A case report of cStage IIIB squamous cell lung carcinoma completely resected after downstaging with neoadjuvant therapy.

Background: The most effective method and length of time for administering adjuvant immunotherapy after surgery for non-small cell lung cancer (NSCLC) are still unknown. Various clinical trials have utilized diverse strategies for adjuvant treatment. In this case, we explore the potential benefits of neoadjuvant immunotherapy combined with chemotherapy in managing locally advanced lung squamous carcinoma, which often poses challenges for treatment. This multimodal approach aims to downstage tumors and optimize surgical outcomes. Case Description: Following a diagnosis of stage IIIB lung cancer, the patient underwent three cycles of neoadjuvant therapy using sintilimab, Abraxane, and Lobaplatin, resulting in a significant 45% reduction in tumor size. Subsequently, a right lower lobe lobectomy and systematic lymphadenectomy were performed using a uniportal video-assisted thoracic surgery (VATS) approach. Postoperative analysis revealed negative lymph nodes, with only a 5-mm residual tumor in the tumor bed, downstaging the cancer to IA1. Remarkably, the patient experienced a smooth recovery without any postoperative complications. One cycle of adjuvant therapy was administered following the operation to further support the patient's recovery and minimize the risk of disease recurrence. This comprehensive treatment approach underscores the importance of neoadjuvant therapy in optimizing surgical outcomes and improving long-term prognosis for patients with locally advanced lung cancer. Conclusions: For patients with stage III locally advanced lung squamous carcinoma, the combination of Sintilimab and Platinum-based drugs can be used as a neoadjuvant therapy which can reduce the difficulty of the operation.

Spinosyn A and Its Derivative Inhibit Colorectal Cancer Cell Growth via the EGFR Pathway.

Spinosyn A (SPA), derived from a soil microorganism, Saccharopolyspora spinosa, and its derivative, LM2I, has potential inhibitory effects on a variety of cancer cells. However, the effects of SPA and LM2I in inhibiting the growth of human colorectal cancer cells and the molecular mechanisms underlying these effects are not fully understood. Cell viability was tested by using a 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) assay and a colony formation assay. On the basis of the IC50 values of SPA and LM2I in seven colorectal cancer (CRC) cell lines, sensitive (HT29 and SW480) and insensitive (SW620 and RKO) cell lines were screened. The GSE2509 and GSE10843 data sets were used to identify 69 differentially expressed genes (DEGs) between sensitive and insensitive cell lines. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interactions (PPI) were performed to elucidate the molecular mechanisms of the DEGs. The hub gene of the DEGs was detected by Western blot analysis and verified using the CRISPR/Cas9 system. Our data indicate that SPA and its derivative LM2I have significant antiproliferative activity in seven colorectal cancer cell lines and colorectal xenograft tumors. On the basis of bioinformatics analysis, it was demonstrated that epidermal growth factor receptor (EGFR) was the hub gene of the DEGs and was associated with the inhibitory effects of SPA and LM2I in CRC cell lines. The study also revealed that SPA and LM2I inhibited the EGFR pathway in vitro and in vivo.

Anatomical segmentectomy under uniportal video-assisted thoracoscopic surgery for early staged non-small cell lung cancer: a case report.

