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Lung cancer has a complex etiology involving multiple regulatory systems. Uncertainty about the biology and evolution of lung cancer has made it difficult to improve its poor prognosis. To create efficient therapeutic targets and optimal molecular screening tools for lung cancer, the most important task seems to be to understand how it develops and progresses. The expression and regulation of GTPBP4 in non-small cell lung cancer (NSCLC) are not well understood. Using methods such as knocking down GTPBP4 in lung cancer cells and establishing a mouse lung cancer model, we found that the expression of GTPBP4 was upregulated in human lung adenocarcinoma cells and tissues, and that knocking down the expression of the GTPBP4 gene in A549 and Calu-1 lung adenocarcinoma cells can inhibit the proliferation of lung adenocarcinoma cells and reduce their invasion ability. The results of the mouse lung cancer model showed that the lung weight and the number of lung surface nodules decreased significantly in the LLC-GTPBP4 KO group. The mechanism by which GTPBP4 regulation affects the progression of lung adenocarcinoma may be related to the regulation of EMT. From this study, new research ideas emerge to explore GTPBP4 as a biomarker and therapeutic target for early diagnosis and treatment of lung cancer.

The human commensal microbiome existing in an internal environment is relatively consistent with that of the host. The presence of bacterial dysbiosis, on the other hand, promptly results in the termination of this symbiotic association. The altered microbial structure in the lung may be responsible for the development of lung cancer by controlling the host’s inflammatory response and influencing a variety of immunological pathways. More and more studies have pointed to the fact that the commensal microbiota plays a vital role in both the development of tumors and the body’s response to lung cancer treatment. Microbiome dysbiosis, genotoxicity, virulence effect, and epigenetic dysregulations are some of the potential mechanisms that may lie behind the process of tumorigenesis that is mediated by microbiome. Other potential mechanisms include regulating host immune activity through a variety of pathogenic factors, dysregulating host metabolism as a result of microbiome alterations, and microbiome dysbiosis. In this historical overview, we go through some of the more recent mechanistic discoveries into the biological processes that are involved in lung cancer that are caused by bacteria. Without a question, obtaining a greater knowledge of the dynamic link between the lung microbiome and lung cancer has the potential to inspire the development of innovative early detection and customized treatment methods for lung cancer.

Previously, we generated a novel bispecific antibody (BsAb) targeting c-MET and PD-1 (PDCD1), which can bridge T cells and c-MET positive tumor cells. This study investigated functional mechanisms and antitumor activities of our BsAb against c-MET/PD-L1 (CD274) positive colorectal cancer (CRC). The Cancer Genome Atlas database was used to evaluate c-MET expression in tumor tissues. The expression of plasma exosomal c-MET/PD-L1 in CRC patients was measured by enzyme-linked immunosorbent assays. Western blotting assay was performed to determine expression levels of c-MET and molecular mechanism. The scratch wound healing migration assay and transwell chamber invasion assay were conducted to determine cell migratory and invasive abilities, respectively. A humanized mouse model was used to evaluate antitumor activity of the BsAb in vivo. The BsAb inhibited c-MET/PD-L1+ CRC cell migration and invasion and mediated strong antitumor activity against HCT116 tumors in mice. The BsAb induced the degradation of c-MET protein in a dose and time-dependent manner. The BsAb suppressed the phosphorylation of c-MET downstream proteins GRB2-associated-binding protein 1 (Gab1) and focal adhesion kinase (FAK). The BsAb promoted macrophage phagocytosis. Furthermore, the level of plasma exosomal-c-MET/PD-L1 distinguished CRC patients from healthy controls. The BsAb exhibited potent anti-tumor activities by two distinguished mechanisms: inhibition of c-MET signal transduction and promotion of macrophage-mediated phagocytosis. Our BsAb may provide a novel therapeutic tool for patients with c-MET/PD-L1+ CRC, and the status of exosomal-c-MET/PD-L1 may be predictive responsiveness to treatment with our BsAb.

Background: Lung cancer is one of most common malignancies worldwide. We have previously identified retinoic acid-induced gene G (Rig-G) as a tumor suppressor in not only acute promyelocytic leukemia, but as well in other solid tumors. However, the clinical significance of Rig-G and the underlying mechanism(s) for its biological function in lung cancer remain largely unexplored. Methods: We first compared the expression of Rig-G between lung cancer (n=138) and normal tissues (n=23), from public-available datasets and our patient cohort. We further analyzed the correlation of Rig-G expression with key clinico-pathological features and survival outcomes in a multi-site clinical cohort of 300 lung cancer patients. Functional studies for Rig-G were performed in cell lines, and an animal model to support clinical findings. Findings: We found that Rig-G was frequently downregulated in lung cancer tissues and celllines, and correlated with poor prognosis in lung cancer patients. Overexpression of Rig-G led to significantly reduced cell growth and suppressed migrationin A549 and NCI-H1944 cells, accompanied by reduced epithelial-mesenchymal transition. Likewise, restoration of Rig-G in Lewis lung carcinoma cells permitted development of fewer cancer metastases vs. controls in an animal model. Gene expression profiling results identified p53 pathway as a key downstream target of Rig-G, and p53 inhibition by pifithrin-α caused abrogation of tumor-suppressive effects of Rig-G in lung cancer. Interpretation: We for the first time have identified Rig-G as a novel and important tumor suppressor, which may serve as a potential therapeutic target for restoring p53 expression in lung cancer patients. Funding Statement: This work was supported by the National Cancer Institute, NIH (CA72851, CA184792, CA202797 and CA187956), the National Natural Science Foundation of China (81272603, 81472179 and 81873975), the Excellent Academic Leader Training Program of Shanghai Health System (2018BR31), the Clinical Research and Cultivation Project of Shanghai Tongji Hospital grant [ITJ(ZD)1803]. This work was also supported by the National Natural Science Foundation of China (81672826 and 81874179), the Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai (2017YQ044), the Shanghai Pujiang Talent Plan (18PJD047), the Natural Science Foundation of Shanghai (19ZR1448800), the Medical Guidance Science and Technology Support Project of Shanghai (9411964800) and the National Natural Science Foundation Training Program of Tongji Hospital (GJPY1804). Declaration of Interests: None of the authors have any potential conflicts to disclose. Ethics Approval Statement: A written informed consent was obtained from all patients and the study was approved by ethics committee of Shanghai Tongji Hospital. All animal experiments were approved by the Tongji Hospital of Tongji University Ethics Committee on the Use and Care of Animals.

