Identification and validation of the surface proteins FIBG, PDGF-ß, and TGF-ß on serum extracellular vesicles for non-invasive detection of colorectal cancer: experimental study.

OBJECTIVES: The absence of non-invasive biomarkers for the early diagnosis of colorectal cancer (CRC) has contributed to poor prognosis. Extracellular vesicles (EVs) have emerged as promising candidates for cancer monitoring using liquid biopsy. However, the complexity of EVs isolation procedures and absence of clear targets for detecting serum-derived EVs have hindered the clinical application of EVs in early CRC diagnosis. METHODS: In the discovery phase, we conducted a comprehensive 4D-DIA proteomic analysis of serum-derived EVs samples from 37 individuals, performing an initial screening of EVs surface proteins. In the technical validation phase, we developed an extraction-free CRC-EVArray microarray to assess the expression of these potential EVs surface proteins in a multicenter study comprising 404 individuals. In the application phase, we evaluated the diagnostic efficacy of the CRC-EVArray model based on machine-learning algorithms. RESULTS: Through 4D-DIA proteomic analysis, we identified 7 potential EVs surface proteins showing significantly differential expression in CRC patients compared to healthy controls. Utilizing our developed high-throughput CRC-EVArray microarray, we further confirmed the differential expression of 3 EVs surface proteins, FIBG, PDGF-ß and TGF-ß, in a large sample population. Moreover, we established an optimal CRC-EVArray model using the NNET algorithm, demonstrating superior diagnostic efficacy with an AUC of 0.882 in the train set and 0.937 in the test set. Additionally, we predicted the functions and potential origins of these EVs-derived proteins through a series of multi-omics approaches. CONCLUSIONS: Our systematic exploration of surface protein expression profiles on serum-derived EVs has identified FIBG, PDGF-ß, and TGF-ß as novel diagnostic biomarkers for CRC. And the development of CRC-EVArray diagnostic model based on these findings provided an effective tool for the large-scale CRC screening, thus facilitating its translation into clinical practice.

Extracellular vesicles-derived CXCL4 is a candidate serum tumor biomarker for colorectal cancer.

Extracellular vesicles (EVs) were promising circulating biomarkers for multiple diseases, but whether serum EVs-derived proteins could be used as a reliable tumor biomarker for colorectal cancer (CRC) remained inconclusive. In this study, we identified CXCL4 by a 4D data-independent acquisition-based quantitative proteomics assay of serum EVs-derived proteins in 40 individuals and subsequently analyzed serum EVs-derived CXCL4 levels by ELISA in 2 cohorts of 749 individuals. The results revealed that EVs-derived CXCL4 levels were dramatically elevated in CRC patients than in benign colorectal polyp patients or healthy controls (HC). Furthermore, receiver operating characteristic curves revealed that EVs-derived CXCL4 exhibited superior diagnostic performance with area under the curve of 0.948 in the training cohort. Additionally, CXCL4 could effectively distinguish CRC in stage I/II from HC. Notably, CRC patients with high levels of EVs-derived CXCL4 have shorter 2-year progression-free survival than those with low levels. Overall, our findings demonstrated that serum EVs-derived CXCL4 was a candidate diagnostic and prognostic biomarker for CRC.

Diagnostic value of aberrant decreased 5-Methylcytosine RNA modification in leukocytes for non-small cell lung cancer.

