High expression of HMBOX1 promotes the progression of lung squamous cell carcinoma.

This study aimed to explore the expression of homeobox-containing 1 (HMBOX1) and its clinical significance in lung squamous cell carcinoma (LSCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of HMBOX1 in LSCC tissues. The relationship between HMBOX1 expression and clinical pathological data was analyzed by Chi-square or Fisher's exact test. Furthermore, the role of HMBOX1 in LSCC in vitro was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), flow cytometry and Western blot (WB) assays. HMBOX1 was highly expressed in LSCC tissues when compared with para-cancer tissues (P<0.05). According to the median expression of HMBOX1, the patients were divided into two groups, including high-expression group and low-expression group. HMBOX1 expression was correlated with tumor size, differentiation and clinical stage (P<0.05). Subsequent experiments indicated that LSCC cells with low expression of HMBOX1 exhibited significantly inhibited proliferation, G0/G1 block and promoted apoptosis (P<0.05). HMBOX1 expression was positively correlated with the expression of VEGF, elaborating that HMBOX1 probably promoted the growth of LSCC cells by affecting VEGF. HMBOX1 might contribute to the development of LSCC.

MPZL1 suppresses the cancer stem-like properties of lung cancer through ß-catenin/TCF4 signaling.

This study was designed to explore the influence of myelin protein zero-like protein 1 (MPZL1) on the stem-like properties of cancer cells and the underlying mechanism in lung adenocarcinoma. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to evaluate mRNA expression level. CCK8, wound healing, and transwell assays were applied to assess cell proliferation, migration, and invasion. Tumorsphere-formation assay was utilized to assess cancer stem cell-like properties. LF3 was used to block the ß-catenin/Transcription factor 4 (TCF-4) signaling. Xenograft nude mouse model was conducted; tumor weight and volume were recorded. Western blot assay was utilized to detect the expression levels of CD44, CD133, ß-catenin, TCF-4, and MPZL1. Following MPZL1 knockdown, the mRNA expression levels of MPZL1, ß-catenin, and TCF-4 were inhibited, while the mRNA expression levels of the above genes were increased after the MPZL1 overexpression. MPZL1 knockdown suppressed cell proliferation, migration, and invasion, reduced the tumorsphere-formation capacity, and restrained the expression levels of CD44 and CD133. However, MPZL1 overexpression promoted the cell proliferation, migration, and invasion, enhanced the tumorsphere-formation capacity, and increased the expression levels of CD44 and CD133. Interestingly, LF3 treatment partially revised the effect of MPZL1 overexpression. These findings were further corroborated by in vivo experiments. We concluded that MPZL1 could suppress the lung adenocarcinoma cells' proliferation, migration, invasion, and lung cancer stem cells characteristics. The underlying mechanism is involved in the activation of ß-catenin/TCF-4 signaling.

Autophagic tumor-associated macrophages promote the endothelial mesenchymal transition in lung adenocarcinomas through the FUT4/p-ezrin pathway.

BACKGROUND: Lung adenocarcinoma is one of the most common malignant tumors with high morbidity and mortality, but the effect of Tumor-associated macrophages (TAMs) on lung adenocarcinoma has not been studied clearly now. METHODS: In this study, TAMs were stably transfected with Atg5 silence or overexpression lentiviral vectors to inhibit or induce autophagy of TAMs. In addition, the expression of fucosyltransferase IV (FUT4) or Ezrin were interfered in TAMs with autophagy. The above treated TAMs were then co-cultured with A549 or H1299 cells. The expressions of genes were detected by qPCR, western blotting, cell immunofluorescence, and enzyme-linked immunosorbent assay. Meanwhile, cell migration and invasion were analyzed by Transwell assay and wound healing assay. Furthermore, the effects of TAMs with autophagy were explored in lung adenocarcinoma xenograft model of mice. RESULTS: The results showed that overexpression of autophagy-related gene 5 (ATG5) induced autophagy in TAMs, which increased the expression of FUT4, TGF-ß1, and p-ezrin, and promoted epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. However, FUT4 silencing partially reversed the effects of TAM autophagy, specifically, the expression of TGF-ß1 and p-ezrin was inhibited and EMT in lung adenocarcinoma cells was suppressed. Notably, ezrin deletion in autophagic TAMs induced by rapamycin reduced TGF-ß1 expression and suppressed EMT in lung adenocarcinoma cells. Consistently, in vivo experiments also revealed that autophagic TAMs increased the expression of FUT4, TGF-ß1, and p-ezrin, and promoted EMT in lung adenocarcinomas. Similarly, FUT4 silencing partially reversed the effects of autophagic TAMs on EMT in lung adenocarcinomas. CONCLUSIONS: In conclusion, autophagic TAMs promoted TGF-ß1 secretion through the FUT4/p-ezrin pathway and induced EMT in co-cultured lung adenocarcinoma cells.