Background Colorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Exosome shave emerged as crucial regulators of intercellular communication and that abundant Circular RNAs (circRNAs) are enriched within exosomes. CircRNAs are novel members of noncoding RNAs regulating cancer proliferation and progression. However, the function and regulatory mechanism of cancer-derived exosomal circRNAs in CRC remains unclear. Methods CRC cells-derived exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot. CCK-8, wound healing and transwell assays, and flow cytometry assays were conducted to assess whether exosomes would affect the proliferation, metastasis, and apoptosis of CRC cells, respectively. Moreover, we performed the RNA sequencing and RT-qPCR to identify circRNAs in exosome-stimulated CRC cells. Fluorescence in situ hybridization (FISH) assay was used to detect the cellular distribution of circPACRGL. Bioinformatic analyses (StarBase 2.0) were used to pool the miRNA targets of circPACRGL. Luciferase assays were performed to verify the direct interaction. Finally, flow cytometry was used to detect the differentiation of N1-N2 neutrophils. Results Our study identified a novel CRC-derived exosomal circRNA, circPACRGL. We found circPACRGL was significantly upregulated in CRC cells after tumor-derived exosomes addition. Moreover, circPACRGL serves as a sponge for miR-142-3p/miR-506-3p to facilitate the transforming growth factor-β1 (TGF-β1) expression. As a result, circPACRGL promoted CRC cell proliferation, migration and invasion, as well as differentiation of N1 to N2 neutrophils via miR-142-3p/miR-506-3p-TGF-β1 axis. Conclusion Our study, the first to reveal that cancer-derived exosomal circPACRGL plays an oncogenic role in CRC proliferation and metastasis, providing mechanistic insights into the roles of circRNAs in CRC progression and a valuable marker for CRC treatment.

Exosomes play important roles in proliferation and microenvironment modulation of many types of cancers, including colorectal cancer (CRC). However, the inhibitory effect of CRC cells-derived exosomes in angiogenesis has not been fully discussed. In this study, the roles of microRNA-183-5p (miR-183-5p) in abundant in exosomes secreted from the CRC cells were investigated. Initially, microarray analysis was employed to determine the differentially expressed miRNAs. Exosomes isolated from CRC cells were co-cultured with HMEC-1 cells to explore the role of exosomes in angiogenesis. Further, the effects of CRC cell-derived exosomal miR-183-5p on proliferation, invasion and tube formation abilities of HMEC-1 cells were assessed. The preventative effect of exosomal miR-183-5p in vivo was measured in nude mice. Initially, it was found that FOXO1 was downregulated while miR-183-5p was upregulated in CRC. Additionally, the inhibition of miR-183-5p was suggested to suppress proliferation, invasion and tube formation abilities of HMEC-1 cells through upregulating FOXO1. Then, in vitro assays demonstrated that CRC cell-derived exosomes overexpressing miR-183-5p contributed to an enhanced proliferation, invasion and tube formation abilities of HMEC-1 cells. Furthermore, in vivo experiments confirmed the tumor-promotive effects of CRC cell-derived exosomal miR-183-5p. Collectively, our study demonstrates that the CRC cell-derived exosomes overexpressing miR-183-5p aggravates CRC through the regulation of FOXO1. Exosomes overexpressing miR-183-5p might be a potential treatment biomarker for CRC.

Treatment of cancer in humans requires a thorough understanding of the multiple pathways by which it develops. Recent studies suggest that nuclear receptor coactivator 4 (NCOA4) may be a predictive biomarker for renal cancer. In the present work, TCGA, GEPIA, and several bioinformatics approaches were used to analyze the NCOA4 expression patterns, prognostic relevance, and association between NCOA4 and clinicopathological features and immune cell infiltration. We investigated NCOA4 expression in malignancies. Low NCOA4 expression was associated with poor overall survival in individuals with malignancies such as cholangiocarcinoma, colon adenocarcinoma, and clear cell renal carcinoma. We also analyzed NCOA4 DNA methylation in normal and primary tumor tissues and investigated possible functional pathways underlying NCOA4-mediated oncogenesis. In conclusion, downregulation of NCOA4 is associated with poor prognosis, and NCOA4 may be a predictive biomarker for COAD.

The PLAC8 expression in lung cancer tissues and in vitro grown lung cancer cells, as well as the involvement of the Wnt/

Early diagnosis and treatment of colorectal cancer (CRC) significantly improves the survival rate and quality of life. Here we screened for differences in glycoproteins associated with tumor-derived exosomes and validated their clinical value to serve as liquid biopsy biomarkers to diagnosed early CRC. Exosomes were extracted from paracancerous tissues, cancer tissues, and plasma. LC-MS/MS proteomic and glycoproteomics analyses were performed using an LTQ-Orbitrap Elite mass spectrometer. The differences in glycoproteins associated with exosomes of paracancerous tissues and cancer tissue were determined, and their levels in plasma exosomes were determined. Statistical analysis was performed to evaluate the diagnostic efficacy of exosome-associated glycoproteins for CRC. We found that the levels of fibrinogen beta chain (FGB) and beta-2-glycoprotein 1 (β2-GP1) in the exosome of CRC tissue were significantly higher compared with those of paracancerous tissues exosome. The areas under the receiver operating characteristic (ROC) curves of plasma exosomal FGB and β2-GP1 as biomarkers for CRC were 0.871 (95% CI = 0.786–0.914) and 0.834 (95% CI = 0.734–0.901), respectively, compared with those of the concentrations of carcinoembryonic antigen concentration [0.723 (95% CI = 0.679–0.853)] and carbohydrate antigen19-9 concentration [0.614 (95% CI = 0.543–0.715)]. Comprehensive proteomics analyses of plasma exosomal biomarkers in CRC identified biomarkers with significant diagnostic efficacy for early CRC, which can be measured using relatively non-invasive techniques.

Rationale: Antimicrobial peptides, such as cathelicidin LL-37/hCAP-18, are important effectors of the innate immune system with direct antibacterial activity. In addition, LL-37 is involved in the regulation of tumor cell growth. However, the molecular mechanisms underlying the functions of LL-37 in promoting lung cancer are not fully understood. Methods: The expression of LL-37 in the tissues and sera of patients with non-small cell lung cancer was determined through immunohistological, immunofluorescence analysis, and enzyme-linked immunosorbent assay. The animal model of wild-type and Cramp knockout mice was employed to evaluate the tumorigenic effect of LL-37 in non-small cell lung cancer. The mechanism of LL-37 involving in the promotion of lung tumor growth was evaluated via microarray analyses, recombinant protein treatment approaches in vitro, tumor immunohistochemical assays, and intervention studies in vivo. Results: LL-37 produced by myeloid cells was frequently upregulated in primary human lung cancer tissues. Moreover, its expression level correlated with poor clinical outcome. LL-37 activated Wnt/β-catenin signaling by inducing the phosphorylation of protein kinase B and subsequent phosphorylation of glycogen synthase kinase 3β mediated by the toll-like receptor-4 expressed in lung tumor cells. LL-37 treatment of tumor cells also decreased the levels of Axin2. In contrast, it elevated those of an RNA-binding protein (tristetraprolin), which may be involved in the mechanism through which LL-37 induces activation of Wnt/β-catenin. Conclusion: LL-37 may be a critical molecular link between tumor-supportive immune cells and tumors, facilitating the progression of lung cancer.