Background: Non-small cell lung cancer (NSCLC) was a disease with poor outcomes, partly because there were no high-efficiency non-invasive diagnostic biomarkers. The RNA modification status of 5-Methylcytosine (m5C) has been shown to be a biomarker for various diseases, but its potentiality to be a diagnostic biomarker for NSCLC remained inconclusive. Methods: In this research, we collected peripheral leukocyte samples from 141 patients with NSCLC and 90 normal people as controls to evaluate the extent of m5C RNA modification. Results: We found that the m5C modification levels in leukocytes of NSCLC patients were decreased dramatically, which were compared to the normal controls, and levels of m5C modification decreased progressively with tumor stage. Importantly, m5C modification exhibited superior diagnostic value compared to carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), cytokeratin 19 fragment (Cyfra21-1), and carbohydrate antigen 125 (CA125), which demonstrated area under the curves (AUCs) of 0.912, 0.773, 0.669, 0.754, and 0.732, respectively. The combination of m5C modification with these serum tumor biomarkers further improved the AUC to 0.960. A nomogram model incorporating m5C modification also provided an effectively diagnostic tool for NSCLC. Conclusion: Collectively, our findings suggested that m5C modification in leukocytes held promise as a prospective biomarker for NSCLC diagnosis.

Inhibition of extranodal NK/T-cell lymphoma by Chiauranib through an AIF-dependent pathway and its synergy with L-asparaginase.

Extranodal NK/T-cell lymphoma (NKTL) is a rare and aggressive form of extranodal lymphoma with a poor prognosis. Currently, there are very limited treatment options for patients with advanced-stage disease or those with relapsed/recurrent disease. Here we show that Chiauranib, an orally small molecule inhibitor of select serine-threonine kinases (aurora B, VEGFRs, PDGFR, CSF1R, c-Kit), inhibited NKTL cell proliferation, induced cell cycle arrest, as well as suppressed the microvessel density in vitro and in vivo similar as in other types of cancer cells. Surprisingly, Chiauranib unfolded a new effect to induce apoptosis of NKTL cells by triggering AIF-dependent apoptosis other than the traditional cyt-c/caspase mitochondrial apoptosis pathway. The knockdown of AIF in vitro and in vivo dramatically blocked the efficacy of Chiauranib on NKTL. Mechanistically, the release of AIF from mitochondria is due to the upregulation of VDAC1 by the AKT-GSK3ß pathway and activation of calcium-dependent m-calpain, which promotes the cleavage of VDAC1 and therefore permits the release of AIF. Notably, the low expression of Bax in both NKTL cells and patient tissues restrained the cyt-c release. It resulted in the inhibition of cyt-c/caspase mitochondrial pathway, suggesting that drugs targeting this traditional pathway may not be effective in NKTL. Furthermore, we found that L-asparaginase triggered CD95 (Fas/Apo-1)-caspase 8-caspase 3 apoptotic pathway in NKTL cells, and combination of Chiauranib and L-asparaginase exhibited a synergistic effect, suggesting a feasibility to combine these two drugs for effective treatment of NKTL. This study demonstrates Chiauranib's positive efficacy toward NKTL through the activation of the AIF-dependent apoptosis pathway for the first time. The novel and multi-targets of Chiauranib and the synergistic effect with L-asparaginase may provide a promising therapy for NKTL patients.

Increased levels of N6-methyladenosine in peripheral blood RNA: a perspective diagnostic biomarker and therapeutic target for non-small cell lung cancer.

OBJECTIVES: Due to lack of effective biomarkers for non-small cell lung cancer (NSCLC), many patients are diagnosed at an advanced stage, which leads to poor prognosis. Dysregulation of N6-methyladenosine (m6A) RNA contributes significantly to tumorigenesis and tumor progression. However, the diagnostic value of m6A RNA status in peripheral blood to screen NSCLC remains unclear. METHODS: Peripheral blood samples from 152 NSCLC patients and 64 normal controls (NCs) were applied to assess the m6A RNA levels. Bioinformatics and qRT-PCR analysis were performed to identify the specific immune cells in peripheral blood cells and investigate the mechanism of the alteration of m6A RNA levels. RESULTS: Robust elevation of m6A RNA levels of peripheral blood cells was exhibited in the NSCLC group. Moreover, the m6A levels increased as NSCLC progressed, and reduced after treatment. The m6A levels contained area under the curve (AUC) was 0.912, which was remarkably greater than the AUCs for CEA (0.740), CA125 (0.743), SCC (0.654), and Cyfra21-1 (0.730). Furthermore, the combination of these traditional biomarkers with m6A levels elevated the AUC to 0.970. Further analysis established that the expression of m6A erasers FTO and ALKBH5 were both markedly reduced and negatively correlated with m6A levels in peripheral blood of NSCLC. Additionally, GEO database and flow cytometry analysis implied that FTO and ALKBH5 attributes to peripheral CD4+ T cells proportion and activated the immune functions of T cells. CONCLUSIONS: These findings unraveled that m6A RNA of peripheral blood immune cells was a prospective biomarker for the diagnosis of NSCLC.