Lysosome Fe2+ release is responsible for etoposide- and cisplatin-induced stemness of small cell lung cancer cells.

Iron metabolism has been shown to hand over cancer stem cell, which is regarded as the root of tumor progression, recurrence and chemoresistance. This study aims to explore whether iron metabolism is involved in etoposide- and cisplatin-induced stemness in small cell lung cancer (SCLC) cells. Here, analysis on tumor-sphere formation and stemness marker expression is performed to determine whether etoposide and cisplatin can induce SCLC cell stemness. Online dataset analysis is constructed to determine the correlation between iron transportation and the survival of lung cancer patients. Chromatin immunoprecipitation combined with rescuing experiments are carried out to reveal the underlying mechanisms. Additionally, the non-lethal doses of etoposide and cisplatin can induce SCLC cell stemness in a concentration-dependent manner and reduce the lysosome iron concentration dependent on Ferritin expression, which is positively regulated by HIF-1α/ß. Moreover, HIF-1α/ß can directly bind to Ferritin promoter region. This HIF/Ferritin axis is responsible for etoposide- and cisplatin-induced iron reduction in lysosomes and stemness of SCLC cells. This work demonstrates that iron in lysosomes is essential for etoposide and cisplatin-induced stemness of SCLC cells, which is regulated by the HIF/Ferritin axis.

Acetylation may strengthen the antitumor activity of low molecular heparin.

BACKGROUND: To synthesize acetylated low anticoagulant low molecular weight heparin (ALMWH) and to detect its antineoplastic activity. METHODS: We obtained Low anticoagulant low molecular weight heparin (LMWH) by splitting unfractionated heparin (UFH) with sodium periodate oxidation and sodium borohydride reduction, then the LMWH was subjected to acetylate catalyzed by dicyclohexylcarbodiimide and dimethylaminopyridine to produce ALMWH. The anti-proliferative activities were determined on MDA-MB-231 human breast cancer cells in vitro. RESULTS: ALMWH exhibited stranger anti-proliferative activity Compared with LMWH, In the MDA-MB-231 cell line, the growth of MDA-MB-231 cells with IC50 of 22.16 µM at 48 h in a concentration-dependent and time-dependent manner, ALMWH produced stronger inhibitory effects especially when it was used in low concentrations. By the use of bulky catalysts, the acetylation site in the molecular chain of low molecular weight heparin with a high selectivity, the synthesis process of Low anticoagulant low molecular weight heparin can be easily controlled. Therefore, large scale industrial production can be carried out. CONCLUSIONS: The synthesized ALMWH possesses a high anti-proliferative activity, Chemical modification of structure can endow LMWH with a high antiproliferative activities. ALMWH is expected to enter clinical trials due to its high druggability. Simultaneously, this study provides a basic method for screening of antineoplastic drug with low toxicity.

lncRNA ZEB2-AS1 Aggravates Progression of Non-Small Cell Lung Carcinoma via Suppressing PTEN Level.