Retinoic acid inducible gene G (RIG-G) is an inducible gene produced during the treatment of acute promyelocytic leukemia with all-trans retinoic acid (ATRA). However, it is unclear the expression level of RIG-G gene in the peripheral blood of healthy subjects and patients with acute promyelocytic leukemia (APL or AML-M3). In the present study, we established the TaqMan-MGB fluorescent probe qPCR (real-time polymerase chain reaction) method for the first time to detect the expression of RIG-G gene in APL. Twenty APL patients were selected, and their RIG-G expression levels were quantified to assess the correlation between the expression of peripheral blood and bone marrow samples. U test was used to analyze the expression level of RIG-G in the peripheral blood of 40 normal specimens and 20 APL patients to observe the prognostic monitoring effect of RIG-G gene in the ATRA treatment process. ROC (receiver operating characteristic curve) was used to analyze and test the diagnostic efficiency of RIG-G gene for APL patients. There is a strong positive correlation between the expression of RIG-G in peripheral blood and bone marrow of APL patients. The expression level of RIG-G in peripheral blood of APL patients is significantly lower than that in healthy controls (p &lt, 0.001). The changes in the expression level of RIG-G in peripheral blood changed indicates the remission and recurrence of APL patients after ATRA treatment, and the ROC curve shows that it has a better diagnostic power for APL. In summary, the TaqMan-MGB real-time PCR method we have established has successfully run. The detection of RIG-G gene expression in peripheral blood can effectively monitor the disease changes of APL patients and avoid harmful bone marrow puncture injury.

Numerous genetic polymorphisms and clinical laboratory parameters are associated with ischemic stroke (IS). However, the results of such studies have frequently been inconsistent. The aim of the present study was to evaluate associations between clinical laboratory parameters with genetic polymorphisms that influence the risk of IS in a Chinese Han population. Clinical laboratory parameters were measured by an automatic biochemical analyzer. Genotype and allele frequencies of the polymorphisms angiotensin-converting enzyme (ACE) D/I, methylene tetrahydrofolate reductase (MTHFR) C677T and β-fibrinogen (β-Fg) A/G, 455/148T/C were characterized by restriction fragment length polymorphism-PCR. Furthermore, the gene polymorphisms plasminogen activator inhibitor (PAI)-1-4G/5G and apolipoprotein E (ApoE) ε2,3,4 were characterized by allele-specific PCR. The associations of genotype and allele frequencies of the six risk genes in different groups with clinical laboratory parameters were analyzed by chi-square tests. The distribution maps of the polymorphisms of the six genes and clinical laboratory parameters were compared between a control group of 336 healthy individuals and 762 patients with IS. Certain laboratory parameters were associated with ACE I/D, β-Fg-455 A/G and PAI-1 4G/5G. The D allele of ACE I/D was associated with high levels of total cholesterol and low-density lipoprotein cholesterol (LDL-C). Furthermore, high levels of fasting blood glucose, triglyceride and LDL-C were risk factors for IS. There were significant differences in the genotype frequencies of ACE I/D, β-Fg-455 A/G and β-Fg-148 T/C between the IS and the control group. In conclusion, clinical laboratory parameters were associated with the risk of polymorphisms of IS-related genes. The present results support the determination of a range of control values of clinical laboratory parameters for common genotypes in patients with diabetes and hyperlipidemia as a strategy for the early prevention of IS.

Additional file of Correction to: Long non-coding RNA HOTTIP enhances IL-6 expression to potentiate immune escape of ovarian cancer cells by upregulating the expression of PD-L1 in neutrophils

Background Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. The current study aimed to elucidate the mechanism by which exosomes carrying KRAS mutant contribute to neutrophil recruitment as well as the formation of the neutrophil extracellular trap (NET) in CRC. Methods APC-WT and APC-KRASG12D mouse models were initially developed. Peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) were isolated to detect neutrophil content. Then, APC-WT and APC-KRASG12D mice were injected with exosomes isolated from APC-WT and APC-KRASG12D mice. The ratio of neutrophils, NETs formation and IL-8 protein content were subsequently quantified in colon tissues. DKs-8 (wild type) and DKO-1 (KRAS mutant) cells were employed for in vitro experimentation. Then, DKs-8 cells were cultured with exosome-treated PMA stimulated neutrophil-forming NETs culture medium, with cell viability, invasion, migration, and adhesion evaluated. Results Compared with APC-WT mice, the numbers of polyps and neutrophils in the peripheral blood, spleen and mLNs were increased in APC-KRASG12D mice, accompanied with increased NET formation, IL-8 expression and exosomes. Meanwhile, IL-8 upregulation, neutrophil recruitment and NET formation were observed in the mice injected with exosomes derived from APC-KRASG12D. The in vitro investigation results revealed that more NETs were formed in the presence of DKO-1-Exos, which were inhibited by DNAse. In addition, DKs-8- and DKO-1 cells-derived exosomes could adhere to NETs under static conditions in vitro. Exosomal KRAS mutants were noted to exert stimulatory effects on the IL-8 production and NET formation to promote the growth of CRC cells. Conclusion The results provide evidence suggesting that exosomes may transfer mutant KRAS to recipient cells and trigger increases in IL-8 production, neutrophil recruitment and formation of NETs, eventually leading to the deterioration of CRC.

Background Ginkgolide B (GKB) is a major active component of the extracts of Ginkgo biloba leaves, and it has been used as an anti-cancer agent. However, it is unknown whether GKB has the therapeutic effects on lung cancer. Here, we studied the effects of GKB on lung cancer cells. Methods The effects of GKB on lung cancer cell proliferation and invasion were analyzed by cell counting kit (CCK-8) and cell invasion assays, respectively. Apoptosis was detected by flow cytometry. Western blot analysis was used to confirm the expression of autophagy-associated proteins in GKB-treated cells. Immunofluorescence analysis was used to analyze the level of light chain 3B (LC3B). Results Treatment with GKB time-dependently inhibited the proliferation and decreased the invasive capacity of A549 and H1975 cells. GKB induced apoptosis of these cells, but there was no significant effect on apoptosis compared to the control treatment. GKB-induced inhibition of cell proliferation and GKB-induced cell death were due to autophagy rather than apoptosis. GKB-induced autophagy of lung cancer cells was dependent on beclin-1, and autophagy-induced inhibition of the NLRP3 inflammasome contributed to the anti-tumor effect of GKB. Conclusions GKB-mediated autophagy of lung cancer cells is beclin-1-dependent and results in inhibition of the NLRP3 inflammasome. Therefore, GKB might be a potential therapeutic candidate for the treatment of lung cancer.