Personalized immune subtypes based on machine learning predict response to checkpoint blockade in gastric cancer.

Immune checkpoint inhibitors (ICI) show high efficiency in a small fraction of advanced gastric cancer (GC). However, personalized immune subtypes have not been developed for the prediction of ICI efficiency in GC. Herein, we identified Pan-Immune Activation Module (PIAM), a curated gene expression profile (GEP) representing the co-infiltration of multiple immune cell types in tumor microenvironment of GC, which was associated with high expression of immunosuppressive molecules such as PD-1 and CTLA-4. We also identified Pan-Immune Dysfunction Genes (PIDG), a conservative PIAM-derivated GEP indicating the dysfunction of immune cell cooperation, which was associated with upregulation of metastatic programs (extracellular matrix receptor interaction, TGF-ß signaling, epithelial-mesenchymal transition and calcium signaling) but downregulation of proliferative signalings (MYC targets, E2F targets, mTORC1 signaling, and DNA replication and repair). Moreover, we developed 'GSClassifier', an ensemble toolkit based on top scoring pairs and extreme gradient boosting, for population-based modeling and personalized identification of GEP subtypes. With PIAM and PIDG, we developed four Pan-immune Activation and Dysfunction (PAD) subtypes and a GSClassifier model 'PAD for individual' with high accuracy in predicting response to pembrolizumab (anti-PD-1) in advance GC (AUC = 0.833). Intriguingly, PAD-II (PIAMhighPIDGlow) displayed the highest objective response rate (60.0%) compared with other subtypes (PAD-I, PIAMhighPIDGhigh, 0%; PAD-III, PIAMlowPIDGhigh, 0%; PAD-IV, PIAMlowPIDGlow, 17.6%; P = 0.003), which was further validated in the metastatic urothelial cancer cohort treated with atezolizumab (anti-PD-L1) (P = 0.018). In all, we provided 'GSClassifier' as a refined computational framework for GEP-based stratification and PAD subtypes as a promising strategy for exploring ICI responders in GC. Metastatic pathways could be potential targets for GC patients with high immune infiltration but resistance to ICI therapy.

5-Methylcytosine (m5C) modification in peripheral blood immune cells is a novel non-invasive biomarker for colorectal cancer diagnosis.

Current non-invasive tumor biomarkers failed to accurately identify patients with colorectal cancer (CRC), delaying CRC diagnosis and thus leading to poor prognosis. Dysregulation of 5-Methylcytosine (m5C) RNA has gradually been reported in various cancers, but their role in tumor diagnosis is rarely mentioned. Our study aimed to determine the role of m5C methylation modification in blood immune cells for the diagnosis of CRC. Peripheral blood samples were obtained from a total of 83 healthy controls and 196 CRC patients. We observed that m5C RNA contents in blood immune cells of CRC patients were markedly enhanced in both training set and validation set. Moreover, levels of m5C increased with CRC progression and metastasis but reduced after treatment. Compared with common blood tumor biomarkers, m5C levels in peripheral blood immune cells had superior discrimination and reclassification performance in diagnosing CRC. Besides, bioinformatics and qRT-PCR analysis identified increased expression of m5C-modified regulators NSUN5 and YBX1 in CRC patients' blood. A series of animal models and cell co-culture models further demonstrated that CRC tumor cells could increase immune cells' m5C levels and m5C-modified regulators. Monocyte was the predominant m5C-modified immune cell type in CRC patients' blood by Gene set variation analysis (GSVA). Taken together, m5C methylation modification in peripheral blood immune cells was a promising biomarker for non-invasive diagnosis of CRC.