Background: Anatomical segmentectomy has become more and more universal in thoracic surgery because of the increasing detection of pulmonary nodules with ground-glass opacity (GGO), most of which proved early staged non-small cell lung cancer (NSCLC) postoperative. With the advantage of preservation of normal lung tissues, segmentectomy may be performed by surgeons when computed tomography (CT) scan shows pure GGO or multiple GGOs appearing. Especially when the patients with poor cardiopulmonary function or severe comorbidities or in the circumstance of bilateral pulmonary GGOs, segmentectomy can provide opportunities to radically resect all lesions. With the development of minimally invasive surgery technology, uniportal video-assisted thoracoscopic surgery (VATS) has become the regular operative route in many medical centers because it can provide less access trauma, less stress response, less pain, shorter hospital stays, and a lower postoperative complication rate and corresponds well with the idea of "minimally invasive". However, all of the procedures must be performed in one tiny portal, so uniportal VATS anatomical segmentectomy not only needs the skill and patience of surgeons but the effective cooperation of assistants, nurses and anesthetists, and plenty of details must be paid special attention. Case Description: Here we present a video of a patient undergoing S1 segmentectomy of right upper lobectomy (RUL) under uniportal VATS. The chief complaints of the patients was that two pure GGOs in the bilateral upper lobe were found by physical examination for 26 months and he had no symptoms. We performed S1 segmentectomy of RUL under uniportal first time and performed trisegmentectomy of left upper lobectomy (LUL) 3 months later. With routinely follow-up, no evidence of relapse and metastasis disease was found. Conclusions: We think anatomical segmentectomy under uniportal VATS can be a feasible and safe procedure that reduces trauma and has equivalent oncology outcomes to lobectomy in early-stage lung cancer but need a more experienced medical center to perform. Keywords: Uniportal video-assisted thoracoscopic surgery (uniportal VATS); segmentectomy; non-small cell lung cancer (NSCLC); case report.

ZLM-7 Blocks Breast Cancer Progression by Inhibiting MDM2 via Upregulation of 14-3-3 Sigma.

Breast cancer is one of the most prevalent malignancies with poor prognosis. Inhibition of angiogenesis is becoming a valid and evident therapeutic strategy to treat cancer. Recent studies uncovered the antiangiogenic activity of ZLM-7 (a combretastain A-4 derivative), but the regulatory mechanism is unclear. ZLM-7 treatment was applied in estrogen receptor-positive cell MCF-7, triple-negative breast cancer cell MDA-MB-231 and xenograft models. Transfections were conducted to overexpress or knockdown targeted genes. The gene and protein expressions were measured by qPCR and Western blotting assay, respectively. Cell proliferation and apoptosis were evaluated using the CCK8 method, clone formation assay and flow cytometry. We found that ZLM-7 upregulated 14-3-3 sigma expression but downregulated MDM2 expression in breast cancer cells. ZLM-7 delayed cell proliferation, promoted apoptosis and blocked cell-cycle progression in human breast cancer cells in vitro, while those effects were abolished by 14-3-3 sigma knockdown; overexpression of 14-3-3 sigma reproduced the actions of ZLM-7 on the cell cycle, which could be reversed by MDM2 overexpression. In xenograft models, ZLM-7 treatment significantly inhibited tumor growth while the inhibition was attenuated when 14-3-3 sigma was silenced. Collectively, ZLM-7 could inhibit MDM2 via upregulating 14-3-3 sigma expression, thereby blocking the breast cancer progression.

Ginsenoside Rh2 inhibits breast cancer cell growth via ERß-TNFα pathway.

Ginsenoside Rh2 is one of rare panaxidiols extracted from Panax ginseng and a potential estrogen receptor ligand that exhibits moderate estrogenic activity. However, the effect of Rh2 on growth inhibition and its underlying molecular mechanism in human breast cells are not fully understood. In this study, we tested cell viability by MTT and colony formation assays. Cell growth and cell cycle were determined to investigate the effect of ginsenoside Rh2 by flow cytometry. The expressions of estrogen receptors (ERs), TNFα, and apoptosis-related proteins were detected by qPCR and western blot analysis. The mechanisms of ERα and ERß action were determined using transfection and inhibitors. Antitumor effect of ginsenoside Rh2 against MCF-7 cells was investigated in xenograft mice. Our results showed that ginsenoside Rh2 induced apoptosis and G1/S phase arrest in MCF-7 cells. Treatment of cells with ginsenoside Rh2 down-regulated protein levels of ERα, and up-regulated mRNA and protein levels of ERß and TNFα. We also found that ginsenoside Rh2-induced TNFα over-expression is through up-regulation of ERß initiated by ginsenoside Rh2. Furthermore, ginsenoside Rh2 induced MCF-7 cell apoptosis via estrogen receptor ß-TNFα pathway in vivo. These results demonstrate that ginsenoside Rh2 promotes TNFα-induced apoptosis and G1/S phase arrest via regulation of ERß.