Background Many genetic polymorphisms and clinical laboratory parameters have been shown to be associated with ischemic stroke (IS). However, these results are often inconsistent. Therefore, the aim of the study was to evaluate the correlation between clinical laboratory parameters, the risk genetic polymorphisms and IS in Shanghai population.Methods The clinical laboratory parameters were measured by automatic biochemical analyzer. The genotype frequencies and allelic frequencies of the polymorphisms gene encoding angiotensin-converting enzyme ( ACE D/I), gene encoding methylene tetrahydrofolate reductase ( MTHFR C677T), gene encoding β-fibrinogen 455/148 ( β-Fg -455/148) were characterized by restricted fragment length polymorphisms-polymerase chain reaction (RFLP-PCR). The gene encoding plasminogen activator inhibitor ( PAI -1 4G/5G) and gene encoding apolipoprotein E 2,3,4 ( ApoE ε2,3,4) were characterized by allele specific PCR. The genotype frequency and allele frequency of the 6 risk genes of IS were analyzed by chi-square test in different groups of laboratory indexes. The distribution maps of the polymorphisms ACE D/I, MTHFR C677T, β-Fg -455 A/G, β-Fg -148 T/C, PAI -1 4G/5G, ApoE ε2,3,4 and clinical laboratory paramaters were compared between healthy people and patients with IS.Results The laboratory parameters were related to ACE I/D, β-Fg -455 A/G, and PAI -1 4G/5G genes. D allele of ACE I/D was associated with high levels of total cholesterol (TC) and Low-density lipoprotein (LDL). High levels of fasting blood glucose (FBG), triglyceride (TG), and LDL were risk factors for IS. There were significant differences in genotype frequencies of ACE I/D, β-Fg -455 A/G, and β-Fg- 148 T/C genes between IS and control group.Conclusions Clinical laboratory paramaters are associated with the risk genes polymorphisms of IS. It is suggested that control value of clinical laboratory paramaters should be determined according to the genotype carried by the patients with diabetes and hyperlipidemia when preventing IS.

Long noncoding RNAs (lncRNAs) have been reported to be involved in the occurrence and tumorigenesis of numerous malignant cancers. Microarray expression profiles were used to screen colorectal cancer (CRC)-related differentially expressed genes and lncRNAs, which revealed that insulin receptor substrate 1 (IRS1) and lncRNA plasmacytoma variant translocation 1 (PVT1) were highly expressed in CRC. This study aimed to investigate the regulatory role of lncRNA PVT1 in CRC. Subcellular localization detected by fluorescence in situ hybridization identified that lncRNA PVT1 was primarily located in the cytoplasm. The interaction between lncRNA PVT1 and microRNA-214-3p (miR-214-3p) and IRS1 was predicted using the RNA22 website. Next the dual luciferase reporter gene assay, RNA pull-down, and RNA immunoprecipitation assays verified lncRNA PVT1 to be a competitive endogenous RNA (ceRNA) against miR-214-3p, and IRS1 was found to be a target of miR-214-3p. The expression pattern of lncRNA PVT1, miR-214-3p, IRS1, phosphoinositide 3-kinase (PI3K), and Akt was characterized in response to lncRNA PVT1 silencing or miR-214-3p upregulation. Meanwhile, their regulatory effects on cell proliferation, invasion, and apoptosis were detected in CRC cells. With increased levels of miR-214-3p and decreased levels of lncRNA PVT1 in CRC cells, the expression of phosphatidylinositol 3-kinase, putative (PI3K) and Akt was reduced, and consequently, the cell apoptosis was stimulated and cell proliferation and invasion were suppressed. All in all, lncRNA PVT1 competitively binds to miR-214-3p to upregulate the expression of IRS1 thus activating the PI3K/Akt signaling pathway, thus accelerating CRC progression. This study suggests that lncRNA PVT1 might be a potential target of therapeutic strategies for CRC treatment. NEW & NOTEWORTHY This study mainly suggests that long noncoding (lnc)RNA plasmacytoma variant translocation 1 (PVT1) is a downregulated lncRNA in colorectal cancer (CRC), accelerating CRC progression. Strikingly, lncRNA PVT1 acts as a competitive endogenous RNA against microRNA (miR)-214-3p, whereas miR-214-3p targets insulin receptor substrate 1, which draws a comprehensive picture of the potential molecular mechanisms of lncRNA PVT1 in CRC.

Exosomes are nanovesicles produced by a number of different cell types and regarded as important mediators of cell-to-cell communication. Although bronchoalveolar lavage fluid (BALF) has been shown to be involved in the development of tumors, its role in lung cancer (LC) remains unclear. In this article, we systemically studied BALF-derived exosomes in LC. C57BL/6 mice were injected with Lewis lung carcinoma cells and exposed to non-typeable Haemophilus influenza (NTHi) lysate. The analysis showed that the growth of lung tumors in these mice was significantly enhanced compared with the control cohort (only exposure to air). Characterization of the exosomes derived from mouse BALF demonstrated elevated levels of tumor necrosis factor alpha and interleukin-6 in mice exposed to NTHi lysates. Furthermore, abnormal BALF-derived exosomes facilitated the development of LC in vitro and in vivo. The internalization of the BALF-derived exosomes contributed to the development of LC tumors. Collectively, our data demonstrated that exosomes in BALF are a key factor involved in the growth and progression of lung cancer.

HOXA transcript at the distal tip (HOTTIP), belonging to the family of long non-coding RNA (lncRNA), is an important biomarker for ovarian cancer (OC) prognosis. Nevertheless, the function of HOTTIP in immune evasion of OC cells remains undecided. In the current study, the effect of HOTTIP on the development of OC was assessed and the mechanism was discovered as well. The neutrophils were isolated from the clinical samples, and the correlation between programmed death-ligand 1 (PD-L1) and interleukin-6 (IL-6) in neutrophils was analyzed. The relationships among HOTTIP, c-jun and IL-6 were investigated by RIP, CHIP, and the dual luciferase reporter gene assay. The roles of PD-L1, IL-6 and HOTTIP on the T cell proliferation and infiltration were measured in vitro and in vivo. Moreover, the association between HOTTIP and IL-6 in OC tissue and the connection between HOTTIP and OC prognosis were evaluated. Positive relationships were observed between IL-6 expression in peripheral blood and the PD-L1 expression in neutrophils of OC patients, and between the HOTTIP and IL-6 expression in OC tissues. HOTTIP could induce the release of IL-6 via c-jun. Besides, HOTTIP boosted the PD-L1 expression in neutrophils and inhibited the T cell proliferation, thus potentiating the immune evasion of OC cells. The prognosis of patients with higher expression of HOTTIP was worse. In summary, the HOTTIP/IL-6/PD-L1 axis was demonstrates as a novel regulatory signaling pathway involving in the progression of OC. Funding Statement: This work is supported by National Natural Science Foundation of China (81472179, 81873975, 81808084), the Excellent Academic Leader Training Program of Shanghai Health System (2018BR31), the Fundamental Research Funds for the Central Universities (22120170071) and the Clinical Research And Cultivation Project of Shanghai Tongji Hospital (ITJ(ZD)1803). We would like to acknowledge the reviewers for their helpful comments on this paper. Declaration of Interests: The authors declare: "None." Ethics Approval Statement: This study was permitted by the Ethics Committee of Tongji Hospital of Tongji University and The Sixth People’s Hospital of Yancheng City. Written informed consents were provided by all participants. All animal experiments were conducted under the review from the Animal Ethical and Experimental Committee of Tongji Hospital of Tongji University and The Sixth People’s Hospital of Yancheng City.