Elevated N6-Methyladenosine RNA Levels in Peripheral Blood Immune Cells: A Novel Predictive Biomarker and Therapeutic Target for Colorectal Cancer.

Effective biomarkers for the diagnosis of colorectal cancer (CRC) are essential for improving prognosis. Imbalance in regulation of N6-methyladenosine (m6A) RNA has been associated with a variety of cancers. However, whether the m6A RNA levels of peripheral blood can serve as a diagnostic biomarker for CRC is still unclear. In this research, we found that the m6A RNA levels of peripheral blood immune cells were apparently elevated in the CRC group compared with those in the normal controls (NCs) group. Furthermore, the m6A levels arose as CRC progressed and metastasized, while these levels decreased after treatment. The area under the curve (AUC) of the m6A levels was 0.946, which was significantly higher than the AUCs for carcinoembryonic antigen (CEA; 0.817), carbohydrate antigen 125 (CA125; 0.732), and carbohydrate antigen 19-9 (CA19-9; 0.771). Moreover, the combination of CEA, CA125, and CA19-9 with m6A levels improved the AUC to 0.977. Bioinformatics and qRT-PCR analysis further confirmed that the expression of m6A modifying regulator IGF2BP2 was markedly elevated in peripheral blood of CRC patients. Gene set variation analysis (GSVA) implied that monocyte was the most abundant m6A-modified immune cell type in CRC patients' peripheral blood. Additionally, m6A modifications were negatively related to the immune response of monocytes. In conclusion, our results revealed that m6A RNA of peripheral blood immune cells was a prospective non-invasive diagnostic biomarker for CRC patients and might provide a valuable therapeutic target.

FUBP1 promotes colorectal cancer stemness and metastasis via DVL1-mediated activation of Wnt/ß-catenin signaling.

Distant metastasis is, unfortunately, the leading cause of death in colorectal cancer (CRC). Approximately 50% of CRC patients develop liver metastases, while 10-30% of patients develop pulmonary metastases. The occurrence of metastasis is considered to be almost exclusively driven by cancer stem cells (CSCs) formation. However, the key molecules that confer the transformation to stem cells in CRC, and subsequent metastasis, remain unclear. Far upstream element-binding protein 1 (FUBP1), a transcriptional regulator of c-Myc, was screened in CSCs of CRC by mass spectrometry and was examined by immunohistochemistry in a cohort of CRC tissues. FUBP1 was upregulated in 85% of KRAS-mutant and 25% of wild-type CRC patients. Further, whether in KRAS-mutant or wild-type patients, elevated FUBP1 was positively correlated with CRC lymph node metastasis and clinical stage, and negatively associated with overall survival. Overexpression of FUBP1 significantly enhanced CRC cell migration, invasion, tumor sphere formation, and CD133 and ALDH1 expression in vitro, and tumorigenicity in vivo. Mechanistically, FUBP1 promoted the initiation of CSCs by activating Wnt/ß-catenin signaling via directly binding to the promoter of DVL1, a potent activator of ß-catenin. Knockdown of DVL1 significantly inhibited the transformation to stem cells in, as well as the tumorigenicity of, CRC. Activation of Wnt/ß-catenin signaling by DVL1 increased pluripotent transcription factors, including c-Myc, NANOG, and SOX2. Moreover, FUBP1 was upregulated at the post-transcriptional level. Elevated FUBP1 levels in KRAS wild-type CRC patients is due to the decrease in Smurf2, which promotes ubiquitin-mediated degradation of FUBP1. In contrast, FUBP1 was upregulated in KRAS-mutant patients through both inhibition of caspase 3-dependent cleavage and decreased Smurf2. Our results demonstrate, for the first time, that FUBP1 is an oncogene, initiating the development of CSCs, as well as a new powerful endogenous Wnt-signaling agonist that could provide an important prognostic factor and therapeutic target for metastasis in both KRAS-mutant and wild-type CRC.

