Neuronal SNAP-23 is critical for synaptic plasticity and spatial memory independently of NMDA receptor regulation.

SNARE-mediated membrane fusion plays a crucial role in presynaptic vesicle exocytosis and also in postsynaptic receptor delivery. The latter is considered particularly important for synaptic plasticity and learning and memory, yet the identity of the key SNARE proteins remains elusive. Here, we investigate the role of neuronal synaptosomal-associated protein-23 (SNAP-23) by analyzing pyramidal-neuron specific SNAP-23 conditional knockout (cKO) mice. Electrophysiological analysis of SNAP-23 deficient neurons using acute hippocampal slices showed normal basal neurotransmission in CA3-CA1 synapses with unchanged AMPA and NMDA currents. Nevertheless, we found theta-burst stimulation-induced long-term potentiation (LTP) was vastly diminished in SNAP-23 cKO slices. Moreover, unlike syntaxin-4 cKO mice where both basal neurotransmission and LTP decrease manifested changes in a broad set of behavioral tasks, deficits of SNAP-23 cKO are more limited to spatial memory. Our data reveal that neuronal SNAP-23 is selectively crucial for synaptic plasticity and spatial memory without affecting basal glutamate receptor function.

Association between anesthesia technique and complications after hip surgery in the elderly population.

BACKGROUND: Spinal anesthesia is superior to general anesthesia for postoperative recovery in older patients (≥ 65 age). However, evidence for this is lacking. AIM: To evaluate the effect of anesthesia on postoperative complications in older patients undergoing hip surgery. METHODS: This is a retrospective, propensity score-matched, cohort study. Patients ≥ 65-years-old who underwent hip surgery at the Traditional Chinese Medicine of Guangdong Provincial Hospital in China from October 2016 to June 2020 were included. The operative methods were femoral fracture's internal fixation and hip replacement. The orthopedic doctors in different hospitals of our group have varied requirements for patients' out-of-bed time after surgery. Therefore, spinal anesthesia or general anesthesia was selected according to the requirements of the orthopedic doctors. The primary outcome of this study was complications during the hospitalization of the postoperative patient. The length of hospital stay, postoperative blood transfusion, routine blood analysis, renal function, coagulation function, and inflammatory correlations were secondary outcomes. Propensity score matching (PSM) was performed utilizing logistic regression. RESULTS: Among the 864 patients identified from the electronic medical record data database, we screened out those with incomplete medical record data. After PSM of the baseline values of the two groups of patients, data of 309 patients (206 patients in spinal anesthesia group and 103 patients in general anesthesia) were utilized in this study. 67/309 patients had complications, including postoperative limb dysfunction, pulmonary infection, delirium, lower extremity venous thrombosis, and shock. The incidence of complications was not related to anesthesia methods (P > 0.05), but the levels of D-Dimer (P = 0.017), fibrinogen (P = 0.005), and high-sensitivity C-reactive protein (hsCRP) (P = 0.002) in the spinal anesthesia group were significantly higher than those in the general anesthesia group. CONCLUSION: Anesthesia technology is not a risk factor for postoperative complications of hip surgery. The levels of D-Dimer and hsCRP were higher in the spinal anesthesia group.

Long Noncoding RNA ASLNC07322 Functions in VEGF-C Expression Regulated by Smad4 during Colon Cancer Metastasis.

