Celastrol acts synergistically with afatinib to suppress non-small cell lung cancer cell proliferation by inducing paraptosis.

Non-small cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is intrinsic resistance to EGFR-tyrosine kinase inhibitors (TKIs), such as afatinib. Celastrol, a natural compound with antitumor activity, was reported to induce paraptosis in cancer cells. In this study, intrinsic EGFR-TKI-resistant NSCLC cell lines H23 (EGFR wild-type and KRAS mutation) and H292 (EGFR wild-type and overexpression) were used to test whether celastrol could overcome primary afatinib resistance through paraptosis induction. The synergistic effect of celastrol and afatinib on survival inhibition of the NSCLC cells was evaluated by CCK-8 assay and isobologram analysis. The paraptosis and its modulation were assessed by light and electron microscopy, Western blot analysis, and immunofluorescence. Xenografts models were established to investigate the inhibitory effect of celastrol plus afatinib on the growth of the NSCLC tumors in vivo. Results showed that celastrol acted synergistically with afatinib to suppress the survival of H23 and H292 cells by inducing paraptosis characterized by extensive cytoplasmic vacuolation. This process was independent of apoptosis and not associated with autophagy induction. Afatinib plus celastrol-induced cytoplasmic vacuolation was preceded by endoplasmic reticulum stress and unfolded protein response. Accumulation of intracellular reactive oxygen species and mitochondrial Ca2+ overload may be initiating factors of celastrol/afatinib-induced paraptosis and subsequent cell death. Furthermore, Celastrol and afatinib synergistically suppressed the growth of H23 cell xenograft tumors in vivo. The data indicate that a combination of afatinib and celastrol may be a promising therapeutic strategy to surmount intrinsic afatinib resistance in NSCLC cells.

miR­139­5p enhances cisplatin sensitivity in non­small cell lung cancer cells by inhibiting cell proliferation and promoting apoptosis via the targeting of Homeobox protein Hox­B2.

The development of chemotherapeutic dug resistance hinders the clinical treatment of cancer. MicroRNAs (miRNAs/miRs) have been revealed to serve essential roles in the drug resistance of numerous types of cancer. miR­139­5p was previously reported to be associated with cisplatin (DDP) sensitivity in human nasopharyngeal carcinoma cells and colorectal cancer cells. However, the effect and underlying mechanism of miR­139­5p in DDP sensitivity in non­small cell lung cancer (NSCLC) cells has not yet been fully elucidated. In the present study, the expression of miR­139­5p and Homeobox protein Hox­B2 (HOXB2) in NSCLC tissues was examined by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. Subsequently, the effect of miR­139­5p on the DDP sensitivity of NSCLC cells in vitro was investigated. Cell proliferation was examined using a Cell Counting Kit­8 assay. Western blotting was used to evaluate the protein expression of HOXB2, phosphorylated (p)­PI3K, p­AKT, caspase­3 and cleaved­caspase­3, and RT­qPCR was used to evaluate the expression of miR­139­5p, and the mRNA expression levels of HOXB2, PI3K, AKT and caspase­3. The apoptotic rate of the cells was detected using flow cytometry. miR­139­5p expression in NSCLC tissues was shown to be significantly lower compared with that in adjacent tissues. Additionally, miR­139­5p increased cell apoptosis and inhibited NSCLC cell proliferation induced by DDP in vitro via modulating the PI3K/AKT/caspase­3 signaling pathway. Furthermore, HOXB2 was identified to be a target of miR­139­5p, and miR­139­5p was revealed to sensitize NSCLC cells to DDP via the targeting of HOXB2. Taken together, the results of the present study demonstrated that regulating the expression of miR­139­5p could provide a novel approach to reverse DDP resistance and increase chemosensitivity in the treatment of NSCLC.

Long Non-Coding RNA (lncRNA) CRNDE Regulated Lipopolysaccharides (LPS)-Induced MRC-5 Inflammation Injury Through Targeting MiR-141.