PEDF inhibits lymphatic metastasis of nasopharyngeal carcinoma as a new lymphangiogenesis inhibitor.

Nasopharyngeal carcinoma (NPC) is one of the most malignant tumors in southern China and Asia, and lymph node metastasis is an important cause for treatment failure. Lymphangiogenesis is a crucial step in lymphatic metastasis of NPC, while little is known about lymphangiogenesis in NPC. Similar to angiogenesis, lymphangitic neovascularization is a process of balance between pro-lymphangiogenesis and anti-lymphangiogenesis factors, but there are few studies on endogenous lymphangiogenesis inhibitors. Pigment epithelium-derived factor (PEDF) is a well-known effective endogenous angiogenesis inhibitor. However, the relationship between PEDF and lymphangiogenesis remains unknown. Our present study reveals that PEDF is lowly expressed in human NPC tissues with poor prognosis and is negatively correlated with lymphatic vessel density (LVD). Consistently, PEDF inhibits lymphangiogenesis and lymphatic metastasis of NPC in vivo experiments. Mechanistically, PEDF inhibits the proliferation, migration, and tube formation of lymphatic endothelial cells and promotes cell apoptosis. On the other hand, PEDF reduces the expression and secretion of vascular endothelial growth factor C (VEGF-C) of NPC cells through the nuclear factor-κB (NF-κB) signaling pathway. Our findings indicate that PEDF plays a vital role in lymphatic metastasis by targeting both lymphatic endothelial cells and NPC cells, and PEDF may represent a novel therapeutic target for NPC.

Adipose triglyceride lipase promotes the proliferation of colorectal cancer cells via enhancing the lipolytic pathway.

Abnormal lipid metabolism is the sign of tumour cells. Previous researches have revealed that the lipolytic pathway may contribute to the progression of colorectal cancer (CRC). However, adipose triglyceride lipase (ATGL) role in CRC cells remains unclear. Here, we find that elevated ATGL positively correlates with CRC clinical stages and negatively associates with overall survival. Overexpression of ATGL significantly promotes CRC cell proliferation, while knockdown of ATGL inhibits the proliferation and promotes the apoptosis of CRC cells in vitro. Moreover, in vivo experiments, ATGL promotes the growth of CRC cells. Mechanistically, ATGL enhances the carcinogenic function of CRC cells via promoting sphingolipid metabolism and CoA biosynthesis pathway-related gene levels by degrading triglycerides, which provides adequate nutrition for the progression of CRC. Our researches clarify for the first time that ATGL is a novel oncogene in CRC and may provide an important prognostic factor and therapeutic target for CRC.

Overexpression of FUBP1 is associated with human cervical carcinoma development and prognosis.