BACKGROUND The aim of this study was to assess the involvement of lncRNA ZEB2-AS1 in the development of NSCLC and to explore the potential mechanism involved. MATERIAL AND METHODS ZEB2-AS1 expressions in 48 paired NSCLC tissues and paracancerous tissues were examined by qRT-PCR. ZEB2-AS1 level in NSCLC patients affected by tumor staging and lymphatic metastasis was examined as well. Regulatory effects of ZEB2-AS1 on proliferative, migratory, and invasive properties of NCI-H1650 and HCC827 cells were evaluated. The interaction between ZEB2-AS1 and EZH2 was identified through RIP assay. Subsequently, the binding of EZH2 on PTEN promoter region was tested by ChIP. Finally, rescue experiments were conducted to assess the involvement of PTEN in the development of NSCLC. RESULTS ZEB2-AS1 was upregulated in NSCLC tissues and cell lines. Its level was higher in NSCLC patients with T3-T4 or accompanied with lymphatic metastasis relative to those with T1-T2 or without metastatic loci. Knockdown of ZEB2-AS1 suppressed proliferative, migratory, and invasive properties of NCI-H1650 and HCC827 cells. PTEN level was elevated after knockdown of ZEB2-AS1 or EZH2 in HCC827 cells. Subsequently, RIP assay proved the interaction between ZEB2-AS1 and EZH2. Knockdown of ZEB2-AS1 markedly reduced the binding of EZH2 on the PTEN promoter region. Notably, knockdown of PTEN reversed the effects of EZB2-AS1 on regulating proliferative, migratory, and invasive properties of NSCLC cells. CONCLUSIONS lncRNA ZEB2-AS1 is upregulated in NSCLC, which elevates the viability and malignant degree of NSCLC cells by downregulating PTEN, thus aggravating the progression of NSCLC.

LEF1-AS1 contributes to proliferation and invasion through regulating miR-544a/ FOXP1 axis in lung cancer.

Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulators in tumor development. This study aims to investigate the potential role oflncRNALEF1-AS1, in the progression of lung cancer. Quantitative real-time PCR (qRT-PCR) and western blot assays showed that LEF1-AS1 was upregulated while miR-544a was downregulated in lung cancer specimens and cells. Overexpression of LEF1-AS1 led to the enhancement of cell proliferation and invasion, revealed by CCK-8 assay and transwell assay. A negative correlation was found between LEF1-AS1 and miR-544a. BLAST analysis and dual-luciferase assay confirmed that FOXP1 is a downstream effector of miR-544a. Therefore, the LEF1-AS1/miR-544a/FOXP1 axis is an important contributor to lung cancer progression. Collectively, our novel data uncovers a new mechanism that governs tumor progression in lung cancer and provides new targets that may be used for disease monitoring and therapeutic intervention of lung cancer.

Effects of mesenchymal stem cells harboring the Interferon-ß gene on A549 lung cancer in nude mice.

Interferon-ß (IFN-ß) exhibits a tumor-killing effect; however, injection of IFN-ß alone for lung cancer is often accompanied by side effects. This study investigated the possibility of using umbilical cord mesenchymal stem cells (MSCs) as cellular carriers of IFN-ß. Isolated umbilical cord MSCs were transfected with a lentivirus packaging IFN-ß-overexpression plasmid. A549 cells were subcutaneously injected into nude mice to establish a non-small cell lung cancer (NSCLC) mouse model. A total of 50 mice were randomly assigned to 5 different groups: a control group, IFN-ß group, IFN-ß-MSCs group, MSCs-lentivirus group, and MSCs group. Next, the IFN-ß-MSCs, MSCs-lentivirus, and MSCs were injected into the A549 lung cancer-bearing mice in the IFN-ß-MSCs, MSCs-lentivirus and MSCs groups, respectively. Mice in the control and IFN-ß groups were injected with solvent or IFN-ß solution. The tumors in nude mice in the IFN-ß and IFN-ß-MSCs groups grew at significantly slower rates than tumors in the control group, and tumors in the MSCs-lentivirus and MSC groups also grew slowly. The rates of tumor cell apoptosis in the IFN-ß and IFN-ß-MSCs groups were significantly higher than those in the MSCs-lentivirus and MSCs groups. The livers, lungs, and kidneys of nude mice in the IFN-ß group displayed hyperemia, exudation, and pathological lesions, while those of nude mice in the IFN-ß-MSCs group showed no abnormal changes. Both INF-ß-MSCs and INF-ß inhibited the growth of subcutaneously implanted lung tumors; however, INF-ß-MSCs specifically targeted the tumor cells, and did not produce the damage to internal organs caused by the use of INF-ß alone.

