Claudin-1/4 as directly target gene of HIF-1α can feedback regulating HIF-1α by PI3K-AKT-mTOR and impact the proliferation of esophageal squamous cell though Rho GTPase and p-JNK pathway.

Immunohistochemical microarray comprising 80 patients with esophageal squamous cell carcinoma (ESCC) and discovered that the expression of CLDN1 and CLDN4 were significantly higher in cancer tissues compared to para-cancerous tissues. Furthermore, CLDN4 significantly affected the overall survival of cancer patients. When two ESCC cell lines (TE1, KYSE410) were exposed to hypoxia (0.1% O2), CLDN1/4 was shown to influence the occurrence and development of esophageal cancer. Compared with the control culture group, the cancer cells cultured under hypoxic conditions exhibited obvious changes in CLDN1 and CLDN4 expression at both the mRNA and protein levels. Through genetic intervention and Chip, we found that HIF-1α could directly regulate the expression of CLDN1 and CLDN4 in cancer cells. Hypoxia can affect the proliferation and apoptosis of cancer cells by regulating the PI3K-Akt-mTOR pathway. Molecular analysis further revealed that CLDN1 and CLDN4 can participate in the regulation process and had a feedback regulatory effect on HIF-1α expression in cancer cells. In vitro cellular experiments and vivo experiments in nude mice further revealed that changes in CLDN4 expression in cancer cells could affect the proliferation of cancer cells via regulation of Rho GTP and p-JNK pathway. Whether CLDN4 can be target for the treatment of ESCC needs further research.

Application of the CRISPR/Cas9-based gene editing technique in basic research, diagnosis, and therapy of cancer.

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for the development of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease9 (CRISPR/Cas9) gene editing technology that provided new tools for precise gene editing. It is possible to target any genomic locus virtually using only a complex nuclease protein with short RNA as a site-specific endonuclease. Since cancer is caused by genomic changes in tumor cells, CRISPR/Cas9 can be used in the field of cancer research to edit genomes for exploration of the mechanisms of tumorigenesis and development. In recent years, the CRISPR/Cas9 system has been increasingly used in cancer research and treatment and remarkable results have been achieved. In this review, we introduced the mechanism and development of the CRISPR/Cas9-based gene editing system. Furthermore, we summarized current applications of this technique for basic research, diagnosis and therapy of cancer. Moreover, the potential applications of CRISPR/Cas9 in new emerging hotspots of oncology research were discussed, and the challenges and future directions were highlighted.

Afatinib induces pro-survival autophagy and increases sensitivity to apoptosis in stem-like HNSCC cells.

Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its antitumor effects in head and neck squamous cell carcinoma (HNSCC) by inducing intrinsic apoptosis through suppression of mTORC1. However, the detailed mechanism and biological significance of afatinib-induced autophagy in HNSCC remains unclear. In the present study, we demonstrated that afatinib induced mTORC1 suppression-mediated autophagy in HNSCC cells. Further mechanistic investigation revealed that afatinib stimulated REDD1-TSC1 signaling, giving rise to mTORC1 inactivation and subsequent autophagy. Moreover, ROS generation elicited by afatinib was responsible for the induction of the REDD1-TSC1-mTORC1 axis. In addition, pharmacological or genetic inhibition of autophagy sensitized HNSCC cells to afatinib-induced apoptosis, demonstrating that afatinib activated pro-survival autophagy in HNSCC cells. Importantly, in vitro and in vivo assays showed that afatinib caused enhanced apoptosis but weaker autophagy in stem-like HNSCC cells constructed by CDH1 knockdown. This suggested that blocking autophagy has the potential to serve as a promising strategy to target HNSCC stem cells. In conclusion, our findings suggested that the combination treatment with afatinib and autophagy inhibitors has the potential to eradicate HNSCC cells, especially cancer stem cells in clinical therapy.

