apparatus secretory pathway blocks the activation of EGFR through phosporalting its threonine 406 and has an anti-tumor effect.

BACKGROUND: Protein kinase G type II (PKG II) is a serine/threonine-protein kinase that was originally isolated from the small intestinal mucosa with primary functions in the secretion of small intestinal mucosal cells, secretion of renin and aldosterone, and chondrocyte activities. Recent studies have shown that PKG II exerts anti-tumor effects, while a previous study by our group confirmed that PKG II inhibited the proliferation and migration of cancer cells. Interestingly, PKG II, which was typically bound to the intracellular side of the membrane, was detected in the serum and cell culture medium as a diagnostic biomarker of tumor growth. Thus, the aim of the present study was to elucidate the function and the targets of PKG II, and the mechanism underlying the secretion of this kinase. METHODS: Construction of peptides and plasmids, RNA interference, Immunoelectron microscopy, Co-immunoprecipitation, N-glycosylation assay and Isolation of the Golgi apparatus were applied to investigate the secretory mechanism, and the targets and function of PKG II. RESULTS: PKG II was secreted by enterochromaffin (EC) cells, which were components of the endocrine system in the gastrointestinal tract. Myristoylation of glycine 2 and the N-terminal sequence, especially the amino acids 3-30, acted as a signal peptide to induce the secretion of PKG II via the conventional protein secretory pathway. Moreover, recombinant PKG II inhibited the epidermal growth factor (EGF)-induced activation of the EGF receptor via phosphorylating the T406 of the extracellular domain and blocked EGF-triggered proliferation of various cancer cells. CONCLUSIONS: These results revealed a correlation between the endocrine system and the secretion of protein kinase, suggesting a novel protein secretory pathway. The resuls also indicated that secreted PKG II was a potential diagnostic biomarker and an inhibitor of tumor.

Polyphyllin I promotes cell death via suppressing UPR-mediated CHOP ubiquitination and degradation in non-small cell lung cancer.

Polyphyllin I (PPI) purified from Polyphyllarhizomes displays puissant cytotoxicity in many kinds of cancers. Several researches investigated its anti-cancer activity. But novel mechanisms are still worth investigation. This study aimed to explore PPI-induced endoplasmic reticulum (ER) stress as well as the underlying mechanism in non-small cell lung cancer (NSCLC). Cell viability or colony-forming was detected by MTT or crystal violet respectively. Cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were assessed by flow cytometry. Gene and protein levels were evaluated by qRT-PCR and immunoblotting respectively. Protein interaction was determined by immunoprecipitation or immunofluorescence assay. Gene overexpression or silencing was carried out by transient transfection with plasmids or small interfering RNAs. The Cancer Genome Atlas (TCGA) database was used for Gene Set Enrichment Analysis (GSEA), survival analysis, gene expression statistics or pathway enrichment assay. PPI inhibited the propagation of NSCLC cells, increased non-viable apoptotic cells, arrested cell cycle at G2/M phase, induced ROS levels but failed to decrease mitochondrial membrane potential. High levels of GRP78 indicates poor prognosis in NSCLC patients. PPI selectively suppressed unfolded protein response (UPR)-induced GRP78 expression, subsequently protected CHOP from GRP78-mediated ubiquitination and degradation. We demonstrated that the natural product PPI, obtained from traditional herbal medicine, deserves for further study as a valuable candidate for lead compound in the chemotherapy of NSCLC.

EGFR mutation mediates resistance to EGFR tyrosine kinase inhibitors in NSCLC: From molecular mechanisms to clinical research.

With the development of precision medicine, molecular targeted therapy has been widely used in the field of cancer, especially in non-small-cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a well-recognized and effective target for NSCLC therapies, targeted EGFR therapy with EGFR-tyrosine kinase inhibitors (EGFR-TKIs) has achieved ideal clinical efficacy in recent years. Unfortunately, resistance to EGFR-TKIs inevitably occurs due to various mechanisms after a period of therapy. EGFR mutations, such as T790M and C797S, are the most common mechanism of EGFR-TKI resistance. Here, we discuss the mechanisms of EGFR-TKIs resistance induced by secondary EGFR mutations, highlight the development of targeted drugs to overcome EGFR mutation-mediated resistance, and predict the promising directions for development of novel candidates.

Gastric cancer mesenchymal stem cells inhibit natural killer cell function by up-regulating FBP1.