RGD peptide-modified, paclitaxel prodrug-based, dual-drugs loaded, and redox-sensitive lipid-polymer nanoparticles for the enhanced lung cancer therapy.

One approach to improve the targeted therapeutic efficiency of lung cancer is to deliver drugs using nano-scaled systems. In this study, RGD peptide-modified, paclitaxel (PTX) prodrug-based, dual-drugs loaded, and redox-sensitive lipid-polymer nanoparticles were developed and the in vitro and in vivo antitumor efficiency was evaluated in lung cancer cells and tumor bearing animal models. RGD-modified PTX and cisplatin (CDDP) loaded LPNs (RGD-ss-PTX/CDDP LPNs) have sizes around 190 nm, and zeta potentials of -35 mV. The half-maximal inhibitory concentration (IC50) values were 26.7 and 75.3 µg/mL for drugs loaded LPNs and free drugs combination, which indicates significantly higher antitumor activity of LPNs than free drugs. RGD-ss-PTX/CDDP LPNs also exhibited the best antitumor efficiency in vivo, which inhibited the tumor size of mice from 1486 mm3 to 263 mm3. The results illustrated that the system could successfully load drugs and achieve synergistic combination lung cancer treatment efficiency with lower systemic toxicity compared with free drugs counterparts. The resulting system could be facilitated as a promising targeted nanomedicine for the treatment of lung cancer.

Reduced RKIP expression levels are associated with frequent non-small cell lung cancer metastasis and STAT3 phosphorylation and activation.

The current study examined the role of Raf kinase inhibitor protein (RKIP) in non-small cell lung cancer (NSCLC) metastasis. A total of 100 patients with NSCLC were recruited following pathological diagnosis in the First Affiliated Hospital of Bengbu Medical College. The patients were classified and statistically analyzed according to their clinicopathological characteristics and tumor-node-metastasis stage. Paired tumor tissue and adjacent non-tumor tissue samples were subject to pathological diagnosis and western blot analysis. Transient transfection and lentivirus particle vector-mediated RKIP overexpression, small interfering RNA-mediated silencing, Transwell assays and immunocytochemistry methods were employed to elucidate the role and underlying mechanisms of RKIP and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in NSCLC metastasis. Furthermore, in order to examine the in vivo effects of RKIP, recombinant lentivirus particles containing the RKIP gene were administrated in a mouse NSCLC tumor model via tail vein injection. The results revealed reduced RKIP expression levels in NSCLC tissue compared with corresponding non-cancer tissue. Additionally, RKIP expression levels were inversely associated with NSCLC intra-lung, lymph node and long-distance metastasis. The results also indicated that RKIP was able to block STAT3 activation via phosphorylation and inhibit NSCLC-cell metastasis in vitro. Furthermore, RKIP knockdown was able to promote STAT3 phosphorylation and cell metastasis in NSCLC cell lines. During in vivo experiments, RKIP overexpression was able to suppress xenograft tumor metastasis in nude mice. Therefore, RKIP may be an important factor in cancer cell metastasis in patients with NSCLC, and RKIP may inhibit NSCLC-cell invasion by blocking the activation of the JAK/STAT3 signaling pathway.

[Establishment of a porcine model of congenital heart defect with decreased pulmonary blood flow].