Deletion and mutation of the Smad4 gene are favorable events for the progression of colon cancer, which is related to the negative regulation of vascular endothelial growth factor C (VEGF-C). However, the regulatory mechanism between Smad4 and VEGF-C remains unclear. We reported first that Smad4 can increase the transcription of miR-128-3p, a microRNA targeting VEGF-C mRNA, resulting in a negative correlation between Smad4 and VEGF-C. Moreover, we found that Smad4 combined with Smad3 can positively regulate VEGF-C during colon cancer metastasis through binding to VEGF-C gene promoter. Further, results revealed a mechanism that long noncoding RNA (lncRNA) ASLNC07322 increased specifically in metastatic colon cancer and decreased miR-128-3p as a sponge, leading to a subsequent elevation of VEGF-C. In a word, there are two pathways in the progression of colon cancer, including Smad4/miR-128-3p/VEGF-C and Smad4/VEGF-C pathways in non-metastatic and metastatic colon cancer, respectively. ASLNC07322 crucially controlled this negative and positive regulatory transformation between them. Additionally, ASLNC07322 knockdown combined with Smad4 overexpression could efficiently inhibit lymphatic endothelial cells (LECs) proliferation and tube formation in vitro, as well as tumor growth and lymphangiogenesis in vivo. These data explained the underlying mechanism of Smad4 contribution on VEGF-C expression during metastasis where ASLNC07322 functions vitally as a switch in colon cancer.

The association of semaphorin 5A with lymph node metastasis and adverse prognosis in cervical cancer.

BACKGROUND: Semaphorin 5A has been linked to tumor growth, invasion, and metastasis in pancreatic cancer. However, the role of semaphorin 5A in cervical cancer is not known. Our aim is to investigate the prognostic value of semaphorin 5A and its potential role in lymphangiogenesis and invasion in cervical cancer. METHODS: In this study, pathological features and clinical data of 232 cervical cancer patients were retrospectively reviewed. Semaphorin 5A protein and mRNA expression was detected by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction, respectively. In vitro, we determined the role and mechanistic pathways of semaphorin 5A in tumor progression in cervical carcinoma cell lines. RESULTS: Semaphorin 5A expression was significantly higher in stage IIb tumors than in stage Ia, Ib, and IIa tumors. High semaphorin 5A expression was significantly associated with pelvic lymph node metastasis, lymphovascular permeation, and poor survival. Semaphorin 5A induced lymphangiogenesis through a plexin-B/Met/vascular endothelial growth factor-C pathway. Semaphorin 5A also increased cervical cancer cell invasion by stimulating the expression and activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 via PI3K/AKT and plexin-B3. CONCLUSION: Our findings indicate that semaphorin 5A may represent a poor prognostic biomarker and anti-metastasis therapeutic target in cervical cancer.

Smad4 Inhibits VEGF-A and VEGF-C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer.

Smad4 is a critical factor in the TGF-ß pathway and is involved in tumor progression and metastasis, but the role of Smad4 in colon cancer cells is unclear. The aim of this study is to explore the effect and the underlying mechanism of Smad4 on the growth, migration and apoptosis of colon cancer cells as well as vascular endothelial growth factor (VEGF)-A and VEGF-C secreted by these cells. In this study, we showed that Smad4, VEGF-A, and VEGF-C are independent prognostic factors of colon cancer, and Smad4 expression was negatively correlated with VEGF-A and -C in samples. We found that Smad4 mRNA and protein levels in colon cancer cells, particularly in HCT-116 cells, were significantly lower than those in the human intestinal epithelial cell line (HIEC). Smad4 overexpression promoted tumor cell apoptosis, inhibited VEGF-A and -C expression in vitro and in vivo, but had no effect on cell proliferation and migration. Tail vein injection of the virus inhibited xenograft growth in nude mice. Importantly, we also demonstrated that Smad4 could increase the phosphorylation level of Smad3, but not Smad2, which may be one of the mechanisms underlying these effects of Smad4 in colon cancer. Therefore, Smad4 may be a new target for the treatment of colon cancer. Anat Rec, 300:1560-1569, 2017. © 2017 Wiley Periodicals, Inc.

Serum miR-21 and miR-125b as markers predicting neoadjuvant chemotherapy response and prognosis in stage II/III breast cancer.