MTDH in macrophages promotes the vasculogenic mimicry via VEGFA-165/Flt-1 signaling pathway in head and neck squamous cell carcinoma.

Vasculogenic mimicry (VM) refers to vessel-like structures formed by aggressive tumor cells and is closely associated with cancer invasion and metastasis. Here, we investigated the effect of macrophage-derived MTDH on VM formation in head and neck squamous cell carcinoma (HNSCC) and its underlying mechanism. Macrophages with MTDH overexpression (Mac-MTDH) promoted cancer cell VM formation, migration, and invasion in vitro. Moreover, MTDH overexpression triggered macrophage polarization into M2 type tumor-associated macrophages. Analysis of HNSCC clinical samples revealed that MTDH+ macrophages were predominantly located in the tumor-stromal region in proximity to VM and correlated with lymph node metastasis. Mechanistically, Mac-MTDH enhanced the expression and secretion of VEGFA-165 rather than other VEGFA isoforms via ß-catenin. The VEGFA-165/Flt-1 axis was responsible for Mac-MTDH's effects in cancer cells through p-STAT3/Twist1/VE-cadherin pathway. Using mouse model, we further confirmed that Mac-MTDH increased VM formation and cancer metastasis in vivo. Furthermore, in subcutaneous xenograft mouse model, HN6 + Mac-MTDH tumor exhibited elevated expression of p-STAT3 and Twist1 than HN6 + Mac-NC tumors. This study revealed that Mac-MTDH promoted VM formation, cancer cell migration and invasion, and cancer metastasis through VEGFA-165/Flt-1 axis, and that macrophage-derived MTDH could be a potential therapeutic target in HNSCC.

PHF20 inhibition promotes apoptosis and cisplatin chemosensitivity via the OCT4­p­STAT3­MCL1 signaling pathway in hypopharyngeal squamous cell carcinoma.

Cisplatin is a widely used platinum­based chemotherapeutic agent for hypopharyngeal squamous cell carcinoma (HSCC). However, resistance to cisplatin limits its use for the treatment of HSCC, and the underlying molecular mechanism requires further investigation. The present study performed functional assays to determine whether the expression of plant homeodomain finger protein 20 (PHF20) may be involved in the apoptosis and cisplatin resistance of HSCC. The expression levels of PHF20 were higher in cisplatin­resistant HSCC cells compared with those in cisplatin­sensitive cells. The inhibition of PHF20 suppressed cell viability but did not affect the migratory and invasive abilities of HSCC cells compared with those of negative control­transfected cells. Furthermore, PHF20 inhibition reduced cell viability by enhancing apoptosis compared with those in the control cells in vitro. Notably, the inhibition of PHF20 sensitized HSCC cells to cisplatin, thus increasing apoptosis via the signal transducer and activator of transcription 3 (STAT3)­myeloid cell leukemia­1 (MCL1) pathway. Octamer­binding transcription factor 4 (OCT4) overexpression restored phosphorylated STAT3­MCL1­mediated apoptosis induced by PHF20 inhibition. In vivo experiments confirmed that PHF20 silencing induced tumor growth and increased apoptosis in HSCC cells compared with those in the control cells. Thus, PHF20 inhibition may promote apoptosis and improve cisplatin chemosensitivity via the OCT4­p­STAT3­MCL1 signaling pathway in HSCC.

Evaluation of Left Ventricular Diastolic Function by 2-D Speckle Tracking Echocardiography in Patients with Connective Tissue Disease-Associated Pulmonary Artery Hypertension.