BACKGROUND Pneumonia is a common disease with high morbidity and even death. In our country, pneumonia is the leading cause of child death. Therefore, research on the pathogenesis of pneumonia can help improve the treatment of pneumonia. Long non-coding RNA (lncRNA) is an important regulator of disease development, and its regulatory mechanism is closely related to cellular processes. However, the function and regulatory network of lncRNA is not fully elucidated in pneumonia. MATERIAL AND METHODS Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of CRNDE and miR-141 in lipopolysaccharides (LPS)-induced MRC-5 cells and pneumonia tissues. MTT (3-(4,5-dimethylthiazol-2-yl)-2 5-diphenyl-2-tetrazolium) assay was used to assess cell proliferation. Flow cytometry assay was performed to detect cell apoptosis in LPS-induced MRC-5 cells. Enzyme-linked immunosorbent assay and western blot were used to measure the levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-alpha, respectively. In addition, luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to prove the relationship between CRNDE and miR-141. RESULTS In this study, we found that CRNDE expression was induced in LPS-induced MRC-5 cells and pneumonia tissues. Moreover, miR-141 expression was low in LPS-induced MRC-5 cells and was verified was a target miRNA of CRNDE by using luciferase reporter assay and RIP assay. The downregulation of CRNDE and upregulation of miR-141 promoted cell viability, inhibited cell apoptosis, as well as decreased the levels of IL-1ß, IL-6, and TNF-alpha. Moreover, we demonstrated that si-CRNDE transfection increased cell viability and suppressed cell apoptosis and the levels of IL-1ß, IL-6, and TNF-alpha, which were alleviated by anti-miR-141 transfection in LPS-induced MRC-5 cells. CONCLUSIONS In this study, we found that downregulation of CRNDE and upregulation of miR-141 inhibited cell apoptosis and inflammation response and promoted cell viability in LPS-induced MRC-5 cells. Low CRNDE expression increased cell growth and suppressed inflammation response, which was impaired by inhibition of miR-141. These results suggested that a novel therapeutic target was found in pneumonia treatment.

LncRNA-ATB promotes apoptosis of non-small cell lung cancer cells through MiR-200a/ß-Catenin.

PURPOSE: Long non-coding RNAs (lncRNAs) play important roles in cancer, but the effects of lncRNA-ATB on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells remain unclear. Therefore, the present study was conducted to explore the role of this lnc-RNA in NSCLC. METHODS: The NSCLC NCI-H838 cell line cultured in vitro were used as the objects of study. First, the expressions of lncRNA-ATB, miR-200a and ß-catenin in cells were detected. Then, the expression of lncRNA-ATB was knocked down using siRNA, and the effects of low expression of lncRNA-ATB on miR-200a/ß-catenin pathway and apoptosis were studied. RESULTS: Compared with normal lung epithelial cells BEAS-2B, NCI-H838 cells had a significantly increased level of lncRNA-ATB (p<0.01), a significantly decreased level of miR-200a (p<0.01) and also a significantly increased level of ß-catenin (p<0.01). After knockdown of lncRNA-ATB using si-ATB, the expression level of miR-200a was significantly increased, while that of ß-catenin was significantly decreased. Besides, si-ATB remarkably increased the expression of Bcl-2 (p<0.01), and reduced the expressions of cleaved-caspase3 and Cytochrome C (p<0.01) and the apoptosis. In addition, the miR-200a mimic lowered obviously the expression of ß-catenin (p<0.05) and reduced apoptosis. CONCLUSIONS: This study suggests that lncRNA-ATB promotes the apoptosis of NSCLC cells through inhibiting the expression of miR-200a and reversely promoting the expression of ß-catenin.

[Experimental Research of Hg2+Removal by TiO2/Bentonite Composite].