The predictive value of serum miRNAs (ser-miRNA) for the response to neoadjuvant chemotherapy (NCT) and the prognosis of breast cancer patients were investigated in the current study. The study included 118 stage II/III breast cancer patients and 30 healthy adult women. Peripheral blood was drawn from participants before the start (baseline [BL]), at the end of the second cycle (first evaluation during NCT [FEN]), and at the end of NCT (second evaluation during NCT [SEN]). The expression of ser-miRNAs was examined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and their association with chemotherapy response and prognosis was analyzed. MiR-19a, miR-21, miR-125b, miR-155, miR-205, and miR-373 were significantly up-regulated in the serum of breast cancer patients at BL, miR-451 was significantly down-regulated, and miR-122 was unchanged compared with the levels in healthy women. The expression of ser-miR-125b and the changes of ser-miR-21 expression during NCT were associated with chemotherapy response and disease-free survival (DFS). In chemotherapy responders, ser-miR-125b expression was lower than that of non-responders at BL, FEN, and SEN, and ser-miR-21 levels decreased from BL to FEN and from BL to SEN. Survival analysis showed that patients with lower ser-miR-125b expression at BL, FEN, and SEN had favorable DFS, and those with decreased ser-miR-21 expression from BL to FEN and from BL to SEN had better DFS. In conclusion, ser-miR-21 and ser-miR-125b were identified as novel, noninvasive predictive markers for NCT response and prognosis in breast cancer.

A novel seven-long non-coding RNA signature predicts survival in early stage lung adenocarcinoma.

Increasing evidence has revealed the significant association between dysregulated lncRNA expression and cancers. The prognostic value of lncRNAs in predicting the risk of disease recurrence and identifying high-risk subgroup of early stage lung adenocarcinoma (LUAD) is still unclear. In this study, we analyzed lncRNA expression profiles of 415 early-stage LUAD patients from Gene Expression Omnibus and identified a novel seven-lncRNA signature that was significantly associated with survival in patients with early-stage LUAD (HR = 2.718, CI = 2.054-3.597, p < 0.001). Based on the seven-lncRNA signature, we constructed a risk score model which is able to classify patients of training dataset into the high-risk group and the low-risk group with significantly different clinical outcome (p < 0.001). The robustness of the seven-lncRNA signature was successfully validated through application in other two independent patient datasets. Furthermore, the prognostic value of seven-lncRNA signature was independent of other clinicopathological factors including age, gender, stage and smoking status. Functional analysis suggested that the seven-lncRNA signature may be involved in a variety of biological pathways including cell cycle, ECM-receptor interaction, Focal adhesion and p53 signaling pathway. Taken together, our study not only provides insights into the lncRNA association with LUAD, but also provide alternative molecular markers in prognosis prediction for early-stage LUAD patients.

Expression of VEGF-D, SMAD4, and SMAD7 and Their Relationship with Lymphangiogenesis and Prognosis in Colon Cancer.

AIM: The vascular endothelial growth factor (VEGF) and TGF-ß1 pathways play important roles in cancer. However, few studies have evaluated the expression and roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, and the conclusions remain controversial. To clarify the roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, we examined their expression and evaluated correlations with lymphangiogenesis, prognosis, and chemotherapeutic outcome. METHODS: The expression of VEGF-D, SMAD4, and SMAD7 was immunohistochemically examined in 251 primary colon cancer samples obtained from the Harbin Medical University. RESULTS: The expression of VEGF-D, SMAD4, and SMAD7 was identified in 71.7, 41.0, and 69.7 % of samples, respectively. Positive expression of VEGF-D and SMAD7 and lost expression of SMAD4 were significantly correlated with lymph node metastasis and high lymphatic vessel density. VEGF-D and SMAD7 were found to be independent indicators of prognosis and chemotherapy outcome, and positive expression of either VEGF-D or SMAD7 was associated with significantly shorter overall survival and disease-free survival (OS and DFS) than negative expression in all 251 patients (P < 0.001 for OS and DFS) and patients following chemotherapy (P < 0.001 for OS and DFS). CONCLUSION: VEGF-D, SMAD4, and SMAD7 were involved in lymphangiogenesis and lymph node metastasis. VEGF-D and SMAD7 can serve as predictors of prognosis and chemotherapeutic outcome in colon cancer.