The purpose of this study was to evaluate the role of 2-D speckle tracking imaging in assessing left ventricular diastolic function in patients with connective tissue disease (CTD). A total of 98 CTD patients and 32 healthy controls were prospectively recruited. Early (E) and late (A) diastolic velocities of the transmitral flow were measured by pulsed Doppler echocardiography. Peak early diastolic myocardial velocity (E') was calculated on tissue Doppler echocardiography. The longitudinal strain rate (SR) was calculated as the average of three apical views, while circumferential and radial SRs were measured in three short-axis views. Pulmonary arterial hypertension (PAH) was defined as systolic pulmonary arterial pressure (sPAP) >36 mm Hg. Compared with the control group, CTD patients exhibited significant impairment of left ventricular diastolic function, manifested as lower global SR during early diastole (SRe) in the longitudinal deformation and higher E/SRe in both longitudinal and radial deformation. CTD-PAH patients had significantly lower SRe and higher E/SRe values in both the longitudinal and radial deformation compared with the patients with CTD without PAH. Pearson's correlation analysis revealed that sPAP levels correlated positively with E/E', longitudinal E/SRe, circumferential E/SRe and radial SRe, and it correlated negatively with septal E' and radial E/SRe. Receiver operating characteristic curve analysis suggested that E/E', longitudinal E/SRe and radial SRe could be used to predict PAH. The present study indicates that 2-D speckle tracking imaging is a useful method for evaluation of left ventricular diastolic function, and these derived parameters can serve as good predictors of PAH, but it may not be superior to the commonly used E/E' in CTD patients.

Molecular Mechanisms of Cardiac Injury Associated With Myocardial SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread around the world. The development of cardiac injury is a common condition in patients with COVID-19, but the pathogenesis remains unclear. The RNA-Seq dataset (GSE150392) comparing expression profiling of mock human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and SARS-CoV-2-infected hiPSC-CMs was obtained from Gene Expression Omnibus (GEO). We identified 1,554 differentially expressed genes (DEGs) based on GSE150392. Gene set enrichment analysis (GSEA), Gene ontology (GO) analysis, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that immune-inflammatory responses were activated by SARS-CoV-2, while muscle contraction, cellular respiration, and cell cycle of hiPSC-CMs were inhibited. A total of 15 hub genes were identified according to protein-protein interaction (PPI), among which 11 upregulated genes were mainly involved in cytokine activation related to the excessive inflammatory response. Moreover, we identified potential drugs based on these hub genes. In conclusion, SARS-CoV-2 infection of cardiomyocytes caused a strong defensive response, leading to excessive immune inflammation, cell hypoxia, functional contractility reduction, and apoptosis, ultimately resulting in myocardial injury.

The diagnostic value of GDF-15 for myocardial involvement in idiopathic inflammatory myopathy.

BACKGROUND: Cardiac involvement is a serious complication of idiopathic inflammatory myopathy (IIM). GDF-15 can predict the risk and the prognosis of cardiovascular disease, but its value is unclear in IIM. OBJECTIVE: To investigate the diagnostic value of GDF-15 for myocardial involvement in IIM. METHODS: A total of 77 IIM patients from May 2018 to August 2020 were included in this retrospective study. Of these, 43 patients underwent cardiac magnetic resonance (CMR) examination. There were 33 SLE patients and 16 healthy people were used as the control group. The concentration of GDF-15 of these groups was measured by ELISA. RESULTS: There were significant differences in GDF-15 levels in patients with IIM, SLE and healthy controls (H = 45.291, P<0.001). GDF-15 levels were statistically significant different between IIM patients with the myocardial injury [1484.88(809.07 2835.50) pg/ml] and without myocardial injury [593.26(418.61 784.59) pg/ml, P =0.001]. After adjusted for age, renal function, the risk of myocardial injury in IIM patients increased an average of 0.3% by per increased unit of GDF-15 (odds ratio=1.003, 95% CI: 1.000, 1.007). The level of GDF-15 was positively correlated with extra-cellular volume (ECV) (rs = 0.348, P =0.028). GDF-15 ≥ 929.505 pg/ml (area under the curve=0.856, 95% CI: 0.744, 0.968) predicted myocardial injury in IIM with a sensitivity of 0.75 and specificity of 0.90. CONCLUSION: GDF-15 could serve as a potential biomarker to predict myocardial injury in IIM patients.

