Lotus leaf flavonoids induce apoptosis of human lung cancer A549 cells through the ROS/p38 MAPK pathway.

BACKGROUND: Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. METHODS: In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. RESULTS: LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaempferitrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. CONCLUSIONS: We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.

Lotus leaf flavonoids induce apoptosis of human lung cancer A549 cells through the ROS/p38 MAPK pathway

BACKGROUND: Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. METHODS: In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. RESULTS: LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaemp-feritrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. CONCLUSIONS: We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.

Effect of Malus asiatica Nakai Leaf Flavonoids on the Prevention of Esophageal Cancer in C57BL/6J Mice by Regulating the IL-17 Signaling Pathway.

BACKGROUND: The aim of this study was to observe the preventive effect of flavonoids extracted from Malus asiatica Nakai leaves (FMANL) on esophageal cancer in mice, especially the ability of FMANL to regulate the interleukin 17 (IL-17) signaling pathway during this process. MATERIALS AND METHODS: The C57BL/6J mice were treated with 4-nitroquinoline N-oxide (4NQO) to induce esophageal cancer, and the visceral tissue index and the serum and esophageal tissue indexes of mice were used to verify the effect of FMANL. RESULTS: The experimental results showed that FMANL can effectively control the changes in visceral tissue caused by esophageal cancer. FMANL could increase the cytokine levels of interleukin 10 (IL-10), monocyte chemotactic protein 1 (MCP-1) and decrease the cytokine levels of tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ), interleukin 6 (IL-6), and interleukin 12p70 (IL-12p70) in serum of mice with esophageal cancer. FMANL could also reduce CD3+, CD4+, and CD8+ and enhance CD19+ mouse peripheral blood lymphocytes. The results of qPCR and Western blot analysis showed that FMANL could down-regulate the mRNA and protein expression levels of IL-17, interleukin 23 (IL-23), interleukin 1 beta (IL-1ß), chemokine (C-X-C) ligand 1 (CXCL1), chemokine (C-X-C) ligand 2 (CXCL2), S100 calcium-binding protein A8 (S100A8), S100 calcium-binding protein A9 (S100A9), matrix metalloprotein 9 (MMP-9), and matrix metalloprotein 13 (MMP-1) in mice with esophageal cancer. High-performance liquid chromatography (HPLC) detection showed that FMANL contained 10 chemicals, including rutin, hyperoside, isoquercitrin, dihydroquercetin, quercitrin, hesperidin, myricetin, baicalin, neohesperidin dihydrochalcone, and quercetin. CONCLUSION: It could be concluded that FMANL can effectively prevent experimentally induced esophageal cancer in mice, and its effects might be obtained from 10 compounds present in FMANL.

Berberine displays antitumor activity in esophageal cancer cells in vitro.

AIM: To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS: Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS: Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 µmol/L berberine for 48 h, the number of cells in G2/M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION: Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.

MicroRNA-506 inhibits esophageal cancer cell proliferation via targeting CREB1.

MicroRNAs (miRNAs) act as key regulators of multiple cancers. MicroRNA-506 (miR-506) functions as a tumor suppressor in various types of cancers. However, its role in esophageal cancer remains unclear. In our study, we found that miR-506 was significantly down-regulated in esophageal cancer tissues and cell lines. In vitro assay, our results showed that ectopic over-expression of miR-506 inhibited esophageal cancer cells proliferation, meanwhile, cells proliferation was promoted by miR-506 inhibition. In exploring mechanisms underlying the inhibitive role, we found that miR-506 significantly decreased the expression and transcription activity of cAMP responsive element binding protein 1 (CREB1). CREB1, tumor oncogene, exhibited significantly promote effect on esophageal cancer cell proliferation. Taken together, our data identify a new role of miR-506 in esophageal cancer involving CREB1 suppression.

Expression of long non-coding RNA ZEB1-AS1 in esophageal squamous cell carcinoma and its correlation with tumor progression and patient survival.

BACKGROUND: LncRNA ZEB1-AS1 has been identified as a tumor oncogene in hepatocellular carcinoma. However, the clinical significance in esophageal squamous cell carcinoma (ESCC) is still unknown. The aim of this study was to explore ZEB1-AS1 expression levels and evaluated its clinical significance in ESCC patients. METHODS: LNCRNA ZEB1-AS1 expression was determined by quantitative real-time PCR (QRT-PCR) in 87 pairs of ESCC specimens and adjacent non-tumor tissues. Then, the association of ZEB1-AS1 expression with clinicopathological factors or survival of ESCC patients were determined. RESULTS: LNCRNA ZEB1-AS1 was found up-regulated in ESCC tissues compared to adjacent non-tumor tissues. Increased lncRNA ZEB1-AS1 expression was significantly associated with tumor grade, depth of invasion, and lymph node metastasis. Kaplan-Meier analysis revealed that ESCC patients with high ZEB1-AS1 expression had a poorer overall survival and disease-free survival. Furthermore, multivariate analysis suggested that ZEB1-AS1 expression was identified as an independent prognostic factor in patients with ESCC. CONCLUSION: These results indicated that lncRNA ZEB1-AS1 was associated with tumor progression and could be an independent prognostic factor for ESCC patients.