Association between VEGF-A, C and D expression and lymph node involvement in breast cancer: a meta-analysis.

BACKGROUND: Metastasis is the primary cause of death in patients with breast cancer. Although VEGF-A, C and D are considered to be prime factors in lymph node metastasis in breast cancer, the published studies have conflicting conclusions. METHODS: To resolve this conflict, we conducted a meta-analysis of 37 studies (n = 5,001 patients) evaluating the correlation between VEGF-A, C and D immunohistochemical expression and lymph node metastasis (LNM). The meta-analysis included 22 studies of VEGF-A, 17 of VEGF-C, and 6 of VEGF-D. The relationships between VEGF-A, C and D and clinicopathological parameters were also examined. RESULTS: The results showed a significant association between VEGF-A or VEGF-C overexpression and LNM (risk ratio [RR] = 1.28 [95% CI 1.04-1.58], p = 0.02; and RR = 1.36 [95% CI 1.07-1.72], p = 0.01, respectively). Subgroup evaluation showed a significant association between VEGF-A, C and D overexpression and LNM when analyses were limited to Asian patients (RR = 1.78 [95% CI 1.28-2.46], p = 0.0005; RR = 1.38 [95% CI 1.04-1.84], p = 0.03, and RR = 2.62 [95% CI 1.35-5.09], p = 0.004, respectively). VEGF-A overexpression was significantly associated with lymph vessel invasion (RR = 1.86 [95% CI 1.33-2.60], p = 0.0003). Overexpression of VEGF-C or VEGF-D was significantly associated with HER-2 positivity (RR = 1.30 [95% CI 1.06-1.59], p = 0.01; and RR = 1.75 [95% CI 1.01-3.03], p = 0.05, respectively). CONCLUSIONS: With some limitations, our meta-analysis indicated that VEGF-A and C could predict LNM in patients with breast cancer, particularly Asian patients.

Semaphorin 4D expression is associated with a poor clinical outcome in cervical cancer patients.

Lymphangiogenesis is thought to be essential for cancer progression, making it an important target in cancer therapy. Lymphangiogenic factors (VEGF-C and VEGF-D) are upregulated in various tumors/cancers, and play an important role in lymphangiogenesis and lymph node metastasis. Similarly, semaphorin 4D (Sema4D) is a potent inducer of angiogenesis, and its overexpression is associated with tumor progression and poor prognosis in a variety of malignancies. However, little is known regarding the functional relationship between Sema4D and VEGF-C/VEGF-D and in the mediation of lymphangiogenesis and lymph node metastasis and clinical outcome. The current study aimed to evaluate the effect of Sema4D expression on outcome in patients with cervical cancer, and to explore the molecular mechanism of Sema4D in tumor progression. We evaluated Sema4D expression, density of lymphatic vessels, and invasion of lymphatic vessels with immunohistochemical methods in 232 human cervical cancers with long-term follow-up. Sema4D expression was correlated with patho-clinical parameters and patients' outcome. A cervical cancer cell line was used to investigate the contribution of sema4D to tumor progression by studying the role of Sema4D in VEGF-C/-D and cell migration using reverse transcription-polymerase chain reaction and Western blotting. We observed that Sema4D expression was higher in metastatic cervical cancer than in nonmetastatic cervical cancer (P<0.001). CD34-positive or D2-40-positive lymphatic vessel density was significantly increased in cases with lymph node metastasis compared with those without lymph node metastasis. The increased Sema4D expression was associated with VEGF-C/-D, the presence of lymphatic invasion, the occurrence of lymph node metastasis, and FIGO stage. We also observed a novel association between Sema4D upregulation and poor prognosis in cervical cancer. In vitro, the Sema4D inhibitory antibody and Sema4D-shRNA suppressed VEGF-C and VEGF-D in the human cervical carcinoma cell lines HeLa, Siha, and Caski cells. Invasiveness assay demonstrated that Sema4D could augment the invasive potential of the tumor cells in the cervical cancer lines and induction of cellular invasiveness by Sema4D stimulation could be inhibited by knockdown of plexinB1 by siRNA. Further mechanistic investigations of tumor cell invasiveness showed that Sema4D could induce activation of GTPase Ras homolog gene family, member A (RhoA), MAPK and AKT. In addition, plexinB1 knockdown by siRNA could suppress the Sema4D signal transmitted to MAPK and Akt. Taken together, these results suggest that Sema4D autocrine within tumor cells contributes to enhanced invasion and tumor progression through increased motility of cervical cancer and VEGF-C/-D-mediated lymphangiogenesis. Sema4D might be useful as a molecular marker of poor prognosis in cervical cancer.