PDK1 promotes metastasis by inducing epithelial-mesenchymal transition in hypopharyngeal carcinoma via the Notch1 signaling pathway.

Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare malignancy and has the worst prognosis among head and neck cancer. Metastasis is the major cause of poor prognosis in HSCC patients. In this study, we found that 3-phosphoinositide-dependent protein kinase 1 (PDK1 or PDPK1) was overexpressed in HSCC. The overexpression was positively correlated lymph node metastasis, clinical stage, and distant metastasis and indicated poor outcome. Loss and gain-of-function revealed that PDK1 increased cell proliferation, migration and invasion in vitro as well as tumor growth and metastasis in vivo. Mechanically, PDK1 induced epithelial-mesenchymal transition and promoted metastasis by activating the Notch1 signaling pathway. We further illustrated that PDK1 bound with the Notch1 intracellular domain, thereby inhibiting its ubiquitin-mediated degradation in a protein kinase B (Akt-) independent manner. In summary, PDK1/Notch1 axis played an important role in HSCC metastasis, and this investigation provided a new perspective on potential therapeutic targets for HSCC.

Upregulation of fascin-1 is involved in HIF-1α-dependent invasion and migration of hypopharyngeal squamous cell carcinoma.

Fascin­1 is an actin­bundling protein, which specifically interacts with F­actin to form parallel actin bundles, and participates in the regulation of cell adhesion, interactions and migration. However, the expression and regulatory mechanisms of fascin­1 in hypopharyngeal squamous cell carcinoma (HSCC) remain poorly understood. The present study investigated the effects and underlying molecular mechanism of fascin­1 on the invasion and metastasis of HSCC. The results demonstrated that fascin­1 was overexpressed and correlated with lymph node metastasis and tumor­node­metastasis stage in HSCC tissues. Further in vitro study revealed that fascin­1 promoted cell morphology polarization to increase the motility of FaDu cells. In addition, fascin­1 significantly promoted the migration and invasion of FaDu cells. At the molecular level, fascin­1 promoted cell invasion and migration by upregulating matrix metalloproteinase­2 (MMP­2) expression in FaDu cells. Immunohistochemical analysis revealed that a correlation existed between hypoxia inducible factor (HIF)­1α and fascin­1 expression in the HSCC tissues. Furthermore, the results from a cobalt chloride­induced hypoxia model demonstrated that fascin­1 may be upregulated by HIF­1α in FaDu cells. Further analysis revealed that fascin­1 knockdown significantly decreased the invasion of cells under hypoxia and partially reversed hypoxia­induced MMP­2 expression under hypoxia in FaDu cells. In conclusion, fascin­1 was upregulated by HIF­1α, and promoted the invasion and migration of HSCC cells; therefore, fascin­1 may provide a potential target for the treatment of invasion and metastasis in HSCC.

Angio-associated migratory cell protein interacts with epidermal growth factor receptor and enhances proliferation and drug resistance in human non-small cell lung cancer cells.

Angio-associated migratory cell protein (AAMP) is expressed in some human cancer cells. Previous studies have shown AAMP high expression predicted poor prognosis. But its biological role in non-small cell lung cancer (NSCLC) cells is still unknown. In our present study, we attempted to explore the functions of AAMP in NSCLC cells. According to our findings, AAMP knockdown inhibited lung cancer cell proliferation and inhibited lung cancer cell tumorigenesis in the mouse xenograft model. Epidermal growth factor receptor (EGFR) is a primary receptor tyrosine kinase (RTK) that promotes proliferation and plays an important role in cancer pathology. We found AAMP interacted with EGFR and enhanced its dimerization and phosphorylation at tyrosine 1173 which activated ERK1/2 in NSCLC cells. In addition, we showed AAMP conferred the lung cancer cells resistance to chemotherapeutic agents such as icotinib and doxorubicin. Taken together, our data indicate that loss of AAMP from NSCLC inhibits tumor growth and elevates drug sensitivity, and these findings have clinical implications to treat NSCLC cancers.