INTRODUCTION: The dysfunction of natural killer (NK) cells has been widely reported in malignancies, including in solid tumours. Gastric cancer mesenchymal stem cells (GCMSCs) are one of the vital elements of stromal cells in the tumour environment (TME) which possess immunosuppressive activity. This study aimed to determine whether GCMSCs are involved in the inhibition of NK cell immune function and explore its underlying mechanism. MATERIAL AND METHODS: CD107a and perforin expression of GCMSCs conditioned medium (GCMSCs-CM)-primed NK cells were detected by flow cytometry. To determine NK cell cytotoxicity, the CytoTox96 Non-Radioactive Cytotoxicity Assay kit was used. Glucose uptake and lactate production assay were performed to evaluate the metabolism state of NK cells treated with GCMSCs-CM. The expression of FBP1 in NK cells was analysed by immunoblotting. RESULTS: GCMSCs inhibited the degranulation capacity, perforin production and cytotoxicity of NK cells. GCMSCs-CM restrained NK cell glucose uptake and lactate production, thus weakening their glycolytic metabolism. FBP1 expression of NK cells was upregulated in the presence of GCMSCs-CM. Using FBP1 inhibitor could reverse the dysfunctional state of NK cells. CONCLUSIONS: This study indicated that GCMSCs could exert immunosuppressive effects on NK cells by up-regulating FBP1 expression, opening up new avenues for NK cell-based GC immunotherapy.

Combination of Tertiary Lymphoid Structure and Neutrophil-to-Lymphocyte Ratio Predicts Survival in Patients With Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is the most common pathological type of primary liver cancer. The lack of prognosis indicators is one of the challenges in HCC. In this study, we investigated the combination of tertiary lymphoid structure (TLS) and several systemic inflammation parameters as a prognosis indicator for HCC. Materials and Methods: We retrospectively recruited 126 postoperative patients with primary HCC. The paraffin section was collected for TLS density assessment. In addition, we collected the systemic inflammation parameters from peripheral blood samples. We evaluated the prognostic values of those parameters on overall survival (OS) using Kaplan-Meier curves, univariate and multivariate Cox regression. Last, we plotted a nomogram to predict the survival of HCC patients. Results: We first found TLS density was positively correlated with HCC patients' survival (HR=0.16, 95% CI: 0.06 - 0.39, p < 0.0001), but the power of TLS density for survival prediction was found to be limited (AUC=0.776, 95% CI:0.772 - 0.806). Thus, we further introduced several systemic inflammation parameters for survival analysis, we found neutrophil-to-lymphocyte ratio (NLR) was positively associated with OS in univariate Cox regression analysis. However, the combination of TLS density and NLR better predicts patient's survival (AUC=0.800, 95% CI: 0.698-0.902, p < 0.001) compared with using any single indicator alone. Last, we incorporated TLS density, NLR, and other parameters into the nomogram to provide a reproducible approach for survival prediction in HCC clinical practice. Conclusion: The combination of TLS density and NLR was shown to be a good predictor of HCC patient survival. It also provides a novel direction for the evaluation of immunotherapies in HCC.

Gastric cancer mesenchymal stem cells regulate PD-L1-CTCF enhancing cancer stem cell-like properties and tumorigenesis.

Rationale: Mesenchymal stem cells (MSCs) have been the focus of many studies because of their abilities to modulate immune responses, angiogenesis, and promote tumor growth and metastasis. Our previous work showed that gastric cancer MSCs (GCMSCs) promoted immune escape by secreting of IL-8, which induced programmed cell death ligand 1 (PD-L1) expression in GC cells. Mounting evidence has revealed that PD-L1 expression is related to intrinsic tumor cell properties. Here, we investigated whether GCMSCs maintained a pool of cancer stem cells (CSCs) through PD-L1 signaling and the specific underlying molecular mechanism. Methods: Stem cell surface markers, aldehyde dehydrogenase (ALDH) activity, migration and sphere formation abilities were tested to evaluate the stemness of GC cells. PD-L1-expressing lentivirus and PD-L1 specific siRNA were used to analyze the effects of PD-L1 on GC cells stemness. Annexin V/PI double staining was used to assess apoptosis of GC cells induced by chemotherapy. Co-Immunoprecipitation (Co-IP) and Mass spectrometry were employed to determine the PD-L1 binding partner in GC cells. PD-L1Negative and PD-L1Positive cells were sorted by flow cytometry and used for limiting dilution assays to verify the effect of PD-L1 on tumorigenic ability in GC cells. Results: The results showed that GCMSCs enhanced the CSC-like properties of GC cells through PD-L1, which led to the resistance of GC cells to chemotherapy. PD-L1 associated with CTCF to contribute to the stemness and self-renewal of GC cells. In vivo, PD-L1Positive GC cells had greater stemness potential and tumorigenicity than PD-L1Negative GC cells. The results also indicated that GC cells were heterogeneous, and that PD-L1 in GC cells had different reactivity to GCMSCs. Conclusions: Overall, our data indicated that GCMSCs enriched CSC-like cells in GC cells, which gives a new insight into the mechanism of GCMSCs prompting GC progression and provides a potential combined therapeutic target.