JAM-C promotes lymphangiogenesis and nodal metastasis in non-small cell lung cancer.

This study aims to investigate lymphatic metastasis-related genes in non-small cell lung carcinomas (NSCLC). NSCLC tissue was analyzed for expression of junctional adhesion molecule-C (JAM-C) protein. Our data revealed novel associations between JAM-C overexpression in primary tumors and lymphatic microvessel density (LMVD), lymph node metastasis, and poorer overall survival and recurrence-free survival. We used the highly metastatic human lung adenocarcinoma cell line Anip973 and its parental line AGZY83-a, which has a low metastatic capacity, in vivo and vitro. We found that JAM-C played an important role in different metastasis capacity of lymph node. JAM-C affected tumor growth, LNM, JAM-C, VEGF-C, vasculature, and ERK1/2 phosphorylation (p-ERK1/2). ß1 integrin was involved in lymph node metastasis. Moreover, JAM-C knockdown in highly metastatic Anip973 decreased cell migration in scratch-wound assays. The JAM-C knockdown in Anip973 cells and JAM-C cDNA in AGZY83-a cells regulated the vascular endothelial growth factor C (VEGF-C) expression. Immunofluorescence showed that blocked VEGF-C expression in JAM-C shRNA Anip973 cells were restored after JAM-C treatment. JAM-C-induced VEGF-C in JAM-C cDNA AGZY83-a cells was also effectively inhibited by treatment with an antibody specifically against JAM-C. Use of media from Anip973 cells, AGZY83-a, and A549cells lung cancer cells that overexpressed or downregulated JAM-C was demonstrated to affect activity of VEGF-C-induced ß1 integrin subunit or ERK activity in human dermal lymphatic endothelial cells (HDLEC) treated with VEGF-C or inhibitory antibody to JAM-C. Overall, these results indicate that JAM-C could mediate metastasis as it contributes to VEGF-C expression in cancer cells. JAM-C affects ß1and ERK activation in HDLEC, thus promoting lymphangiogenesis and nodal metastasis. Our findings indicate that JAM-C may be a therapeutic target for preventing and treating lymphatic metastases.

Arctigenin anti-tumor activity in bladder cancer T24 cell line through induction of cell-cycle arrest and apoptosis.

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 µmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer.

4-aminopyridine induces apoptosis of human acute myeloid leukemia cells via increasing [Ca2+]i through P2X7 receptor pathway.

4-AP, a voltage-gated potassium channel blocker, was identified to exert critical pro-apoptotic properties in various types of cancer cells. The present study aims to explore the effect of 4-AP on the apoptosis of human AML cells and the underlying mechanism. We found 4-AP inhibited the proliferation and induces apoptosis in both AML cell lines and primary cultured human AML cells. The apoptosis of AML cells after 4-AP treatment was further confirmed by the disruption of mitochondrial membrane potential (MMP) and activation of caspase 3 and 9. 4-AP inhibited Kv currents in NB(4), HL-60 and THP-1 cells. Furthermore, 4-AP induced significant increment in [Ca(2+)](i), which were inhibited by KN-62, a specific blocker of P(2)X(7) receptors. KN-62 also abrogated 4-AP induced apoptosis. Knockdown of P(2)X(7) receptor by small interfering RNA blocked the effect of 4-AP. Conclusively, this study indicated that 4-AP promotes apoptosis in human AML cells via increasing [Ca(2+)](i) through P(2)X(7) receptor.