Loss of CDH1 promotes the metastasis of hypopharyngeal squamous cell carcinoma through the STAT3-MMP-9 signaling pathway.

BACKGROUND: Distant metastasis is the major cause of death in patients with hypopharyngeal squamous cell carcinoma (HSCC). CDH1 is correlated with tumor invasion and metastasis; however, its function in HSCC remains unclear. METHODS: We used immunohistochemistry (IHC) staining to evaluate the expression of CDH1 in 31 and 78 specimens from primary HSCC patients with and without postoperative lung metastases respectively. Sulforhodamine B (SRB) and CCK-8 assays were used to test the proliferation of HSCC cells. Motility of HSCC cells was investigated by migration and invasion assays. Western blot analysis was used to measure the levels of CDH1 and other proteins. RESULTS: We found that the low expression of CDH1 was significantly associated with postoperative lung metastasis in HSCC (P<0.001). Moreover, CDH1 was reduced concomitantly with the upregulation of MMP-9 in the same HSCC sample. Further mechanistic investigation showed that silencing CDH1 elevated the level of MMP-9, which was coupled with the phosphorylation of STAT3. Subsequently, inhibiting STAT3 either by siRNA transfection or by pharmacological suppression with AG490 attenuated MMP-9 upregulation and prevented the enhanced proliferation and invasion caused by CDH1 loss in FaDu cells. CONCLUSIONS: CDH1 plays vital roles in HSCC metastasis and might serve as a potential therapeutic target for the clinical treatment of HSCC.

Are we speaking the same language? an argument for the consistent use of terminology and definitions for childhood vaccination indicators.

Vaccination indicators are used to measure the health status of individuals or populations and to evaluate the effectiveness of vaccination programs or policies. Ensuring that vaccination indicators are clearly and consistently defined is important for effective communication of outcomes, accurate program evaluation, and comparison between different populations, times, and contexts. The purpose of this commentary is to describe commonly used vaccination indicators and to highlight inconsistencies in how childhood vaccine researchers use and define these terms. The indicators we describe are vaccine coverage, uptake, and rate; vaccination status, initiation, and completion; and up-to-date, timely, partial, and incomplete vaccination. We conclude that many vaccination indicators are not explicitly defined within published research studies and/or are used quite differently across studies. We also note that the choice of indicator in a given study is often driven by program or vaccine specific factors, may be constrained by data availability, and should be chosen to best reflect the outcome of interest. We conclude that the use of consistent language and definitions would promote more effective communication of research findings. We also propose some standardized definitions for common indicators, with the goal of provoking discussion and debate on the issue.

Notch1 serves as a prognostic factor and regulates metastasis via regulating EGFR expression in hypopharyngeal squamous cell carcinoma.

OBJECTIVE: Hypopharyngeal squamous cell carcinoma (HSCC) remains one of the most lethal malignancies in head and neck. Notch1 has been validated to play prominent roles in the occurrence and development of various types of cancer. The aim of this study was to explore the function and underlying mechanism of Notch1 in HSCC. PATIENTS AND METHODS: Seventy-one cancer tissue samples and adjacent noncancerous formalin-fixed paraffin embedded tissue specimens were analyzed by immunohistochemistry. As Notch1 is overexpressed in HSCC, we further questioned whether there was a relationship between Notch1 and the clinicopathological characteristics. After confirming the successful knockdown of Notch1 by siRNA, the migration and invasion after gene knockdown were investigated by Transwell chambers. We then tried to identify YBX1 and EGFR expression using real-time PCR (RT-PCR) and Western blot analyses. To further determine whether the downexpression of EGFR was caused by YBX1 and the overexpression of YBX1 was caused by gene amplification, the expression of EGFR was detected by RT-PCR and Western blot assays. RESULTS: We found that the expression of Notch1 and EGFR in HSCC tissues was upregulated compared with those in the adjacent noncancerous tissues. Further clinicopathological characteristics analysis revealed that the expression of Notch1 was positively correlated with distant metastasis (P=0.003) and tumor differentiation (P=0.031). The high expression of Notch1 is an independent prognostic factor for a poor overall survival in patients with HSCC (P=0.015, χ 2=10.403). Knocking down of Notch1 significantly inhibits the migration and invasion of FaDu cells in vitro. Mechanistic investigation reveals that Notch1 knockdown is found suppressing the expression of EGFR at transcriptional level. Interestingly, we further found that Notch1 knockdown also decreased the expression of YBX1, which is a transcription factor of EGFR. Moreover, the upregulation of YBX1 reverses the suppression of Notch1 on EGFR. Furthermore, forced overexpression of YBX1 induced the invasion of FaDu cells. CONCLUSION: Taken together, we found a positively cross-linked role of Notch1 signaling in the outcome of HSCC, providing a novel valuable prognostic marker and potential therapeutic target for the treatment of HSCC patients. Notch1 is a core signaling molecule for regulating migration and invasion via interplaying with EGFR in HSCC cells.