AIMS: Far upstream element-binding protein 1 (FUBP1) has been shown to involve in the tumorigenesis and tumor progression of various cancers. However, the expression and function of FUBP1 in cervical carcinoma remains unknown. MAIN METHODS: Transcriptional expression of FUBP1 was initially evaluated using the Oncomine database, followed by evaluation of FUBP1 protein levels using immunohistochemistry in 119 cervical carcinoma patient tissues. In vitro experiments were performed to assess the tumorigenic role of FUBP1. Besides, Gene Set Enrichment Analysis, EnrichmentMap analysis, and protein-protein interaction (PPI) networks were used to evaluate the potential mechanisms of FUBP1 in promoting cervical cancer progression. KEY FUNDINGS: In this research, we found both FUBP1 mRNA transcription and protein expression levels increased significantly in cervical carcinoma tissues compared with adjacent normal cervical tissues. Furthermore, elevated FUBP1 expression was positively correlated with age, T classification, N classification, tumor recurrence, Ki67 expression, and poor prognosis in cervical carcinoma patients. Besides, elevated FUBP1 expression acted as an independent unfavorable predictor for overall survival and disease-free survival in cervical carcinoma. Overexpression of FUBP1 significantly promoted cervical carcinoma cell proliferation and inhibits cell apoptosis in vitro, while knockdown of FUBP1 showed the opposite effect. Mechanistically, bioinformatics analysis revealed that FUBP1 promoted the biological function of cervical carcinoma cells via enhancing DNA repair signal pathways. Our results demonstrate for the first time that FUBP1 is a novel prognostic factor and therapeutic target for cervical carcinoma.

Chiauranib selectively inhibits colorectal cancer with KRAS wild-type by modulation of ROS through activating the p53 signaling pathway.

Colorectal cancer (CRC) is one of the top three most deadly cancers despite using chemotherapy based on oxaliplatin or irinotecan combined with targeted therapy. Chiauranib has recently been identified to be a promising anticancer candidate with impressive efficacy and safety. However, the role and molecular mechanisms of Chiauranib in the treatment of CRC remain to be elucidated. Our study shows that Chiauranib inhibits cell proliferation and induces apoptosis in KRAS wild-type CRC cells in a dose- and time-dependent manner, but not mutation ones. Meanwhile, Chiauranib increases ROS production in KRAS wild-type CRC cells. Moreover, Chiauranib selectively suppresses KRAS wild-type CRC cells growth in vivo. Mechanistically, Chiauranib inhibits KRAS wild-type CRC cells by triggering ROS production via activating the p53 signaling pathway. Further, KRAS mutation CRC cells are resistant to Chiauranib by increasing Nrf2 to stably elevate the basal antioxidant program and thereby lower intracellular ROS induced by Chiauranib. Our findings provide the rationale for further clinical evaluation of Chiauranib as a therapeutic agent in treating KRAS wild-type CRC.

Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1.

Metastatic colorectal cancer (mCRC) patients have poor overall survival despite using irinotecan- or oxaliplatin-based chemotherapy combined with anti-EGFR (epidermal growth factor receptor) drugs, especially those with the oncogene mutation of KRAS Metformin has been reported as a potentially novel antitumor agent in many experiments, but its therapeutic activity is discrepant and controversial so far. Inspiringly, the median survival time for KRAS-mutation mCRC patients with diabetes on metformin is 37.8 mo longer than those treated with other hypoglycemic drugs in combination with standard systemic therapy. In contrast, metformin could not improve the survival of mCRC patients with wild-type KRAS Interestingly, metformin is preferentially accumulated in KRAS-mutation mCRC cells, but not wild-type ones, in both primary cell cultures and patient-derived xenografts, which is in agreement with its tremendous effect in KRAS-mutation mCRC. Mechanistically, the mutated KRAS oncoprotein hypermethylates and silences the expression of multidrug and toxic compound extrusion 1 (MATE1), a specific pump that expels metformin from the tumor cells by up-regulating DNA methyltransferase 1 (DNMT1). Our findings provide evidence that KRAS-mutation mCRC patients benefit from metformin treatment and targeting MATE1 may provide a strategy to improve the anticancer response of metformin.

The contrary intracellular and extracellular functions of PEDF in HCC development.