The TiO2/bentonite composite was synthesized by modifying calcium-based bentonite with Nano-TiO2. The products before and after modification were characterized via the approach of X-ray power diffraction(XRD) and scanning electron microscope(SEM).The effect of TiO2/bentonite composite on mercury removal from aqueous solutions of HgCl2was studied at different dosage, pH, adsorption time and the initial concentration of Hg2+ was investigated and compared with the bentonite by indoor simulation experiment, as well as the orthogonal experiments to determine the optimal condition of Hg2+ adsorption. The experimental results showed:after modified by TiO2, TiO2/bentonite composite particles were apparently smaller, the basal spacing was increased and with a loose and porous structure. The adsorption rates of TiO2/bentonite composite on Hg2+ were increased compared with bentonite. The Hg2+ adsorption rates were increased with the increasing dosages, pH and adsorption time. The adsorption rates were higher than 98.0% when the dosage was 1.5 g·L-1, pH 7.0, and the adsorption time was 120 min. The adsorption rates became smaller with increasing initial concentration of Hg2+. False secondary dynamic equation could describe the adsorption of TiO2/bentonite composite on Hg2+, and the chemical adsorption was dominant. The adsorption isotherm of Hg2+ conformed to Langmuir equation, indicating that the adsorption of Hg2+ was typical monolayer adsorption. The optimal experimental condition was:dosage of 2.0 g·L-1, pH 8.0, adsorption time of 16 h and the initial Hg2+concentration of 45 mg·L-1. Under this condition, the adsorption rate was 99.9%, and the equilibrium concentration of Hg2+ was 0.034 mg·L-1.

Gene mutation analysis in non-small cell lung cancer patients using bronchoalveolar lavage fluid and tumor tissue as diagnostic markers.

Non-small cell lung cancer (NSCLC) is one of the main causes of cancer death in the world. Early detection of NSCLC can improve its outcome. The aim of this study was to identify the mutations of the KRAS and p53 genes in bronchoalveoar lavage (BAL) fluid for the early detection of peripheral NSCLC. We examined the DNA obtained from the tumor, nearby normal lung tissue, and matched BAL fluid for mutations in the KRAS and p53 genes; the material was obtained from 48 patients with peripheral NSCLC, and was analyzed by PCR-single strand conformation polymorphism and DNA sequencing. BAL fluids from 26 patients with benign lung disease were used as controls. Positive rates of KRAS and p53 mutations were distributed as follows: in NSCLC tissue, 52% and 58%; in BAL fluid of NSCLC patients, 38% and 44%; in normal lung tissue, 6% and 4%; and in BAL fluid of patients with benign lung disease, 8% and 4%. The combined detection of both KRAS and p53 mutations yielded a sensitivity of 66% for the diagnosis of peripheral NSCLC, which is markedly higher than that of cytology plus histology by first bronchoscopy (38%, p=0.008). In each patient with the 2 gene mutations in BAL fluid, mutation type and location were the same as those of the primary tumor. Our study indicates that the detection of the KRAS and p53 mutations in BAL fluids could be a helpful addition to cytology and histology examination for the diagnosis of peripheral NSCLC.

Evaluation of diagnostic value of four tumor markers in bronchoalveolar lavage fluid of peripheral lung cancer.

AIM: The diagnostic role of carcinoembryonic antigen (CEA), squamous cell carcinoma (SCC) antigen, Cyfra 21-1 and neuron-specific enolase (NSE) in the bronchoalveolar lavage fluid (BALF) for lung cancer is still controversial. The aim of this study was to evaluate the diagnostic value of these four tumor markers in BALF for peripheral lung cancer. METHODS: We measured and compared the levels of CEA, SCC, Cyfra21-1 and NSE in BALF in 42 patients with peripheral lung cancer and 22 patients with benign lung disease. In the patients with peripheral lung cancer, the BAL was separately performed in the bronchus of the tumor-bearing lung and in the corresponding bronchus of the opposite healthy lung. RESULTS: The levels of CEA, SCC, Cyfra21-1 and NSE were significantly elevated in BALF from the tumor-bearing lung compared with the opposite healthy lung in the lung cancer patients (P < 0.001) or the benign lung disease patients (P < 0.005). The diagnostic sensitivities of Cyfra21-1 (86 and 76%), with a specificity of 91%, were the highest among the four tumor markers for the tumor-bearing lung versus the opposite healthy lung and benign lung disease. The combination of Cyfra21-1 and CEA increased the sensitivity to 93 and 86 percent, respectively. CONCLUSION: The assay of these tumor markers in BALF may be used as a diagnostic tool to complement a cytological examination in the diagnosis of peripheral lung cancer.