Background: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. The current study aimed to elucidate the mechanism by which exosomes carrying KRAS mutant contribute to neutrophil recruitment as well as the formation of the neutrophil extracellular traps (NETs) in CRC. Methods: APC-WT and APC-KRASG12D mouse models were initially established. The peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) were isolated to detect neutrophil content. Next, exosomes isolated from APC-WT and APC-KRASG12D mice were injected into APC-WT and APC-KRASG12D mice. The neutrophil ratio, NETs formation and IL-8 protein content in colon tissue were subsequently quantified. DKs-8 (wild type) and DKO-1 (KRAS mutant) cells were employed for in vitro experimentation. Then, DKs-8 cells were cultured with exosome-treated PMA stimulated neutrophil-forming NETs culture medium, with cell viability, invasion, migration, and adhesion evaluated. Results: Compared with APC-WT mice, the number of polyps and neutrophils in the peripheral blood, spleen and mLNs increased in APC-KRASG12D mice, with increased NETs formation, IL-8 expression, and exosomes. IL-8 upregulation, neutrophil recruitment and NETs formation were observed in mice injected with exosomes derived from APC-KRASG12D. The in vitro investigation results revealed that more NETs were formed by DKO-1-Exosomes, which were inhibited by DNAse. In addition, DKs-8 and DKO-1 cells-derived exosomes can adhere to NETs under static conditions in vitro. Exosomal KRAS mutants were noted to exert a stimulatory effect on the production of IL-8 while promoting NETs formation as well as CRC promotion. Conclusion: The results obtained provide evidence suggesting that exosomes may transfer mutant KRAS to recipient cells and trigger an increase in IL-8 production, promote neutrophil recruitment and form NETs, ultimately leading to the promotion of CRC. Funding Statement: This work was supported by the National Natural Science Foundation of China (81873975, 81802084, 81974314, 81902984), the Excellent Academic Leader Training Program of Shanghai Health System (2018BR31), the Medical Guidace Science and Technology Support Project of Shanghai (19411964800), the Natural Science Foundation of Shanghai (19ZR1448800), the Clinical Research and Cultivation Project of Shanghai Tongji Hospital [ITJ(ZD)1803, ITJ(ZD)1905, ITJ(QN)1905]. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocol was approved by the Ethics Committee of Tongji Hospital of Tongji University. All subjects have written informed consent. All animal use and experiments were performed in strict accordance with the procedures approved by the National Cancer Institute Animal Care and Use Committee (ACUC).

// Dong Li 1, * , Junjun Sun 1, * , Wenfang Liu 2, * , Xuan Wang 3 , Robert Bals 4 , Junlu Wu 1 , Wenqiang Quan 1 , Yiwen Yao 1 , Yu Zhang 1 , Hong Zhou 1 , Kaiyin Wu 5 1 Department of Clinical Laboratory, Shanghai Tongji Hospital, Tongji University School of Medicine, 200065 Shanghai, China 2 Department of General Surgery, Shanghai Tongji Hospital, Tongji University School of Medicine, 200065 Shanghai, China 3 Department of Pharmacy, Putuo People’s Hospital, 200060 Shanghai, China 4 Department of Internal Medicine V – Pulmonology, Allergology, Respiratory Intensive Care Medicine, Saarland University Hospital, 66424 Homburg, Germany 5 Institute of Pathology, Charite Medical University, 10117 Berlin, Germany * These authors contributed equally as first authors Correspondence to: Dong Li, email: 186ld@163.com Keywords: Rig-G, lung cancer, growth inhibition, NF-κB, STAT3 Received: February 25, 2016 Accepted: August 24, 2016 Published: September 01, 2016 ABSTRACT The expression of the retinoic acid-induced G (Rig-G) gene, an all trans retinoic acid (ATRA)-inducible gene, was observed in multiple cancer cells, including lung cancer cells. However, whether Rig-G is a tumor suppressor in lung cancer is unknown. Here, we found that ectopic expression of Rig-G can lead to a significant decrease in proliferation of lung cancer cells, resulting in an inhibition of tumor growth. Rig-G knockdown results in a modest increase in cell proliferation, as well as confers an increase in colony formation. Furthermore, transcriptome and pathway analyses of cancer cells revealed a fundamental impact of Rig-G on various growth signaling pathways, including the NF-κB pathway. Rig-G inhibits NF-κB activity by suppressing STAT3 in lung cancer cells. The downregulation of miR21 and miR181b-1 and subsequent activation of PTEN/Akt and CYLD/IκB signaling axis leading to decreased NF-κB activity required to maintain the tumor-inhibiting effect of Rig-G.. Our findings contribute to a better understanding of the antitumor effect mechanism of Rig-G, as well as offer a novel strategy for lung cancer therapy.

Objective The purpose of this study was to establish and validate a nomogram to predict the prognosis in patients with non-small cell lung cancer (NSCLC) from multiple perspectives. Results A total of 98,640 eligible patients were randomly divided into a training set (n = 69,048) and a validation set (n = 29,592). The baseline characteristics of the two sets were similar. We used clinical data from patients in the training set for univariate and multivariate Cox regression analyses. Twelve independent risk factors were incorporated for constructed a prognostic nomogram. And the nomogram with a concordance index of 0.777 (95 % CI, 0.775 to 0.779) for overall survival. The calibration curve results showed that the actual survival rate was consistent with the predicted survival rate. The area under curve of the receiver operating characteristic curves demonstrated that the nomogram has a high prediction of the overall survival rate in patients with NSCLC. Conclusion We have developed a nomogram with high prediction accuracy and discrimination ability, which can help clinicians making personalized survival predictions for NSCLC patients.

Placenta-specific 8 (PLAC8) is closely associated with the proliferation, apoptosis and autophagy of several tumor cells. However, the expression and function of PLAC8 in oral squamous cell carcinoma (OSCC) remain unknown. Therefore, the present study investigated the function and mechanism of PLAC8 in OSCC. Reverse transcription-quantitative PCR and western blot analyses were performed to quantify the expression of PLAC8 in OSCC cell lines. The function of PLAC8 in OSCC was investigated via transfection, the Transwell and Cell Counting Kit-8 assays, immunofluorescence staining and western blotting. The results demonstrated that PLAC8 exspression was downregulated in OSCC cell lines. PLAC8 inhibited the cell proliferation in OSCC. In addition, PLAC8 restrained invasion and epithelial-mesenchymal transition of OSCC cells. Furthermore, β-catenin helped to repress PLAC8 expression by regulating the Wnt/β-catenin and PI3K/Akt/GSK3β signaling pathways in OSCC cells. Collectively, the results of the present study suggest that PLAC8 acts as a tumor suppressor in OSCC by downregulating β-catenin.