Pigment epithelium-derived factor (PEDF), a classic angiogenic inhibitor, has been reported to function as a tumor suppression protein and to downregulate in many types of solid tumors. However, the expression level of PEDF and its role in hepatocellular carcinoma (HCC) are contradictory. The present study investigates the expression and different activities of secreted and intracellular PEDF during HCC development, as well as the underlying mechanism of PEDF on HCC lipid disorders. We found that PEDF had no association with patients' prognosis, although PEDF was highly expressed and inhibited angiogenesis in HCC tumor tissues. The animal experiments indicated that full-length PEDF exhibited equalizing effects on tumor growth activation and tumor angiogenesis inhibition in the late stage of HCC progression. Importantly, the pro-tumor activity was mediated by the intracellular PEDF, which causes accumulation of free fatty acids (FFAs) in vivo and in vitro. Based on the correlation analysis of PEDF and lipid metabolic indexes in human HCC tissues, we demonstrated that the intracellular PEDF led to the accumulation of FFA and eventually promoted HCC cell growth by inhibiting the activation of AMPK via ubiquitin-proteasome-mediated degradation, which causes increased de novo fatty acid synthesis and decreased FFA oxidation. Our findings revealed why elevated PEDF did not improve the patients' prognosis as the offsetting intracellular and extracellular activities. This study will lead to a comprehensive understanding of the diverse role of PEDF in HCC and provide a new selective strategy by supplement of extracellular PEDF and downregulation of intracellular PEDF for the prevention and treatment of liver cancer.

OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGF­C expression and secretion.

Gastric cancer is one of the most malignant tumor types, and its metastasis is a notable cause of mortality. Among the methods of tumor metastasis, lymphatic metastasis is the predominant one in gastric cancer. A previous study reported that the plasma oxidized low­density lipoprotein (oxLDL) is the risk factor associated with the development of tumors in patients with abnormal lipid metabolism, but the influence of plasma oxLDL in the lymphatic metastasis of gastric cancer remains unclear. In the present study, the concentration of plasma oxLDL from patients with gastric cancer was detected with an ELISA kit, and the lymphatic vessel density in gastric cancer tissues was determined by D2­40 staining. The correlation analysis of oxLDL concentration and lymphatic vessel density demonstrated that plasma oxLDL was positively correlated with lymphatic metastasis in patients with gastric cancer. Subsequently, the popliteal lymph node metastasis animal experiment with nude mice confirmed that oxLDL could promote the lymphatic metastasis of gastric cancer. Following this, the western blotting and ELISA data demonstrated that oxLDL promoted the expression and secretion of vascular endothelia growth factor (VEGF)­C in gastric cancer cell lines. Finally, blocking the lectin­like oxLDL­1 (LOX­1) receptor, a specific receptor for oxLDL, and the nuclear factor (NF)­κB signaling pathway following oxLDL (50 µg/ml) treatment in HGC­27 cells revealed that oxLDL could activate the NF­κB signaling pathway mediated by LOX­1, with subsequent upregulation of VEGF­C expression, and secretion in and from gastric cancer cells, and finally that it could promote the lymphatic metastasis of gastric cancer. These data indicate the association between the plasma oxLDL and the lymphatic metastasis of gastric cancer, and indicate that oxLDL elimination may be a potential therapeutic target for the prevention and intervention of early lymph node metastasis in gastric cancer.

Pigment epithelium-derived factor/vascular endothelial growth factor ratio plays a crucial role in the spontaneous regression of infant hemangioma and in the therapeutic effect of propranolol.