Roles of VEGF-C and Smad4 in the lymphangiogenesis, lymphatic metastasis, and prognosis in colon cancer.

BACKGROUND/AIMS: We combined two different signal pathways on transforming growth factor ß1 (TGF-ß1)-Smad and vascular endothelial growth factor C (VEGF-C)/VEGF receptors for exploring changes in pathway members and their influence on lymphangiogenesis and clinicopathological features. MATERIALS AND METHODS: Expression of TGF-ß1, TGF-ßRII, Smad4, VEGF-C, and VEGFR-3 was immunohistochemically evaluated in 147 colon cancer patients who were followed up for 5 years. RESULTS: Lymphatic vessel density in colon cancer tissues was significantly higher than in normal colonic tissues. Smad4 expression negatively correlated with lymphatic vessel count and VEGF-C expression. VEGF-C expression positively correlated with lymphatic vessel count. Analysis using the Kaplan-Meier method indicated that patients with VEGF-C-positive tumors had significantly shorter overall survival and tumor-free survival time than those with VEGF-C-negative tumors. Patients with Smad4-negative tumors had significantly shorter overall survival and tumor-free survival time than those with Smad4-positive tumors. CONCLUSIONS: Both Smad4 and VEGF-C are involved in lymphangiogenesis and lymphatic metastasis. Smad4 and VEGF-C expression may be clinically useful indicators for prognostic evaluation in colon cancer patients.

COX-2 expression is correlated with VEGF-C, lymphangiogenesis and lymph node metastasis in human cervical cancer.

Lymphangiogenesis has been shown to promote lymph node metastasis in cancers, making it an important target in cancer therapy. Vascular endothelial growth factor (VEGF)-C is upregulated in various tumors/cancers and is one of the most potent growth factors for inducing lymphangiogenesis and promoting lymph node metastasis (LNM). Likewise, cyclooxygenase (COX)-2 plays major roles in carcinogenesis, tumor growth and metastasis via multiple mechanisms including inactivation of host antitumor immunity and promotion of tumor cell migration, tumor cell invasiveness and tumor-associated angiogenesis and lymphangiogenesis. We previously demonstrated an association between COX-2 and VEGF-C in an in vitro model of lung cancer. However, little is known about the regulation of VEGF-C by COX-2 in cervical cancer. In this study, we measured the COX-2 and VEGF-C expressions by immunohistochemistry in 23 LNM-positive and 20 LNM-negative cervical cancer specimens. We then examined the correlations among the expressions and the lymphatic microvessel density (LMVD) and ultrastructural changes to the lymphatic vessel walls by enzyme histochemical staining and electron microscopy. In addition, we used the HeLa cervical cancer cell line to explore the in vitro regulation of VEGF-C by COX-2 and its metabolite, PGE(2), using siRNA-mediated gene silencing and EP receptor blockade. The LNM-positive specimens exhibited significantly higher VEGF-C expression, COX-2 expression and LMVD than the LNM-negative specimens. Furthermore, there were strong correlations between the levels of COX-2 expression and the levels of VEGF-C expression and secretion and a significant positive association between the LMVD and LNM. siRNA-mediated knockdown of COX-2 expression inhibited VEGF-C mRNA expression while EP1 and EP4 receptor antagonists reduced the VEGF-C protein level and tyrosine phosphorylation of Src kinase. Moreover, inhibition of Src kinase with the tyrosine kinase inhibitor PP1 attenuated VEGF-C expression. Collectively, our data provide evidence for a clinical association between COX-2 and VEGF-C expressions in cervical cancer. EP1 and EP4 receptors may be involved in the COX-2-mediated regulation of VEGF-C protein and mRNA expressions. Src may be a downstream mediator of EP1 and EP4 receptors. COX-2 inhibition may diminish LNM by suppressing VEGF-C-mediated lymphangiogenesis.

