Poly (ADP-ribose) polymerase-1 Binding Protein Facilitates Lung Adenocarcinoma Cell Proliferation and Correlates with Poor Prognosis.

OBJECTIVE: This study makes a preliminary inquiry on the biological functions of PARPBP in lung adenocarcinoma progression. METHODS: mRNA expression profiles and clinical data for lung adenocarcinoma and normal lung samples were downloaded from the TCGA database. The expression of PARPBP was analyzed by a student's t test. The survival curve of lung adenocarcinoma patients was drawn using Kaplan-Meier method. A Chi-square test was applied for analyzing the correlation between PARPBP expression and clinical factors. The prognosis value of PARPBP was determined using a cox proportional hazards model. A549 cells with silenced PARPBP were constructed using RNA interference. CCK8 assay and scratch tests were conducted to test the influence of PARPBP on A549 cell proliferation and migration. A Western blot was implemented to examine the changes of p-MEK/MEK and p-ERK/ERK ratios after depleting PARPBP. RESULTS: PARPBP expression is enhanced in lung adenocarcinoma tissues (p=3.51E-35) and correlates with poor prognosis in lung adenocarcinoma patients (p=0.003). High PARPBP expression is closely correlated with pathologic-stages (p=0.033) and can be utilized as an independent predictor in lung adenocarcinoma patients. Moreover, the knockdown of PARPBP significantly suppressed A549 cell viability and migration (p<0.01). In addition, the ratios of p-MEK/MEK and p-ERK/ERK declined after silencing PARPBP (p<0.01). CONCLUSIONS: PARPBP expression is enhanced in lung adenocarcinoma tissues and is a potential factor in the progression of lung adenocarcinoma.

Clinical characteristics and prognosis of ground-glass opacity nodules in young patients.

BACKGROUND: The detection rate of ground-glass opacity (GGO) in young patients has increased year by year with the increasingly widespread use of high-resolution computed tomography (HRCT) and the increased resolution of HRCT imaging. However, no scholars have reported the clinical characteristics and prognosis of GGO in young patients systematically. The purpose of this study is to investigate the clinical characteristics and prognosis presenting as GGO in young patients. METHODS: Clinical data of 127 young patients who were diagnosed as GGO and who underwent video-assisted thoracoscopic surgery (VATS) and had routine pathological examination were collected from January 2016 to January 2017. Nodules were classified according to benign and malignant: 26 benign nodules (Group A) and 115 malignant nodules (Group B). The pathological types, nodules size, surgical methods were analyzed, and the clinical characteristics and prognosis were evaluated. RESULTS: The results of pathological examination of 91 pure ground-glass opacities (pGGOs) revealed 16 adenocarcinoma in situs (AISs), 42 micro invasive adenocarcinomas (MIAs), 13 invasive adenocarcinomas (IAs), 8 atypical adenomatous hyperplasias (AAHs), 1 inflammatory granuloma, 2 pulmonary inflammatory pseudotumors (IPTs) and 9 other benign nodules. The results of pathological examination of 50 mixed ground-glass opacities (mGGOs) revealed 6 AISs, 29 MIAs, 9 IAs, 1 AAH, 2 inflammatory granulomas and 3 other benign nodules. All patients had no lymph nodes invasion. The rates of perioperative complications were 6.30%, compared to 7.63% for long-term complications. None of the patients with GGO experienced a recurrence and death [2-year recurrence-free survival (RFS), 100%; 2-year overall survival (OS), 100%]. CONCLUSIONS: The GGO in young patients that received VATS has a high proportion of malignant, its prognosis is satisfied.

TINCR facilitates non-small cell lung cancer progression through BRAF-activated MAPK pathway.

Long non-coding RNAs are critically involved in oncogenesis in various cancer types. Here we reported a novel lncRNA signature correlated with progression of non-small cell lung cancer (NSCLC). In particular, we identified elevated expression of terminal differentiation-induced noncoding RNA (TINCR) in human NSCLC samples, which were associated with enhanced migration, viability in NSCLC cells in vitro. Higher TINCR level was also correlated with poor survival. Furthermore, TINCR increased xenograft tumor growth in vivo mouse models. Mechanistic study demonstrated that TINCR can interact with BRAF to facilitate its kinase activity, thereby leading to activation of oncogenic mitogen-activated protein kinase (MAPK) pathway. These results suggested that TINCR upregulation may signal through the MAPK pathway to promote NSCLC tumorigenesis. Therefore, TINCR may serve as a potential prognostic marker and therapeutic target for NSCLC patients.