Infantile hemangioma (IH) is a benign tumor that is formed by aberrant angiogenesis and that undergoes spontaneous regression over time. Propranolol, the first-line therapy for IH, inhibits angiogenesis by downregulating activation of the vascular endothelial growth factor (VEGF) pathway, which is hyperactivated in IH. However, this treatment is reportedly ineffective for 10% of tumors, and 19% of patients relapse after propranolol treatment. Both pro-angiogenic and anti-angiogenic factors regulate angiogenesis, and pigment epithelium-derived factor (PEDF) is the most effective endogenous anti-angiogenic factor. PEDF/VEGF ratio controls many angiogenic processes, but its role in IH and the relationship between this ratio and propranolol remain unknown. Results of the present study showed that the PEDF/VEGF ratio increased during the involuting phase of IH compared with the proliferating phase. Similarly, in hemangioma-derived endothelial cells (HemEC), which were isolated with magnetic beads, increasing the PEDF/VEGF ratio inhibited proliferation, migration, and tube formation and promoted apoptosis. Mechanistically, the VEGF receptors (VEGFR1 and VEGFR2) and PEDF receptor (laminin receptor, LR) were highly expressed in both IH tissues and HemEC, and PEDF inhibited HemEC function by binding to LR. Interestingly, we found that propranolol increased the PEDF/VEGF ratio but did so by lowering VEGF expression rather than by upregulating PEDF as expected. Furthermore, the combination of PEDF and propranolol had a more suppressive effect on HemEC. Consequently, our results suggested that the PEDF/VEGF ratio played a pivotal role in the spontaneous regression of IH and that the combination of PEDF and propranolol might be a promising treatment strategy for propranolol-resistant IH.

Kallistatin inhibits lymphangiogenesis and lymphatic metastasis of gastric cancer by downregulating VEGF-C expression and secretion.

BACKGROUND: Tumor-induced lymphangiogenesis and lymphatic metastasis are predominant during the metastasis of many types of cancers. However, the endogenous inhibitors that counterbalance the lymphangiogenesis and lymphatic metastasis of tumors have not been well evaluated. Kallistatin has been recognized as an endogenous angiogenesis inhibitor. METHODS AND RESULTS: Our recent study showed for the first time that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Kallistatin expresses anti-lymphangiogenic activity by inhibiting the proliferation, migration, and tube formation of human lymphatic endothelial cells (hLECs). Therefore, the present study focuses on the relationships of changes in kallistatin expression with the lymphangiogenesis and lymphatic metastasis of gastric cancer and its underlying mechanisms. Our results revealed that the expression of kallistatin in cancer tissues, metastatic lymph nodes, and plasma of gastric cancer patients was significantly downregulated and that the plasma level of kallistatin was negatively associated with the phase of lymph node metastasis. Furthermore, treatment with kallistatin recombinant protein decreased LVD and lymph node metastases in the implanted gastric xenograft tumors of nude mice. Mechanically, kallistatin suppressed the lymphangiogenesis and lymphatic metastasis by downregulating VEGF-C expression and secretion through the LRP6/IKK/IÒ¡B/NF-Ò¡B signaling pathway in gastric cancer cells. CONCLUSIONS: These findings demonstrated that kallistatin functions as an endogenous lymphangiogenesis inhibitor and has an important part in the lymphatic metastasis of gastric cancer.

Kallistatin exerts anti-lymphangiogenic effects by inhibiting lymphatic endothelial cell proliferation, migration and tube formation.

Kallistatin has been recognized as an endogenous angiogenic inhibitor. However, its effects on lymphatic endothelial cells and lymphangiogenesis remain poorly understood. Lymphangiogenesis is involved in tumor metastasis via the lymphatic vasculature in various types of tumors. The aim of this study was to investigate the effects of kallistatin on lymphangiogenesis and the mechanism of action involved. Treatment with kallistatin recombinant protein or overexpression of kallistatin inhibited the proliferation, migration and tube formation of human lymphatic endothelial cells (hLECs), and induced apoptosis of hLECs. Furthermore, our results showed that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Treatment with kallistatin recombinant protein decreased the LVD in the implanted gastric xenograft tumors of nude mice. To the best of our knowledge, the present study is the first to demonstrate that kallistatin possesses anti-lymphangiogenic activity in vitro and in vivo. Moreover, kallistatin inhibited proliferation and migration of hLECs by reducing the phosphorylation of ERK and Akt, respectively. These findings suggested that kallistatin may be a promising agent that could be used to suppress cancer metastasis by inhibiting both angiogenesis and lymphangiogenesis.