COX-2-mediated regulation of VEGF-C in association with lymphangiogenesis and lymph node metastasis in lung cancer.

The mechanisms underlying the effects of COX-2 on tumor lymphangiogenesis remain largely undefined. Here, the human lung cancer cell lines A549, 95D, Anip973, and AGZY83-a with different metastatic capacities were investigated by immunostaining, western blotting, and real-time RT-PCR. We observed increased expressions of COX-2 and VEGF-C in the three highly metastatic cell lines compared with the less metastatic AGZY83-a cell line. The COX-2-specific inhibitor Celecoxib suppressed VEGF-C expression whereas the main COX-2 metabolite PGE(2) elevated VEGF-C expression in Anip973 and AGZY83-a cells in positive and negative experiments. To determine the functional link to COX-2 more specifically and elucidate the mechanistic pathway, we used a siRNA to knock down the high COX-2 expression in Anip973 cells and transfected a COX-2 cDNA to enhance the low COX-2 expression in AGZY83-a cells, and then treated the cells with EP1/EP4 agonists or antagonists, respectively. The results revealed that the EP1/EP4 agonists significantly increased VEGF-C production in the COX-2-knockdown Anip973 cells. In contrast, the EP1/EP4 antagonists diminished VEGF-C production in the COX-2-overexpressing AGZY83-a cells. Furthermore, animal models provided evidence that Celecoxib decreased VEGF-C expression, lymphangiogenesis, and lymph node metastases in Anip973 cells, whereas PGE(2) treatment increased the same factors in the parental AGZY83-a cells. A positive correlation between COX-2 and VEGF-C was also confirmed in vivo. The present data suggest that COX-2 regulates VEGF-C expression via the PGE(2) pathway, and that EP1/EP4 receptors are involved in PGE(2)-mediated VEGF-C production. Thus, COX-2 may represent a candidate gene for blocking tumor lymphangiogenesis and lymph node metastasis.

Inhibition of cyclooxygenase-2 suppresses lymph node metastasis via VEGF-C.

Most experimental work addressing cyclooxygenase-2 (COX-2) inhibitor has focused on suppressing hematogenic spread. Little is known about the mechanism by which this inhibitor can also block lymphatic metastasis. Here, the effects of COX-2 inhibitor on vascular endothelial growth factor-C (VEGF-C) expression, lymphangiogenesis and lymph node metastasis were investigated. Utilizing the highly metastatic human lung adenocarcinoma cell line Anip973 and its parental line AGZY83-a, which has a low metastatic capacity, we found elevated VEGF-C and COX-2 immunoreactivity in Anip973 cells compared with AGZY83-a cells. Celecoxib down-regulated expression of VEGF-C mRNA and protein in Anip973 cells while PGE(2) up-regulated expression of VEGF-C mRNA and protein in AGZY83-a cells in a concentration-dependent manner. The expression of COX-2 and VEGF-C was significantly increased in xenografted Anip973 tumors compared with AGZY83-a tumors. The Anip973 tumors showed more lymphatic vessels and lymph node metastasis than the AGZY83-a tumors. In vivo, celecoxib decreased VEGF-C expression in Anip973 tumor-treated mice to a similar level to that in the AGZY83-a tumor-treated mice. Consistent with this decrease in VEGF-C expression, the density of lymphatic vessels and lymph node metastasis in Anip973 tumor-treated mice were suppressed to approximately that found in the AGZY83-a tumor-treated ones. Taken together, our results suggest that the differential expression of COX-2 and VEGF-C might help explain the different metastasis phenotype of lung adenocarcinoma cancer, and that COX-2 inhibitor mediates VEGF-C to block lymphangiogenesis and lymph node metastasis. Thus, COX-2 may be a potential therapeutic target for blocking lymph node metastasis in lung adenocarcinoma.