MG­132 reverses multidrug resistance by activating the JNK signaling pathway in FaDu/T cells.

Multidrug resistance (MDR) is a major impediment to cancer therapy. MG­132 has been identified to be effective against MDR in several types of cancer. However, the mechanism of MG­132 in head and neck squamous cell carcinomas remains unknown. Based on our previous study, the present detected P­gp and P­gp expression in hypopharyngeal carcinoma FaDu cells, revealing that their expression was lower than that observed in the MDR cell line FaDu/T. To reverse the MDR of FaDu/T cells, the present study introduced MG­132 and demonstrated that the high expression of P­gp/P­gp in FaDu/T cells was attenuated in a time­dependent manner. MG­132 also strengthened the sensitivity of FaDu/T cells to multidrugs. c­Jun N­terminal kinase (JNK) activation was further observed in FaDu/T cells. However, P­gp/P­gp did not decrease when FaDu/T cells were pretreated with SP600125. These results indicated that MG­132 reversed the MDR of hypopharyngeal carcinoma by downregulating P­gp/P­gp, and the underlying mechanism may be associated with the activation the of the JNK signaling pathway.

Epidemiology of pertussis in Alberta, Canada 2004-2015.

BACKGROUND: We describe the epidemiology of pertussis in Alberta, Canada by person, place, and time between 2004 and 2015, identify outbreak years, and examine vaccination coverage and vaccination timeliness. METHODS: We used health data from Alberta's Communicable Disease Registry System for the period of January 1, 2004 through August 31, 2015 to identify unique cases of pertussis. Unique cases were deterministically linked to data in Alberta's immunization repository and health care insurance plan registry. Population estimates and vaccination coverage were extracted from Alberta's online Interactive Health Data Application. We estimated pertussis incidence rates per 100,000 persons by year, age group, gender, and health zone. Outbreak years were identified using a one-sided cumulative sum (CUSUM) analysis by comparing annual incidence rates to baseline rates. RESULTS: Over the period, 3510 cases of pertussis were confirmed by laboratory testing or epidemiological linkage. Incidence rates per 100,000 persons were highest in 2004 (20.5), 2005 (13.6), and 2015 (10.4) for all age groups. Incidence rates were highest among the youngest age groups and decreased as age groups increased. Based on CUSUM analysis, 2008 and 2012 met the criteria for outbreak years. Vaccination coverage was over 90% among the general population, however only 61% of cases received at least one dose. About 60% of cases were diagnosed 5+ years after receiving the vaccine. Approximately 87-91% of vaccinated cases did not receive the first three vaccine doses in a timely manner. CONCLUSION: Pertussis incidence rates fluctuated over the period across all age groups. The majority of cases had no record of vaccination or were delayed in receiving vaccines. CUSUM analysis was an effective method for identifying outbreaks.