Platelets enhance the ability of bone-marrow mesenchymal stem cells to promote cancer metastasis.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been identified to be closely associated with cancer progression. Our previous experimental results showed that BM-MSCs promote tumor growth and metastasis of gastric cancer through paracrine-soluble cytokines or exosomes. However, the elements that affect the role of BM-MSCs in promoting tumor metastasis are not clear. It is known that thrombocytosis in cancer patients is very common. Recently, platelets are recognized to play a critical role in tumor progression. PURPOSE: This study aims to observe the effect of BM-MSCs which were co-cultured with platelets on tumor cell metastasis. METHODS: Platelet aggregation rate and the expression of P-selectin of platelets co-incubated with conditioned medium of SGC-7901 cells and BM-MSCs were detected by flow cytometry and platelet aggregometer. We also analyzed the change of BM-MSCs after co-incubation with platelets or platelets which were treated with SGC-7901 cells using transwell assay and Western blot analysis. The proliferation and migration ability and expression of VEGF, c-Myc, and sall-4 in SGC-7901 cells treated with medium of BM-MSCs which were co-cultured with platelets were detected. SGC-7901 cells were injected into Balb/c nude mice and the extent of lung metastasis was observed. Both in vitro and in vivo assays were used to analyze the effect of platelets on enhancing the ability of BM-MSCs to promote cancer metastasis. RESULTS: Results suggested that BM-MSCs and tumor cells can promote platelet aggregation rate and the expression of P-selectin. The protein levels of α-smooth muscle actin, vimentin, and fibroblast activation protein in BM-MSCs were higher after co-incubation with platelets, and SB431542 was used to confirm the effect of TGF-ß on transdifferentiation of BM-MSCs into cancer-associated fibroblasts. Medium of BM-MSCs treated with platelets enhanced the proliferation and migration ability of SGC-7901 cells. More lung metastases were found in mice which were injected with SGC-7901 cells treated with conditioned medium from BM-MSCs co-incubated with platelets. CONCLUSION: Tumor cells and BM-MSCs activate platelets which can change the characteristics of BM-MSCs through secretion of TGF-ß. Moreover, we found that platelets enhanced the effect of BM-MSCs on tumor metastasis, which suggested a potential target and approach for gastric cancer therapy.

Gastric cancer mesenchymal stem cells derived IL-8 induces PD-L1 expression in gastric cancer cells via STAT3/mTOR-c-Myc signal axis.

The expression of PD-L1 in tumor cells is one of the main causes of tumor immune escape. However, the exact mechanism for regulating PD-L1 expression in gastric cancer (GC) cells remains unclear. Our previous studies have shown that mesenchymal stem cells (MSCs) exert broad immunosuppressive potential, modulating the activity of cells either in innate or adaptive immune system to promote tumor progress. This study aims to investigate whether GCMSCs regulate the PD-L1 expression in GC cells and explore the specific molecular mechanism. The results have shown that GCMSCs enhanced PD-L1 expression in GC cells resulting in the resistance of GC cells to CD8+ T cells cytotoxicity. However, this resistance was attenuated with IL-8 inhibition. Further studies proved that IL-8 derived from GCMSCs induced PD-L1 expression in GC cells via c-Myc regulated by STAT3 and mTOR signaling pathways. Our data indicated that blocking IL-8 derived from GCMSCs may overcome the immune escape induced by PD-L1 in GC cells and provide a potential strategy to enhance the immunotherapy efficiency in GC.

Human Gastric Cancer Mesenchymal Stem Cell-Derived IL15 Contributes to Tumor Cell Epithelial-Mesenchymal Transition via Upregulation Tregs Ratio and PD-1 Expression in CD4+T Cell.