Cathelicidin is an antimicrobial peptide that plays an essential role in cell proliferation, angiogenesis, and also has been indicated in tumor promotion. However, it is unclear how cathelicidin causes tumor growth, and the pathogenic mechanisms based on gain or loss of function have not been proposed. Here, a cathelicidin related antimicrobial peptide (CRAMP) knockout mouse was generated using an A/J background (A/J-CRAMP-/- mice), and lung carcinoma growth was induced using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and Non-typeable Haemophilus influenzae (NTHi). Compared with A/J mice, A/J-CRAMP-/- mice were found to have a lower tumor burden and longer survival times, with a significant reduction in both PCNA and Ki-67 positive cells. However, there was no difference between the number of apoptotic lung-cancer cells between the A/J and A/J-CRAMP-/- mice. This indicated cathelicidin might be a tumor growth factor for lung cancer, which was associated for proliferation of tumor cells. In the future, this animal model will be useful to study the distinct role of cathelicidin in induced-lung cancer development.

Although naloxone has been documented to exert neuroprotection in animal model of cerebral ischemia, the mechanism is not well understood. In this present study we investigated whether naloxone affected the mitochondrial apoptotic pathway in ischemic brain injury of rats. SD rats were subjected to a permanent middle cerebral artery occlusion surgery, and received naloxone (0.5, 1, 2 mg/kg, i.v.) immediately after ischemia. Neurological deficits were evaluated 24 h after ischemia using the McGraw Stroke Index, and then the rats were killed, and the brains were collected for further analyses. We show that naloxone treatment dose-dependently decreased the infarction volume and morphological injury, improved motor behavioral function, and markedly curtailed brain edema. Furthermore, naloxone administration significantly inhibited the nuclear translocation of NF-κB p65 and decreased the levels of nuclear NF-κB p65 in the ischemic penumbra. Naloxone administration also dose-dependently increased the NF-κB inhibitory protein (IκBα) levels and attenuated phosphorylated NIK and IKKα levels in the ischemic penumbra. In addition, naloxone administration dose-dependently increased Bcl-2 levels, decreased Bax levels, stabilized the mitochondrial transmembrane potential, and inhibited cytochrome c release and caspase 3 and caspase 9 activation. These results indicate that the neuroprotective effects of naloxone against ischemic brain injury involve the inhibition of NF-κB activation via the suppression of the NIK/IKKα/IκBα pathway and the obstruction of the mitochondrial apoptotic pathway in neurons.

Red blood cell distribution width (RDW) is a parameter of standard full blood count tests that reflects the size variability of erythrocytes In recent studies, RDW levels have been associated with ischemic heart disease, acute and chronic heart failure, hypertension, and inflammatory bowel disease. However, it is unclear whether RDW is associated with colorectal cancer.Eighty-five patients were diagnosed with colorectal cancer. Fifty-four other patients each diagnosed with colon polyps during the same period served as the control group. The patients were classified according to the seventh edition of the AJCC Cancer Staging Manual of 2009 into groups of different cancer stages, and simultaneously divided into groups with or without metastasis. The multigroup metering data was tested by a non-parametric Kruskal-Wallis H test, and the two subsets of patients formed above were compared using a Mann-Whitney U test. The association between continuous variables was assessed by Spearman correlation analysis while the association between RDW and colorectal cancer metastasis was estimated by receiver operating characteristic (ROC) curve analysis.Increased RDW was observed in patients with colorectal cancer. The RDW was significantly different for each subgroup of colorectal cancer as follows: stage III + IV stage III, T3 + T4 T1 + T2, N1 + N2 N0, and M1 M0 (P 0.05). The area under the receiver-operating characteristic curve of the RDW in the diagnosis of colorectal cancer metastasis was 0.721 (95% confidence interval of 0.612-0.831).The value of RDW is closely related to colorectal cancer metastasis.

// Dong Li 1, * , Wenfang Liu 2, * , Xuan Wang 3 , Junlu Wu 1 , Wenqiang Quan 1 , Yiwen Yao 1 , Robert Bals 4 , Shurong Ji 2 , Kaiyin Wu 5 , Jia Guo 6 , Haiying Wan 1 1 Department of Clinical Laboratory, Tongji Hospital of Tongji University, 200065 Shanghai, China 2 Department of General Surgery, Tongji Hospital of Tongji University, 200065 Shanghai, China 3 Department of Pharmacy, Putuo People’s Hospital, 200060 Shanghai, China 4 Department of Internal Medicine V – Pulmonology, Allergology, Respiratory Intensive Care Medicine, Saarland University Hospital, 66424 Homburg, Germany 5 Institute of Pathology, Charite University Hospital, 12200 Berlin, Germany 6 Tongji University Suzhou Institute, 215000 Suzhou, China * These authors have contributed equally to this work Correspondence to: Dong Li, e-mail: 186ld@163.com Keywords: Cathelicidin, Colon cancer, Macrophage, Wnt/β-catenin, Growth Received: June 27, 2014 Accepted: December 03, 2014 Published: December 15, 2014 ABSTRACT Here we found that levels of cathelicidin, an antimicrobial peptide, were increased in colon cancer tissues compared to noncancerous tissues. Importantly, cathelicidin was mainly expressed in immune cells. Contact with tumor cells caused macrophages to secrete cathelicidin. Neutralization of cathelicidin, in vivo , significantly reduced the engraftment of macrophages into colon tumors, as well as proliferation of tumor cells, resulting in an inhibition of tumor growth. Furthermore, treatment with cathelicidin neutralizing antibody de-activated the Wnt/β-catenin signaling pathway in tumor cells both in vivo and in vitro . Cathelicidin activated Wnt/β-catenin signaling by inducing phosphorylation of PTEN, leading to activation of PI3K/Akt signaling and subsequent phosphorylation of GSK3β, resulting in stabilization and nuclear translocation of β-catenin. These data indicate that cathelicidin, expressed by immune cells in the tumor microenvironment, promotes colon cancer growth through activation of the PTEN/PI3K/Akt and Wnt/β-catenin signaling pathways.