An aberrant anterior ascending segmental pulmonary artery (A3aii) in a patient with lung cancer.

A 60-year-old woman was admitted to our department for a left upper lobe and left lower lobe ground glass nodules. Three-dimensional computed tomography bronchography and angiography showed an aberrant anterior ascending segmental pulmonary artery (A3aii) branching from the lingular artery. We performed left upper division segmentectomy and left lower superior segmentectomy for ground glass opacity lesions, and the A3aii variation artery was safely dissected. The patient received an uneventful recovery, and the final pathologic diagnosis was early-stage multiple primary lung cancers. The A3aii branching of the lingular artery was extremely rare. Preoperative three-dimensional computed tomography bronchography and angiography was important to provide accurate vessel variation information and achieve accurate and safe segmentectomy procedure.

Naturally-occurring spinosyn A and its derivatives function as argininosuccinate synthase activator and tumor inhibitor.

Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.

ZLM-7 inhibits the occurrence and angiogenesis of breast cancer through miR-212-3p/Sp1/VEGFA signal axis.

BACKGROUND: Breast cancer (BC) is a common malignant tumor with poor prognosis. Angiogenesis is related to the growth and progression of solid tumors and associated with prognosis. ZLM-7, SP1, VEGFA and miR-212-3p were associated with BC angiogenesis and proliferation, however the detailed mechanism was not clear. This study aimed to reveal the regulatory mechanism of angiogenesis of BC. METHODS: BC cell lines were treated with 10 nM ZLM-7 for 8 h. We detected protein expression level by western blot and RNA expression level by qRT-PCR. Overexpression or inhibition of miR-212-3p is performed using miR-212-3p mimics or miR-212-3p inhibitor, Sp1 overexpression using pcDNA3.1 vector. Angiogenesis was analyzed by co-culturing BC cell lines and HUVEC cells. To evaluate regulatory relationship between miR-212-3p and Sp1, dual luciferase assay was performed. Besides, the direct interaction between Sp1 and VEGFA was analyzed by ChIP. Migration and invasion were analyzed by transwell assay and proliferation was detected by clone formation assay. In mice xenograft model developed using BC cells, we also detected angiogenesis marker CD31 through immunohistochemistry. RESULTS: ZLM-7 up-regulated miR-212-3p and inhibited invasion, migration, proliferation and angiogenesis of BC, while miR-212-3p inhibitor antagonized such effects. Binding sequence was revealed between miR-212-3p and Sp1, and expression of Sp1 was inhibited by miR-212-3p on both protein and mRNA level. Sp1 could interact with VEGFA and promoted its expression. Overexpression of miR-212-3p inhibited migration, invasion, proliferation and angiogenesis of BC cell lines, while Sp1 overexpression showed the opposite effect and could antagonize these effects of miR-212-3p overexpression. ZLM-7 decreased VEGFA expression, which was rescued by co-transfection with miR-212-3p inhibitor. Similar, ZLM-7 could inhibit tumor growth and angiogenesis through the miR-212-3p/Sp1/VEGFA axis in vivo. CONCLUSIONS: ZLM-7 could directly up-regulate miR-212-3p in BC. MiR-212-3p could inhibit VEGFA expression through Sp1, thereby inhibiting angiogenesis and progression of BC.

Ginsenoside metabolite 20(S)­protopanaxadiol promotes neural stem cell transition from a state of proliferation to differentiation by inducing autophagy and cell cycle arrest.