Several studies show that mesenchymal stem cells (MSCs) homing to tumors not only provide the microenvironment for tumor cells but also promote tumor growth and metastasis. However, the exact mechanism remains unclear. Our study aims to investigate the role of gastric cancer MSCs (GCMSCs)-derived IL15 during GC progression. The effects of IL15 secreted by GCMSCs on GC development were evaluated by detecting the stemness, epithelial-mesenchymal transition (EMT), and migration abilities of GC cell lines. The expression of IL15 in serum and tissues of GC patients was also assessed. We found that IL15 derived from GCMSCs enhanced stemness, induced EMT and promoted migration of GC cell lines. The level of IL15 was higher in GC patients both in serum and tissues compared with that in healthy donors, which was associated with lymph node metastasis. In addition, the results have shown that IL15 in GC microenvironment was mainly produced by GCMSCs. Moreover, IL15 upregulated Tregs ratio through activation of STAT5 in CD4+T cells was accompanied by elevated expression of programmed cell death protein-1 (PD-1). Our data proved that the high concentration of IL15 in tumor microenvironment, which was mainly secreted by GCMSCs, may contribute to tumor cell metastasis and offer a new opportunity to develop effective therapeutics for intercepting tumor progression.

The constitutively active PKG II mutant effectively inhibits gastric cancer development via a blockade of EGF/EGFR-associated signalling cascades.

Type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG II) is a membrane-anchored enzyme expressed mainly in the intestinal mucosa and the brain, and is associated with various physiological or pathological processes. Upregulation of PKG II is known to induce apoptosis and inhibit proliferation and metastasis of cancer cells. The inhibitory effect of PKG II has been shown to be dependent on the inhibition of the activation of epidermal growth factor receptor (EGFR) and blockade of EGFR downstream signal transduction in vitro. However, it remains unclear whether similar phenomena/mechanisms exist in vivo and whether these effects are independent of cGMP or cGMP analogues. In the present work, nude mice with transplanted orthotopic tumours were infected with adenovirus encoding cDNA of constitutively active PKG II mutant (Ad-a-PKG II) and the effect of constitutively active PKG II (a-PKG II) on tumour development was detected. The results showed that a-PKG II effectively ameliorated gastric tumour development through delaying the growth, inducing the apoptosis, and inhibiting the metastasis and angiogenesis. The effect was related to blockade of EGFR activation and abrogation of the downstream signalling cascades. These findings provide novel insight which will benefit the development of new cancer therapies.

Cross-linked hyaluronic acid gel inhibits metastasis and growth of gastric and hepatic cancer cells: in vitro and in vivo studies.

Cross-linked hyaluronic acid gel (CHAG) has been used to prevent postoperative adhesion of abdominal tumorectomy. However, its effect on tumor cells is still unknown. This paper was designed to investigate the effect of CHAG on metastasis and growth of tumor cells. Migration and invasion assays, Western blotting, pull down assay, siRNA interference, and nude mice implantation tumor model were applied in this study. The results of in vitro experiments with gastric cancer cell line AGS and hepatic cancer cell line HepG2 showed that CHAG inhibited the migration and invasion activities, the MAPK and PI3K/Akt mediated signaling, the activation of small G proteins Rac1 and RhoA, and the expression of MMPs and PCNA initiated by EGF, through blocking the activation of EGFR. CHAG also had inhibitory effect on activation of other membrane receptors, including integrin and VEGFR. When the expression of hyaluronic acid receptors (CD44 or RHAMM) was interfered, the above inhibitory effects of CHAG still existed. In vivo experimental results showed that CHAG suppressed colonization, growth and metastasis of gastric cancer cell line SGC-7901 in peritoneal cavity of nude mice. In conclusion, CHAG had inhibitory effect on tumor cells, through covering cell surface and blocking the interaction between extracellular stimulative factors and their receptors.

PKG II reverses HGF-triggered cellular activities by phosphorylating serine 985 of c-Met in gastric cancer cells.

Previous studies showed that type II cGMP-dependent protein kinase G (PKG II) could inhibit the activation of epidermal growth factor receptor (EGFR). Both c-Met and EGFR belong to family of receptor tyrosine kinases (RTKs) and have high molecular analogy. However, the effect of PKG II on c-Met activation is unclear. This study was designed to investigate the inhibitory effect of PKG II on the activation of c-Met and consequent biological activities. The results from CCK8 assay, Transwell assay and TUNEL assay showed that HGF enhanced cell proliferation and migration, and decreased cell apoptosis. Activated PKG II reversed the above changes caused by HGF. Immunoprecipitation and Western blotting results showed that PKG II could bind with c-Met and phosphorylate its Ser985, and thereby inhibited HGF-induced activation of c-Met and MAPK/ERK and PI3K/Akt/mTOR mediated signal transduction. When Ser985 of c-Met was mutated to Alanine for preventing phosphorylation of this site, the blocking effect of PKG II on c-Met activation was annulled. When Ser985 of c-Met was mutated to Aspartic acid for mimicking phosphorylation of this site, HGF-induced activation of c-Met was prevented. In conclusion, the results indicated that PKG II could block c-Met activation via phosphorylating Ser985 of this RTK.