Cordycepin induces autophagy-mediated c-FLIPL degradation and leads to apoptosis in human non-small cell lung cancer cells.

Cordycepin, a main active composition extracted from Cordyceps militaris, has been reported to exert anti-tumor activity in a broad spectrum of cancer types. However, the function of cordycepin on human non-small cell lung cancer cells is still obscure. Our present work showed that cordycepin inhibited cell growth by inducing apoptosis and autophagy in human NSCLC cells. Further study revealed that cordycepin triggered extrinsic apoptosis associated with down-regulation of c-FLIPL which suppresses the activity of caspase-8. And ectopic expression of c-FLIPL dramatically prevented cordycepin-caused apoptosis. Meanwhile, cordycepin stimulated autophagy through suppressing mTOR signaling pathway in lung cancer cells. When autophagy was blocked by Atg5 siRNA or PI3K inhibitor LY294002, the levels of apoptosis caused by cordycepin were obviously attenuated. In addition, suppression of autophagy could also elevate the level of c-FLIPL which indicated cordycepin-triggered autophagy promoted the degradation of c-FLIPL. Therefore, we conclude that cordycepin induces apoptosis through autophagy-mediated downregulation of c-FLIPL in human NSCLC cells. Taken together, our findings provide a novel prospect on the anti-tumor property of cordycepin, which may further prompt cordycepin to serve as a promising therapeutic approach in NSCLC treatment.

Triterpenoid Saponins with Potential Cytotoxic Activities from the Root Bark of Ilex rotunda Thunb.

A new 19-oxo-18,19-seco-ursane-type triterpeonoid saponin, laevigin E (8), together with 17 known compounds (1 - 7 and 9 - 18) were isolated from the root bark of Ilex rotunda Thunb. Their structures were determined by various spectroscopic analysis. Among them, compounds 6, 9, 11, and 18 were isolated from this species for the first time, while compounds 10 and 12 were firstly isolated from the family Aquifoliaceae. Biological activity assay showed that all triterpenoids exhibit moderate cytotoxic activities against MCF7, A549, HeLa and LN229 cell lines. The four triterpenoid saponins (3, 4, 6, and 8) exhibit slightly better activities compared to the four triterpenoid sapogenins (1, 2, 5, and 7). Compound 8 showed the best cytotoxicity against A549, HeLa and LN229 cell lines with IC50 of 17.83, 22.58 and 30.98 µm, respectively.

Elevated AEG-1 expression in macrophages promotes hypopharyngeal cancer invasion through the STAT3-MMP-9 signaling pathway.

Macrophages play a critical role in tumor invasion and metastasis, which remain major causes of mortality in patients with hypopharyngeal cancer. Here we investigate the effect of an oncogene, AEG-1 expressed in macrophages on the invasion of hypopharyngeal cancer cells. AEG-1 is more highly expressed in macrophages of human hypopharyngeal cancer samples compared with adjacent non-tumor controls. Using matrigel invasion assay system, THP-1-derived macrophages with forced AEG-1 overexpression enhance FaDu cell invasion whereas macrophages with AEG-1 silence inhibit. Matrix metalloproteinase 9 (MMP-9), which is important in tumor invasion and metastasis through degrading extracellular matrix, is up-reulated by AEG-1 partly through NF-κB p65 in macrophages. Intriguingly, macrophage AEG-1 also induces MMP-9 up-regulated expression in FaDu cells. Furthermore, macrophage AEG-1 activates signal transducer and activator of transcription 3 (STAT3) in FaDu cells, which is responsible for macrophage AEG-1-induced an increase in MMP-9 expression and invasion of FaDu cells. This is the first to demonstrate that macrophage AEG-1 promotes tumor invasion through up-regulation of MMP-9 in both macrophages and cancer cells. Thus, the results provide evidences that macrophage AEG-1 contributes to promotion of tumor invasion, and represents as a potential target in hypopharyngeal cancer therapy.