Objective: Exosomes acted as important role in tumor proliferation and tumor microenvironment modulation, including colorectal cancer (CRC). However, the inhibitory effect of CRC cells -derived exosomes in angiogenesis in vascular endothelial cell has not been fully discussed. In this study, the microRNA-183-5p (miR-183-5p) that promote the development of CRC, as well as the exosome secreted from the CRC cells were investigated. Methods: Microarray analysis was employed to determine the differentially expressed miRNAs. Exosomes derived from CRC cells were isolated and identified. Meanwhile, exosomes derived from CRC cells were isolated and then co-cultured with HMEC-1 cells to identify the role of exosomes in angiogenesis. Further, the effects of CRC cell-derived exosomal miR-183-5p on proliferation, invasion and tube formation abilities of HMEC-1 cells were assessed. The preventative effect of exosomal miR-183-5p in vivo was measured in nude mice. Results: It was found that FOXO1 was downregulated while miR-183-5p was upregulated in CRC. Additionally, the inhibition of miR-183-5p was suggested to suppress proliferation, invasion and tube formation abilities of HMEC-1 cells through upregulating FOXO1. Then, in vitro experiments demonstrated that CRC cell-derived exosomes overexpressing miR-183-5p contributed to an enhanced proliferation, invasion and tube formation abilities of HMEC-1 cells. Furthermore, in vivo experiments confirmed the promotive effects of CRC cell-derived exosomes overexpressing miR-183-5p. Conclusion: Our study demonstrates that the CRC cell-derived exosomes overexpressing miR-183-5p aggravates CRC through the regulation of FOXO1. Additionally, exosomes overexpressing miR-183-5p might be a potential treatment biomarker for CRC. Citation Format: ANQUAN SHANG, Chenzheng Gu, Junlu Wu, Ping Ji, Zujun Sun, Dong Li. Colorectal cancer cell-derived exosomes containing miR-183-5p regulate angiogenesis via FOXO1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A001. doi:10.1158/1535-7163.TARG-19-A001

To investigate the effect of antimicrobial peptide cathelicidin secreted by non-tumorous cells in lung tumor growth.CRAMP(-/-) mice and WT mice were used to establish a lung cancer model via tail vein injection of Lewis lung carcinoma cells (LLC1). Lung was weighted and tumor number on the lung surface was counted. Kaplan-Meier (K-M) survival curve was used to analyze survival rate of mice. Expression of cathelicidin, Ki-67 and CD68 in the tumor tissue was measured by immunohistochemical analysis. BALF cells were stained with Diff Quik and percentages of leukocyte types were determined by light microscopy.Cathelicidin was high expression in inflammatory cells of tumor tissue, whereas weak expression in tumor cells. The lung weight and number of tumor in CRAMP-/- mice were (0.25±0.04)g and (9.60± 2.25), respectively, which were significantly lower than those of WT mice (0.65±0.05) g and (23.40± 2.68). The difference was statistically significant (t=6.07, 3.95, all P0.05). And Kaplan-Meier survival analysis showed median survival time of CRAMP-/- mice was 49(46-51)d, which was longer than 34(28-39) d of WT mice (χ2=12.00, P0.05). And the positive rate of Ki-67 tumor cells was significant reduced from (35.80±2.96)% in WT mice to (18.80±2.38)% in CRAMP-/- groups (t=4.48, P0.05). The total cell number as well as the number of lymphocytes, neutrophils, and macrophages in BALFs of CRAMP-/- mice were (4.72±0.86)×10(4), (0.08±0.02)×10(4), (0.05±0.02)×10(4) and (4.60±0.84)×10(4), respectively, while of WT mice were (16.18±1.61)×10(4), (0.32±0.05)×10(4), (0.20±0.05)×10(4) and (15.66±1.57)×10(4). All of them had significant difference (t=6.28, 4.39, 3.00, 6.20, all P0.05). In addition, the infiltration of macrophages into lung tumors was decreased in CRAMP-/- mice compared to WT mice, from (15.53±2.28)/high power field to (6.77±3.12)/high power field (t=3.41, P0.05).Non-tumor cells secreted cathelicidin promotes tumor cell proliferation and lung tumor growth. Recruitment of inflammatory cells such as macrophages into the tumor microenvironment may be the main mechanism of action.

To investigate the effect of antibacterial peptide hCAP18/LL-37 on ovarian cancer microenvironment and the regulatory mechanism of its expression.We assessed the effect of macrophage-promoted ovarian cancer cells invasion using BioCoat Matrigel invasion chamber. The expressions of hCAP18/LL-37 and versican V1 were determined by real-time PCR and Western blot analysis. SKOV3 cells were transfected with shRNA plasmid to abrogate the expression of versican V1, and then the expression of hCAP18/LL-37 in macrophages and the invasiveness of SKOV3 cells were assayed.The Matrigel invasion assay showed that after co-culture with macrophages for 4 days, the number of penetrated SKOV3 cells was 112.8±17.1/per high power field, significantly higher than that in the SKOV3 cells cultured alone (8.2±1.9/per high power field) (P0.05). Addition of hCAP/LL-37 neutralizing antibody into the co-cultured macrophage-SKOV3 cells markedly inhibited the macrophage-promoted SKOV3 cells invasion. The penetrated SKOV3 cells was 22.2±5.6/per high power field, significantly lower than the 100.6±25.2/per high power field in the control macrophage- SKOV3 co-cultured cells (P0.05). The expressions of hCAP18/LL-37 mRNA and protein in macrophages were remarkably enhanced upon co-culture with SKOV3 cells, but not changed in SKOV3 cells cultured alone. The expression and secretion of versican V1 in the ovarian cancer cells were also significantly increased after co-cultured with macrophages. Knockdown of versican V1 in SKOV3 cells by small interfering RNA significantly reduced the expression of hCAP18/LL-37 mRNA and protein in the macrophages, as well as decreased the invasiveness of SKOV3 cells (P0.05).In the cancer microenvironment, the macrophage-secreted hCAP18/LL-37 promote the invasiveness of ovarian cancer cells, and the hCAP18/LL-37 expression is regulated by versican V1 protein released by ovarian cancer cells.