OBJECTIVE: To establish an animal model of congenital heart defect with decreased pulmonary blood flow for better understanding the pathophysiology of pulmonary vascular development and related regulatory mechanisms of congenital heart defect with decreased pulmonary blood flow. METHOD: One to two months old pigs were randomly divided into three groups: control group (group C, n = 6) with right chest small incisions induced transient pulmonary blood reduction; light-moderate stenosis groups (group T(1), n = 7): artificial atrial septum defect (ASD) plus controlled pulmonary artery banding to generate a systolic pressure gradient of 20 - 30 mm Hg (1 mm Hg = 0.133 kPa); severe stenosis groups (group T(2), n = 7): similar surgical procedures as group T(1), and controlled pulmonary artery banding to generate a systolic pressure gradient ≥ 30 - 50 mm Hg. 64-slice computed tomography scanning was performed at one month post operation. Arterial blood gas analysis, hemoglobin value, pulmonary vessel, ASD and banding ring diameters and trans-pulmonary artery banding pressure (Trans-PABP) were determined at two months post operation. RESULTS: One pig died due to tracheal intubation accident in the C group, one pig died due to bowel obstruction in the T(1) group and two pigs died due to acute right heart failure and chronic heart failure respectively in T(2) group. 64-slice CT angiography results showed that aortic diameter of T(1) group was significantly lower than that of C group and banding diameter was significantly lower than aortic diameter in the T(1) and T(2) groups at one month post operation. Two months after operation, the size of ASD were (8.0 ± 0.5) mm and (8.9 ± 1.4) mm (P > 0.05) respectively in the T(1) and T(2) groups after operation. The Trans-PABP was significantly higher in the T(1) and T(2) groups than in C group (P < 0.01), and the Trans-PABP was significantly higher in the T(2) group than in T(1) group (P < 0.01). PaO2 and SaO2 in the T(1) and T(2) groups were significantly lower than those in C group. CONCLUSION: Artificial atrial septum defect combined pulmonary artery banding procedures could be successfully used to establish model of congenital heart defect with decreased pulmonary blood flow and this model could help to understand the pathophysiology and monitor therapy efficacy for patients with congenital heart defect with decreased pulmonary blood flow.

[Establishment of a cyanotic congenital heart defect porcine model with decreased pulmonary blood flow].

OBJECTIVE: To establish a porcine model of congenital heart disease with decreased pulmonary blood to explore the morphological changes of immature pulmonary vascular vessels. METHODS: Twenty piglets (one to two-month-old) were randomly divided into three groups: sham-operated group (group S, n = 6), small incisions on the right chest, produced a transient reduction in pulmonary blood; Operation group 1(group T(1), n = 7), small incisions on the right chest, producing artificial atrial defect with self-made dilator and simultaneous banding pulmonary artery to generate a systolic pressure gradient between 20 - 30 mm Hg (1 m Hg = 0.133 kPa); Operation group 2(group T(2), n = 7): operation procedure was similar as group T(1) with systolic pressure gradient between 30 - 50 mm Hg. Lung tissue from right middle lobe (1.0 cm×0.8 cm×0.8 cm) was taken immediately after thoracotomy, at the end of surgery and at 2 months after operation and stained by Weigert (elastic fiber) and van Gieson (collagen) methods to observe the morphological changes. RESULTS: Five animals survived in Group S, 6 animals survived in group T(1) and 5 animals survived in group T(2). The inside diameter of pulmonary arterioles after thoracotomy and at the end of surgery was similar among the three groups (P > 0.05). At 2 months after operation, the inside diameter of pulmonary artery was significantly higher in group T(1) and T(2) than in group S (all P < 0.05) while the number of pulmonary small artery per square centimeter (APSC) of group T(1) and T(2) was significantly lower than that of group S (all P < 0.05). Tunica media of pulmonary artery was thinner and vascular lumen was larger in group T(1) and T(2) compared to those of group S. CONCLUSION: In this piglets model with reduced pulmonary blood, the pulmonary arterioles underwent dysplastic changes. Thus, pulmonary blood flow is an important determinant for the physiological development of pulmonary artery.