20(S)­Protopanaxadiol (PPD) is an active ginseng metabolite and is the final form of protopanaxadiol saponins metabolized by human intestinal microflora. The neuroprotective effects and mechanisms underlying PPD on neural stem cells (NSCs) are not completely understood. The aim of the present study was to assess the effects of PPD on the proliferation and differentiation of neural stem cells. In the present study, following treatment with different concentrations of PPD for 24 h, the percentage of BrdU­positive cells decreased significantly with increasing concentrations of PPD. Moreover, flow cytometric analysis results indicated that PPD treatment increased the proportion of cells in the G0/G1 and G2/M phase and decreased the proportion of cells in the S phase. The activation of autophagy, determined by an increased number of autophagic vacuoles and light chain 3 lipidation, was associated with an increase in the expression of the neuronal marker tubulin­ß3 following PPD treatment. PPD also partially rescued NSCs from the inhibitory effects of the autophagic inhibitor wortmannin, suggesting that the effect of PPD on NSC differentiation was associated with autophagy. Collectively, the results indicated that PPD promoted the transition of NSCs from a state of proliferation to differentiation through the induction of autophagy and cell cycle arrest. Therefore, the present study may provide a basis for the development of regenerative therapies based on ginsenoside, an approved and safe drug.

[Effects of three drying methods on the physical properties and drug delivery in chitosan microspheres].

OBJECTIVE: The purpose of this study is to investigate the effects of different drying methods on the physical properties and drug delivery of chitosan microspheres. METHODS: Three types of drying methods were utilized, including air drying and freeze drying after freezing at -20 ℃ (slow cooling) and at -80 ℃ (fast cooling). The physical properties of microspheres were characterized. Utilizing bovine serum albumin (BSA) as the model drug, the in-vitro release behaviors of drug-loaded beads were investigated. RESULTS: By comparing the physical properties of the different drying methods, the microspheres' diameters, porosities, and surface area were observed to increase successively from air drying and slow cooling to fast cooling, whereas the pore size and the swelling and degradation rates varied. The drug-loading experiments revealed that the loading capacity of air-dried microspheres was the lowest and the release rate was the slowest. Although the loading capacity of fast cooling microspheres was high, an obvious burst release was observed. The loading capacity of slow cooling microspheres was similar to that of the fast cooling microspheres and the loaded BSA can be released continuously. CONCLUSIONS: The results indicate that different drying methods can affect the physical properties of chitosan microspheres, which further influence drug loading and release.

Role of PFKFB3 and CD163 in Oral Squamous Cell Carcinoma Angiogenesis.

6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), an enzyme producing fructose 2, 6-bisphosphate (F-2, 6-BP), serves as a switch to activate phosphofructokinase-1, and is a critical enzyme for endothelial glycolysis, mediating circadian control of carcinogenesis. Also, tumor-associated macrophages (TAMs) play an important role in the progression and prognosis of numerous cancers. However, the role and clinical significance of PFKFB3 and TAMs in oral squamous cell carcinoma (OSCC) have not been elucidated. The present study was designed to investigate the correlation between PFKFB3 expression, CD163+ TAMs infiltration and tumor angiogenesis in OSCC by tissue microarray. Tissue microarrays containing 117 OSCC specimens and 56 matched paracarcinoma tissues were studied by immunohistochemistry. The expression levels of PFKFB3, CD163 and CD31 were significantly increased in OSCC specimens as compared with normal oral mucosa (P<0.05), and PFKFB was signifcantly correlated with tumor differentiation and tumor size (P<0.05), and CD163 was significantly correlated with areca nut chewing habit among OSCC tissues (P<0.05). Furthermore, Pearson's correlation analysis revealed that PFKFB3 was signifcantly correlated with both CD163 and CD31 (P<0.05), meanwhile CD163 was signifcantly correlated with CD31 (P<0.001), suggesting PFKFB3 may promote angiogenesis in tumor progression and metastases by regulating CD163+ TAMs infiltration in OSCC.

Transcriptomics-based identification and characterization of 11 CYP450 genes of Panax ginseng responsive to MeJA.