The aim of this study was to investigate the mechanism of cigarette smoking (CS)-induced lung cancer growth in mice.RelA/p65⁻/⁻ mice and WT mice were used to establish mouse models of lung cancer. Both mice were divided into two groups: air group and CS group, respectively. Tumor number on the lung surface was counted and maximal tumor size was evaluated using HE staining. Kaplan Meier (K-M) survival curve was used to analyze the survival rate of the mice. Expression of Ki-67, TNF-α and CD68 in the tumor tissue was determined by immunohistochemical analysis, and cyclin D1 and c-myc proteins were examined by Western blot. Apoptosis of tumor cells was analyzed using TUNEL staining. The concentrations of inflammatory cytokines TNF-α, IL-6 and KC in the mouse lung tissues were evaluated by ELISA.Compared with the WT air group, the lung weight, lung tumor multiplicity, as well as maximum tumor size in the WT mice exposed to CS were (1.5 ± 0.1)g, (64.8 ± 4.1) and (7.6 ± 0.2) mm, respectively, significantly increased than those in the WT mice not exposed to CS (P0.05 for all). However, there were no statistically significant differences between RelA/p65⁻/⁻ mice before and after CS exposure (P0.05 for all). Kaplan-Meier survival analysis showed that CS exposure significantly shortened the life time of WT mice (P0.05), and deletion of RelA/p65 in myeloid cells resulted in an increased survival compared with that of the WT mice (P0.05 for all). The ratios of Ki-67 positive tumor cells were (43.4 ± 2.9)%, (60.6 ± 5.4)%, (12.8 ± 3.6)% and (15.0 ± 4.2)% in the WT air group, WT CS groups, RelA/p65⁻/⁻ air groups and RelA/p65⁻/⁻ CS groups, respectively. After smoking, the number of Ki-67-positive cells was significantly increased in the WT mice (P0.05). However, there was no significant difference between the RelA/p65⁻/⁻ groups before and after smoking (P0.05). The apoptosis rate of WT air, WT CS, RelA/p65⁻/⁻ air and RelA/p65⁻/⁻ CS groups were (11.6 ± 1.7)%, (13.0 ± 2.0)%, (13.2 ± 2.0)% and (11.0 ± 1.4)%, respectively, with no significant difference among them (P0.05). Expression of cyclin D1 and c-myc was induced in response to CS exposure in lung tumor cells of WT mice. In contrast, their expressions were not significantly changed in the RelA/p65⁻/⁻ mice after smoke exposure. CS exposure was associated with an increased number of macrophages infiltrating in the tumor tissue, in both WT and RelA/p65⁻/⁻ mice (P0.05). The concentrations of IL-6, KC and TNF-α were significantly increased after CS exposure in the lungs of WT mice (P0.05).Cigarette smoking promotes the lung cancer growth in mice. Myeloid cell RelA/p65 mediates CS-induced tumor growth. TNFα regulated by RelA/p65 may be involved in the lung cancer development.

We have previously reported that ginkgolides containing ginkgolides A and B (GKAB) reduce infarct size in a rat model of focal ischemia. c-Jun N-terminal kinase (JNK), also known as stress-activated kinase (SAPK), is a critical stress-responsive kinase activated by various brain insults. Previous studies have demonstrated a brief increase in p-SAPK/JNK levels after focal ischemic brain injuries. In this study, we sought to investigate whether the neuroprotective effects of GKAB in rat models of permanent focal cerebral ischemia are associated with the JNK signaling pathway. Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion by intraluminal suture blockade. GKAB was injected intravenously immediately after ischemia onset. Here we demonstrate in rats that GKAB reduces neuronal apoptosis and blocks the increase of p-SAPK/JNK levels and nuclear translocation after cerebral ischemia in a dose-dependent manner. Furthermore, we report that cerebral ischemia increases ischemia-induced induction of reactive oxygen species, and this effect was blocked by GKAB. In addition, we show that BimL is induced and attenuated by GKAB. GKAB also repressed the ischemia-induced increase in the expression of Bax and reversed the decline in expression of Bcl-2. Likewise, there was a reduction in the release or activation of several mitochondrial proapoptotic molecules, including cytochrome c, caspases 3 and 9, and PARP. Taken together, our findings strongly suggest that GKAB-mediated neuroprotective effects against focal ischemia act through the inhibition of p-SAPK/JNK activation, in which the obstruction of the mitochondrial apoptotic pathway via the JNK signaling pathway is a key downstream mechanism of GKAB.

Tumor-associated macrophages have been shown to promote tumor growth. They may have an obligatory function in angiogenesis, invasion, and metastasis through release of inflammatory mediators. Their presence in ovarian cancer has been correlated with poor prognosis in these patients. The human cationic antimicrobial protein-18 (hCAP18)/LL-37 was originally identified as an effector molecule of the innate immune system. It is released by innate immune cells, such as macrophages, to combat microorganisms. Previous studies have characterized the hCAP18/LL-37 as a growth factor that has been shown to promote ovarian tumor progression. However, the role hCAP18/LL-37 has in macrophage-promoted ovarian tumor development and how its expression is controlled in this context remains poorly understood. Here, we demonstrate in co-culture experiments of macrophages and ovarian cancer cells a significant increase in the in vitro proliferation and invasiveness of the tumor cells is observed. These enhanced growth and invasion properties correlated with hCAP18/LL-37 induction. HCAP18/LL-37 expression was diminished by addition of two neutralizing antibodies, TLR2 or TLR6, as well as Cyp27B1 or VDR inhibitors. Furthermore, either the TLR2 or TLR6 antibody reduced vitamin D3 signaling and tumor cell progression in vitro. Addition of Cyp27B1 or VDR inhibitors abrogated TLR2/6 activation-induced expression of hCAP18/LL-37 in macrophages. Knockdown of tumor-produced versican V1 by RNAi in these tumor cells led to a decreased induction of hCAP18/LL-37 in macrophages. Versican V1 knockdown also inhibited TLR2 and vitamin D3 signaling, as well as growth and invasiveness of these tumor cells in the in vitro co-culture. In summary, we have found that versican V1 enhances hCAP18/LL-37 expression in macrophages through activation of TLR2 and subsequent vitamin D-dependent mechanisms which promote ovarian tumor progression in vitro.

CDA-2 (cell differentiation agent 2), a urinary preparation, has potent anti- proliferative and pro-apoptotic properties in cancer cells. However, the mechanisms of tumor inhibitory action of CDA-2 are far from clear, and especially there was no report on lung cancer. Here we demonstrate that CDA-2 and its main component phenylacetylglutamine (PG) reduce the metastatic lung tumor growth, and increases survival time after inoculation with Lewis lung carcinoma (LLC) cells in a dose-dependent manner in C57BL6 mice. Proliferative program analysis in cancer cells revealed a fundamental impact of CDA-2 and PG on proliferation and apoptosis, including Bcl-2, Bcl-XL, cIAP1, Survivin, PCNA, Ki-67 proteins and TUNEL assays. CDA-2 and PG significantly reduced NF-κB DNA-binding activity in lung cancer cells and in alveolar macrophages of tumor bearing mice and especially decreased the release of inflammatory factors including TNFα, IL-6, and KC. Furthermore, CDA-2 and PG decrease the expressions of TLR2, TLR6, and CD14, but not TLR1, TLR3, TLR4, and TLR9 in bone-marrow-derived macrophages (BMDM) of mice stimulated by LLC-conditioned medium (LLC-CM). Over-expressing TLR2 in BMDM prevented CDA-2 and PG from inhibiting NF-κB activation, as well as induction of TNFα and IL-6. TLR2:TLR6 complexes mediate the effect of NF-κB inactivation by CDA-2. In conclusion, CDA-2 potently inhibits lung tumor development by reduction of the inflammation in lung through suppression of NF-κB activation in myeloid cells, associating with modulation of TLR2 signaling.