Type II cGMP-dependent protein kinase directly inhibits HER2 activation of gastric cancer cells.

Our previous study demonstrated that type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced phosphorylation/activation of epidermal growth factor receptor (EGFR). Since human epidermal growth factor receptor 2 (HER2) has a similar molecular structure to EGFR, the present study was designed to investigate whether PKG II also inhibits HER2 activation. The human gastric cancer cell line HGC­27 was infected with an adenoviral construct encoding cDNA of PKG II (Ad­PKG II) to increase the expression of PKG II and treated with 8­(4­chlorophenylthio)guanosine­3',5'­cyclic monophosphate (8­pCPT­cGMP) to activate the kinase. Western blotting was performed to detect the tyrosine and serine/threonine phosphorylation of HER2. Co­immunoprecipitation was performed in order to determine the binding between PKG II and HER2. In addition, a QuikChange Lightning Site­Directed Mutagenesis kit was used to mutate threonine 686 of HER2 to glutamic acid or alanine. The results demonstrated that EGF treatment increased the tyrosine phosphorylation (activation) of HER2. Increasing the PKG II activity of HGC­27 cells through infection with Ad­PKG II and stimulation with 8­pCPT­cGMP inhibited the EGF­induced tyrosine phosphorylation/activation of HER2. PKG II bound directly with HER2 and caused phosphorylation of threonine 686. When threonine 686 of HER2 was mutated to alanine, which could not be phosphorylated by PKG II, the inhibitory effect of PKG II on the activation of HER2 was eradicated. When threonine 686 of HER2 was mutated to glutamic acid, which mimicked the phosphorylation of this site, treatment with EGF had no stimulating effect on tyrosine phosphorylation/activation of the mutant HER2. The results suggested that PKG II inhibits EGF­induced activation of HER2 through binding with and causing threonine 686 phosphorylation of this oncogenic protein.

Nitric oxide/cyclic guanosine monophosphate inducers sodium nitroprusside and L-arginine inhibit the proliferation of gastric cancer cells via the activation of type II cyclic guanosine monophosphate-dependent protein kinase.

Nitric oxide (NO) may activate soluble guanylyl cyclase (sGC), resulting in the increase of intracellular cyclic guanosine monophosphate (cGMP), a key molecule in the activation of type II cGMP-dependent protein kinase (PKG II). In our previous study, the membrane-permeable cGMP analogue 8-pCPT-cGMP was used to activate PKG II. The aim of the present study was to investigate whether NO/sGC-induced endogenous cGMP is able to activate PKG II and induce the corresponding effects. In the AGS gastric cancer cell line, the expression of PKG II was increased by infecting the cells with an adenoviral construct encoding PKG II cDNA (Ad-PKG II) and the activation of PKG II was induced by 8-pCPT-cGMP (positive control), the NO donor sodium nitroprusside (SNP) and the NO precursor L-arginine. ELISA was performed to detect the concentration of cGMP in AGS cells and the Cell Counting Kit-8 was used to analyze the proliferation of differently treated cells. Western blot analysis was used to detect the expression and phosphorylation of associated proteins. The results demonstrated that the level of cGMP was increased in cells treated with the NO donor or precursor. There was an obvious increase of Ser239 phosphorylation of the vasodilator-stimulated phosphoprotein, representing the increase in the activity of PKG II. The epidermal growth factor (EGF)-induced proliferation of AGS cells was inhibited by infection with Ad-PKG II and treatment with SNP or L-arginine. In addition, EGF-induced tyrosine phosphorylation of the EGF receptor (EGFR) and tyrosine/serine phosphorylation of extracellular signal-regulated kinase (ERK) were also inhibited by infection with Ad-PKG II and treatment with the NO donor or precursor. These data indicated that NO donors and precursors may activate the expression of PKG II, thereby blocking EGF-initiated signaling of the mitogen-activated protein kinase/ERK pathway and inhibiting EGF-induced proliferative activity through preventing the phosphorylation of EGFR at tyr1068.