Cytochromes P450 (CYP450s), a superfamily of mono-oxygenases, are essential to generate highly functionalized secondary metabolites in plants and contribute to the diversification of specialized triterpenoid biosynthesis in eudicots. However, screening and identifying the exact CYP450 genes in ginsenoside biosynthesis is extremely challenging due to existence of large quantities of members in CYP450 superfamily. Therefore, to screen the CYP450 genes involved in ginsenoside biosynthesis, transcriptome dataset of Panax ginseng was created in our previous work using the technique of the next-generation sequencing. On the basis of bioinformatics analysis, 16 putative CYP450 genes with significant differential expression were screened from the dataset and submitted to GenBank, in which 11 of them have been cloned. Methyl jasmonate (MeJA) was used as an elicitor to analyze the expression profiles of candidate CYP450 genes by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The results of qRT-PCR analysis revealed that the expression of some CYP450 genes were strongly induced by MeJA and showed different transcription levels at different treatment time points. Homology analysis indicated that each putative CYP450 protein of P. ginseng has a conserved domain consisting of E-E-R-F-P-R-G. The CYP450 genes were screened and cloned here to enrich the resources of CYP450 genes, and the results of bioinformatics analysis provided a foundation to further identify the function of CYP450s involved in ginsenoside biosynthesis. Furthermore, this study facilitated the construction of microbial cell factories for increasing the production of ginsenosides by means of metabolic engineering.

Two-step method for creating a gastric tube during laparoscopic-thoracoscopic Ivor-Lewis esophagectomy.

AIM: To introduce a two-step method for creating a gastric tube during laparoscopic-thoracoscopic Ivor-Lewis esophagectomy and assess its clinical application. METHODS: One hundred and twenty-two patients with middle or lower esophageal cancer who underwent laparoscopic-thoracoscopic Ivor-Lewis esophagectomy at Liaoning Cancer Hospital and Institute from March 2014 to March 2016 were included in this study, and divided into two groups based on the procedure used for creating a gastric tube. One group used a two-step method for creating a gastric tube, and the other group used the conventional method. The two groups were compared regarding the operating time, surgical complications, and number of stapler cartridges used. RESULTS: The mean operating time was significantly shorter in the two-step method group than in the conventional method group [238 (179-293) min vs 272 (189-347) min, P < 0.01]. No postoperative death occurred in either group. There was no significant difference in the rate of complications [14 (21.9%) vs 13 (22.4%), P = 0.55] or mean number of stapler cartridges used [5 (4-6) vs 5.2 (5-6), P = 0.007] between the two groups. CONCLUSION: The two-step method for creating a gastric tube during laparoscopic-thoracoscopic Ivor-Lewis esophagectomy has the advantages of simple operation, minimal damage to the tubular stomach, and reduced use of stapler cartridges.

Stratified aspartate aminotransferase-to-platelet ratio index accurately predicts survival in hepatocellular carcinoma patients undergoing curative liver resection.

The aspartate aminotransferase-to-platelet ratio index has been reported to predict prognosis of patients with hepatocellular carcinoma. This study examined the prognostic potential of stratified aspartate aminotransferase-to-platelet ratio index for hepatocellular carcinoma patients undergoing curative liver resection. A total of 661 hepatocellular carcinoma patients were retrieved and the associations between aspartate aminotransferase-to-platelet ratio index and clinicopathological variables and survivals (overall survival and disease-free survival) were analyzed. Higher aspartate aminotransferase-to-platelet ratio index quartiles were significantly associated with poorer overall survival (p = 0.002) and disease-free survival (p = 0.001). Multivariate analysis showed aspartate aminotransferase-to-platelet ratio index to be an independent risk factor for overall survival (p = 0.018) and disease-free survival (p = 0.01). Patients in the highest aspartate aminotransferase-to-platelet ratio index quartile were at 44% greater risk of death than patients in the first quartile (hazard ratio = 1.445, 95% confidence interval = 1.081 - 1.931, p = 0.013), as well as 49% greater risk of recurrence (hazard ratio = 1.49, 95% confidence interval = 1.112-1.998, p = 0.008). Subgroup analysis also showed aspartate aminotransferase-to-platelet ratio index to be an independent predictor of poor overall survival and disease-free survival in patients positive for hepatitis B surface antigen or with cirrhosis (both p < 0.05). Similar results were obtained when aspartate aminotransferase-to-platelet ratio index was analyzed as a dichotomous variable with cutoff values of 0.25 and 0.62. Elevated preoperative aspartate aminotransferase-to-platelet ratio index may be independently associated with poor overall survival and disease-free survival in hepatocellular carcinoma patients following curative resection.