Vascular endothelial growth factor D and intratumoral lymphatics as independent prognostic factors in epithelial ovarian carcinoma.

Lymph node metastasis is an important prognostic indicator for disease progression and is crucial for therapeutic strategies of epithelial ovarian carcinoma. Vascular endothelial growth factor (VEGF)-D has been confirmed to have potent lymphangiogenic function in experimental models, but the role in the progression of human ovarian carcinoma remains presently controversial. The purpose of this study was to investigate the prognostic significance of VEGF-D and the presence of intratumoral lymphatics in patients with epithelial ovarian carcinoma. The VEGF-D expression was evaluated by immunohistochemistry in 78 specimens of epithelial ovarian carcinoma and tumoral lymphatic vessels were measured by D2-40. The expression of VEGF-D protein was detected in the cytoplasm of the tumor cells and in stroma occasionally. The high expression of VEGF-D was closely associated with the FIGO stage, intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Univariate and multivariate analysis indicated that VEGF-D, intratumoral lymphatics, and lymphatic invasion were independent prognostic factors for overall survival and disease-free survival in patients with epithelial ovarian carcinoma. We conclude that VEGF-D plays an essential role in tumoral lymphangiogenesis and lymphatic spread, VEGF-D expression, and the intratumoral lymphatics may be clinically useful indicators for prognostic evaluation in patients with epithelial ovarian carcinoma.

Synergistic effect of all-trans-retinoic acid and arsenic trioxide on growth inhibition and apoptosis in human hepatoma, breast cancer, and lung cancer cells in vitro.

AIM: To investigate the effect of all-trans-retinoic acid (ATRA) on arsenic trioxide (As(2)O(3))-induced apoptosis of human hepatoma, breast cancer, and lung cancer cells in an attempt to find a better combination therapy for solid tumors. METHODS: Human hepatoma cell lines HepG2, Hep3B, human breast cancer cell line MCF-7, and human lung adenocarcinoma cell line AGZY-83-a were treated with As(2)O(3) together with ATRA. Cell survival fraction was determined by MTT assay, cell viability and apoptosis were measured by annexin V-fluorescein isothiocyanate (FITC) and PI staining, and intracellular glutathione (GSH) and glutathione-S-transferase (GST) activities were determined using commercial kits. RESULTS: Cytotoxicity of ATRA was low. ATRA (0.1, 1, and 10 micromol/L) could synergistically potentiate As(2)O(3) to exert a dose-dependent inhibition of growth and to induce apoptosis in each of the cell lines. HepG2 and Hep3B with low intracellular GSH or GST activities were remarkably sensitive to As(2)O(3) or As(2)O(3)+ATRA, while AGZY-83-a with higher GSH or GST activities was less sensitive to As(2)O(3) or As(2)O(3)+ATRA. Treatment with 2 micromol/L As(2)O(3) for 72 h significantly decreased intracellular GSH and GST levels in each of the cell lines, and 1 micromol/L ATRA alone reduced minimal intracellular GSH and GST levels. ATRA potentiated the effect of As(2)O(3) on intracellular GSH levels, but intracellular GST levels were not significantly affected by the combination of As(2)O(3) and ATRA for 72 h as compared to As(2)O(3) alone. CONCLUSION: ATRA can strongly potentiate As(2)O(3)-induced growth-inhibition and apoptosis in each of the cell lines, and two drugs can produce a significant synergic effect. The sensitivity to As(2)O(3) or As(2)O(3)+ATRA is inversely proportional to intracellular GSH or GST levels in each of the cell lines. The GSH redox system may be the possible mechanism by which ATRA synergistically potentiates As(2)O(3) to exert a dose-dependent inhibition of growth and to induce apoptosis.