Antitumor activity of cobrotoxin in human lung adenocarcinoma A549 cells and following transplantation in nude mice.

The aim of the present study was to investigate cobra neurotoxin (cobrotoxin) activity in A549 cell lines transplanted into nude mice, and to explore its molecular mechanism. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the growth inhibition rate of cobrotoxin in human lung A549 adenocarcinoma cells and HFL1 lung fibroblasts. Cell colony formation assays were performed to determine the effect of cobrotoxin on A549 cell colony formation, and transmission electron microscopy was used to detect cobrotoxin autophagy. In addition, western blot analysis was performed to determine the effect of 3-methyl adenine (3-MA) activity on the inhibition of autophagy, SB203580 inhibition of the p38-mitogen-activated protein kinase (MAPK) pathway, and Beclin 1, LC3, p62, p38 and phosphorylated (p)-p38 protein expression. Nude mice were injected with human lung A549 cells, and intervention and control groups were compared with regard to tumor suppression. The MTT assay revealed that various concentrations of cobrotoxin inhibited growth of A549 cells, but not HFL1 cells. A549 cell colony formation decreased and autophagosome activity was significantly increased compared with the controls. Following 3-MA administration, SB203580 autophagosome activity decreased, and following cobrotoxin administration, Beclin 1, p-p38, and LC3-II protein expression significantly increased, whereas p62 expression significantly decreased. Following 3-MA inhibition of autophagy, Beclin 1, LC3-II and p62 expression increased. Furthermore, following SB203580 inhibition of the p38-MAPK pathway, Beclin 1, p-p38, LC3-II and p62 protein expression increased. Cobrotoxin exhibited inhibitory activity on the human lung cancer A549 cells transplanted into the nude mice, suppressing the tumor growth rate by 43.4% (cobrotoxin 40 µg/kg group). However, following the addition of 3-MA (10 mmol/kg) and SB203580 (5 mg/kg), the suppression of the tumor growth rate decreased significantly. Cobrotoxin inhibits the growth of human lung cancer A549 cells in vitro and A549 cells transplanted into nude mice. Furthermore, the induction of autophagy may be associated with the activation of the p38-MAPK pathway.

PIK3CA hypomethylation plays a key role in activation of the PI3K/AKT pathway in esophageal cancer in Chinese patients.

AIM: To investigate the role of PIK3CA oncogene in tumorigenesis and development of esophageal cancer in Chinese patients at the levels of genetic mutation and epigenetics. METHODS: Seventy six esophageal tumor samples and corresponding adjacent normal tissues were collected, and the genomic DNA was extracted. Mutations in the 9th and 20th exons of PIK3CA gene were detected using conventional sequencing. PIK3CA methylation rates in two selected CpG islands (CpG island 1 and 2) were detected using sub-bisulfate modified sequencing. P110α and pAKT expression levels were detected with Western blotting. RESULTS: In PIK3CA gene of the tumor tissues, G1633C (E545Q) mutation was detected in the 9th exon with a rate of 3.95% (3/76), whereas mutation was not found in the 20th exon. Nor mutation did occur in PIK3CA gene of the adjacent normal tissues. The methylation rate of the CpG island 1 had no significant difference between the tumor and adjacent tissues (0.77%±0.009% vs 0.89%±0.008%), but the methylation rate of the CpG island 2 in the esophageal tumors was significantly lower than that in the adjacent tissues (6.00%±2.80% vs 10.45%±5.51%). Furthermore, the rate of methylation of the CpG island 2 in TNM stage III and IV esophageal cancer (3.84%±2.08%) was significantly lower than in stage I (8.52%±2.55%) and stage II (6.42%±2.36%). PIK3CA gene hypomethylation in esophageal cancer was significantly correlated with high expression of p110α. CONCLUSION: PIK3CA gene hypomethylation plays a key role in the tumorigenesis and development of esophageal cancer in Chinese patients, while the mutations of PIK3CA gene have little effect on the development of esophageal cancer.

Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice.

AIM: To investigate the antitumor actions of the Crotalus durissus neurotoxin (crotoxin) on human esophageal carcinoma (Eca-109) cells in vitro and transplanted esophageal Eca-109 tumors in nude mice. METHODS: The growth-inhibitory effect was analyzed in Eca-109 cells using MTT assay. Cell morphology changes in nuclei were observed using Hoechst 33342 staining, while apoptosis and cell cycle distribution were examined by flow cytometry. RT-PCR was used to measure the Bcl-2, p15, and caspase-3 p17 gene expression levels. A tumor transplantation model was established by inoculation of Eca-109 cells were into female Balb/c nude mice. Crotoxin (25, 50, and 100 mg/kg) was subcutaneously injected into the transplanted tumors every 2 d for a total of 10 injections. Tumor size and weight were measured. Bcl-2, p15, and caspase-3 p17 protein expression in transplanted tumors was analyzed using Western blotting. RESULTS: Crotoxin (25, 50, and 100 µg/mL) inhibited the growth of Eca-109 cells in a dose-dependent manner with inhibition rates of 22.9%, 35.8%, and 57.2%, respectively. Hoechst 33342 staining revealed apoptotic cells with pyknotic nuclear chromatin after crotoxin treatment. In Eca-109 cells, crotoxin induced apoptosis and G1 block, significantly upregulated the expression of p15 and caspase-3 p17 genes and downregulated the expression of Bcl-2 gene. Furthermore, crotoxin inhibited the growth of Eca-109 tumors in nude mice in a dose-dependent manner. Western blotting showed that crotoxin increased p15 and caspase-3 p17 protein levels and reduced Bcl-2 protein level in tumor specimens. CONCLUSION: Crotoxin inhibits the growth of Eca-109 cells in vitro via apoptosis induction and G1 block. Local administration of crotoxin inhibits the growth of subcutaneously transplanted Eca-109 cells in nude mice, possibly via increasing p15 and caspase-3 p17 protein expression and reducing Bcl-2 protein expression.

Therapeutic potential of Naja naja atra venom in a rat model of diabetic nephropathy.

OBJECTIVE: To study the protective effects of naja naja atra venom (NNAV) in a rat model of diabetic nephropathy (DN). METHODS: The rat diabetes model was induced by intraperitoneal injection of streptozotocin (STZ). Thirty-two model rats were randomly divided into one DN group (n=8) and three treatment groups (n=8 each) that received NNAV at doses of 30, 90, or 270 µg/(kg·day) via oral gavage, another eight rats as normal controls. After 12 weeks, all rats were sacrificed and the changes in serum and urine biological index levels were determined by colorimetric assay. Microalbumin (mALB), N-acetyl-ß- glucosaminidase (NAG) and cystatin C (CysC) concentrations were measured by ELISA. Renal tissues were sliced for pathological and immunohistochemical observations. RESULTS: Comparied with the DN group, serum glucose was decreased by 31.04%, total cholesterol 21.96%, triglyceride 23.78%, serum creatinine 19.83%, blood urea nitrogen 31.28%, urinary protein excretion 45.42%, mALB 10.42%, NAG 20.65%, CysC 19.57%, whereas albumin increased by 5.55%, high-density lipoprotein-cholesterol 59.09%, creatinine clearance 19.05% in the treatment group by NNAV administration at dose of 90 µg/(kg·day). NNAV also reduced the levels of malondialdehyde in serum (22.56%) and kidney tissue (9.79%), and increased superoxide dismutase concentration in serum (15%) and decreased it in renal tissue (8.85%). In addition, under light microscopy kidney structure was improved and glomerular hypertrophy decreased by 8.29%. As shown by immunohistochemistry, NNAV inhibited transforming growth factor-ß1 by 6.70% and nuclear actor-κB by 5.15%. CONCLUSION: NNAV improves biological indexes in DN, and it may exert renoprotective effects in rats with STZ-induced diabetes.