Long noncoding RNA CCAT2 promotes proliferation and metastasis in non-small cell lung cancer through the Wnt pathway.

Colon cancer associated transcript 2 (CCAT2), a long non-coding RNA (lncRNA), was shown to be associated with colon, ovarian and prostate cancer. Recent studies showed CCAT2 was highly expressed in various tumors, including non-small cell lung cancer (NSCLC) and probably being an independent prognostic factor of this disease. However, the physiological and biochemical mechanisms for CCAT2 with NSCLC were still unknown. In this study, we performed the analysis focused on the expression, biological functions and mechanism of CCAT2 in NSCLC and found that CCAT2 was significantly up-regulated in NSCLC tissues compared with corresponding non-cancerous tissues. Knockout of CCAT2 in NSCLC cell lines using lentivirus-mediated RNA interference significantly inhibited the proliferation and metastasis of the NSCLC cells. Most importantly, Wnt/ß-catenin pathway was found to be inactivated in the NSCLC cell lines after CCAT2 knockout experiment. These results indicated that CCAT2 maybe serve as an oncogenic lncRNA that promoted proliferation and metastasis of NSCLC and activated the Wnt/ß-catenin pathway.

Blood neutrophil-lymphocyte ratio predicts survival after hepatectomy for hepatocellular carcinoma: A propensity score-based analysis.

AIM: To investigate whether an elevated preoperative neutrophil-to-lymphocyte ratio (NLR) can predict poor survival in patients with hepatocellular carcinoma (HCC). METHODS: We retrospectively reviewed 526 patients with HCC who underwent surgery between 2004 and 2011. RESULTS: Preoperative NLR ≥ 2.81 was an independent predictor of poor disease-free survival (DFS, P < 0.001) and overall survival (OS, P = 0.044). Compared with patients who showed a preoperative NLR < 2.81 and postoperative increase, patients who showed preoperative NLR ≥ 2.81 and postoperative decrease had worse survival (DFS, P < 0.001; OS, P < 0.001). Among patients with preoperative NLR ≥ 2.81, survival was significantly higher among those showing a postoperative decrease in NLR than among those showing an increase (DFS, P < 0.001; OS, P < 0.001). When elevated, alpha-fetoprotein (AFP) provided no prognostic information, and so preoperative NLR ≥ 2.81 may be a good complementary indicator of poor OS whenever AFP levels are low or high. CONCLUSION: Preoperative NLR ≥ 2.81 may be an indicator of poor DFS and OS in patients with HCC undergoing surgery. Preoperative NLR ≥ 2.81 may be a good complementary indicator of poor OS when elevated AFP levels provide no prognostic information.

The anti-angiogenic effect and novel mechanisms of action of Combretastatin A-4.

Combretastatin A-4 (CA4) is the lead compound of a relatively new class of vascular disrupting agents that target existing tumor blood vessels. Recent studies showed the CA4 might inhibit angiogenesis. However, the underlying molecular mechanisms by which CA4 exerts its anti-angiogenic effects are not fully understood. In this study, we revealed that CA4 inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs). In in vivo assay, CA4 suppressed neovascularization in chicken chorioallantoic membrane (CAM) model and decreased the microvessel density in tumor tissues of a breast cancer MCF-7 xenograft mouse model. In addition, CA4 decreased the expression level and secretion of VEGF both in MCF-7 cells and HUVECs under hypoxia, as well as the activation of VEGFR-2 and its downstream signaling mediators following VEGF stimulation in HUVECs. Moreover, VEGF and VEGFR-2 expression in tumor tissues of the mouse xenograft model were down-regulated following CA4 treatment. Taken together, results from the current work provide clear evidence that CA4 functions in endothelial cell system to inhibit angiogenesis, at least in part, by attenuating VEGF/VEGFR-2 signaling pathway.