Crotoxin enhances the antitumor activity of gefinitib (Iressa) in SK-MES-1 human lung squamous carcinoma cells.

Crotoxin (CrTX), a neurotoxin, is isolated from the venom of South American rattlesnakes and has potent antitumor activity. Here, we investigated the antitumor effect of CrTX on the SK-MES-1 human lung squamous cell carcinoma cell line that has acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. CrTX caused G1 arrest and p-JNK protein upregulation that resulted in apoptosis of SK-MES-1 cells. SP600125, a specific inhibitor of p-JNK, could rescue SK-MES-1 cells from CrTX-induced apoptosis. CrTX and gefinitib (Iressa) both inhibited the viability and proliferation of SK-MES-1 cells in a dose- and time-dependent manner. The combination of CrTX and Iressa significantly enhanced the antitumor activity of Iressa. In vivo studies revealed that CrTX caused increased damage to blood vessels and reduced tumor size when combined with Iressa. The present study showed that the JNK signal transduction pathway mediated the anti-apoptotic effect of CrTX, and furthermore, CrTX could enhance the antitumor effect of tyrosine kinase inhibitors in cells with acquired resistance.

Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549.

AIM: To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms. METHODS: A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo. RESULTS: Treatment of A549 cells with CrTX (25-200 µg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC(50)=78 µg/mL). Treatment with CrTX (25 µg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 µg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 µmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G(1) arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 µg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels. CONCLUSION: CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.

Triptolide inhibits NF-kappaB activation and reduces injury of donor lung induced by ischemia/reperfusion.

AIM: To investigate the protective effect of triptolide (TRI) on ischemia/reperfusion-induced injury of transplanted rabbit lungs and to investigate the mechanisms underlying the actions of TRI. METHODS: We established the rabbit lung transplantation model and studied lung injury induced by ischemia/reperfusion and the inhibitory effect of TRI on NF-kappaB. The severity of lung injury was determined by a gradual decline in PvO2, the degree of lung edema, the increase in the myeloperoxidase (MPO) activity, and the ultrastructural changes of transplanted lungs. The activation of NF-kappaB was measured by immunohistochemistry. The increase in intercellular adhesion molecule-1 (ICAM-1), which is the target gene of NF-kappaB, was evaluated by ELISA. RESULTS: After reperfusion, there was a gradual decline in the PvO2 level in the control group (group I). The level of PvO2 in the group treated with lipopolysaccharide (group II) was significantly decreased, whereas that of the group treated with TRI (group III) was markedly improved (P<0.01). In group III, the activity of MPO was downregulated, and the pulmonary edema did not become severe and the ultrastructure of the donor lung remained normal. The activity of NF-kappaB and the expression of ICAM-1 was significantly increased in the donor lungs. TRI blocked NF-kappaB activation and ICAM-1 expression. CONCLUSION: The effects of TRI on reducing injury to donor lungs induced by ischemia/reperfusion may possibly be mediated by inhibiting the activity of NF-kappaB and the expression of the NF-kappaB target gene ICAM-1. Thus, TRI could be used in lung transplantations for improving the function of donor lungs.

Inhibitory effects of prostaglandin A1 on apoptosis of rat cardiac microvascular endothelial cells was mediated by NF-kappaB.

AIM: To study the effects of prostaglandin A1 (PGA1) on rat cardiac microvascular endothelial cells. METH-ODS: Isolated rat cardiac microvascular endothelial cells were cultured in hypoxia and reoxygen conditions, respectively. Endothelial cell apoptosis was detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining. The activity of NF-kappaB was detected by electrophoretic mobility shift assay. Bcl-2 and Bax protein expression were examined by Western blot and bcl-2 mRNA expression was examined by Northern blot. RESULTS: PGA1 reduced endothelial cell apoptosis markedly, inhibited activity of NF-kappaB, and increased expression of Bcl-2 protein and bcl-2 mRNA. However, PGA1 did not alter Bax protein expression resulting in an increase in the ratio of Bcl-2 to Bax. CONCLUSION: PGA1 can inhibit rat cardiac microvascular endothelial cell apoptosis by inhibiting activity of NF-kappaB.