Downregulation of microRNA-382 is associated with poor outcome of esophageal squamous cell carcinoma.

AIM: To study the potential prognostic role of microRNA-382 (miR-382) in esophageal squamous cell carcinoma (ESCC). METHODS: Forty six patients were divided into 2 groups according to postoperative survival time: the poor outcome group (28 patients), who showed early metastasis but no recurrence, and died within 1 year after surgery, 12 patients of the group received postoperative chemotherapy treatment that was given after early metastasis happening; the good outcome group (18 patients), who had no clinical metastasis and recurrence, and survived 5 years or more after surgery, all patients did not receive any postoperative treatment. Total RNA was extracted from the patients' formalin-fixed and paraffin-embedded esophageal cancer tissues. miR-382 level was evaluated using high-throughput real-time quantitative polymerase chain reaction analysis. The correlation between miR-382 level and clinicopathologic features was analyzed through COX regression model, and Kaplan-Meier analysis was used to analyze the relationship between miR-382 level and patient survival time. RESULTS: miR-382 was differentially expressed in the two groups. Overall the average miR-382 level in the ESCC patients with good outcome was 9.8 ± 3.8, while miR-382 level in the ESCC patients with poor outcome was 3.0 ± 0.8. The differences of miR-382 levels between two groups were significant (P < 0.05). Kaplan-Meier analysis results showed that miR-382 expression level generally had a significant reverse-correlation with ESCC patient survival time (P < 0.001), in which the patients with higher expressions of miR-382 had a longer survival time either among individuals with the same tumor stage or among the overall patients. CONCLUSION: miR-382 levels are reverse-correlated with ESCC poor outcomes, suggesting that miR-382 could be a potential predictive biomarker for both prognosis and treatment of ESCC.

Expression of flotillin-2 in human non-small cell lung cancer and its correlation with tumor progression and patient survival.

INTRODUCTION: Recent studies have revealed that flotillin-2 (FLOT2) played important roles in cancer progression. The aim of this study was to investigate the clinicopathologic and prognostic significance of FLOT2 expression in human non-small cell lung cancer (NSCLC). METHODS: Quantitative real-time PCR (qRT-PCR) was performed to detect FLOT2 mRNA expression in lung cancer cell lines, normal bronchial epithelial cells, 24 pairs of NSCLC tissues and matched adjacent non-tumor tissues. Immunohistochemistry (IHC) was performed to examine FLOT2 protein expression in paraffin-embedded tissues from 90 NSCLC patients. Statistical analyses were performed to evaluate the clinicopathological significance of FLOT2 expression. RESULTS: FLOT2 mRNA expression was evidently up-regulated in lung cancer cell lines and NSCLC tissues compared with normal bronchial epithelial cells and adjacent non-tumor tissues. In the 90 cases of tested NSCLC samples, FLOT2 protein level was positively correlated with tumor stage, and lymph node metastasis. Patients with high FLOT2 expression had shorter overall survival compared with the low FLOT2 expression group. Univariate and multivariate analyses indicated that high FLOT2 expression was an independent poor prognostic factor for NSCLC patients. CONCLUSIONS: Our findings provided that high FLOT2 expression was associated with poor outcomes in NSCLC patients, and FLOT2 could be a potential prognostic biomarker for lung cancer progression.

Expression of miR-32 in human non-small cell lung cancer and its correlation with tumor progression and patient survival.

INTRODUCTION: miR-32 has recently been found to be implicated in many critical processes in various types of human cancer. However, its clinical significance in human non-small cell lung cancer (NSCLC) has not yet been elucidated. In the present study, we investigated the expression of miR-32 in NSCLC and analyzed its association with clinical features and prognosis of NSCLC patients. METHODS: Quantitative real-time PCR (qRT-PCR) was used to measure expression level of miR-32 in lung cancer cell lines, normal bronchial epithelial cells, 90 pairs of tumor samples and adjacent non-tumor tissues. To determine its prognostic value, overall survival was evaluated using the Kaplan-Meier method. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis. RESULTS: The expression of miR-32 was significantly decreased in lung cancer cell lines and NSCLC tissues compared with normal bronchial epithelial cells and adjacent non-tumor tissues (P < 0.05). This reduction of miR-32 was associated with tumor stage and lymph node metastasis (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with low miR-32 expression had shorter overall survival time than those with high miR-32 expression (P < 0.05). Univariate analysis revealed statistically significant correlations between overall survival and miR-32 level, tumor stage and lymph node metastasis (P < 0.05). Furthermore, miR-32 levels, tumor stage and lymph node metastasis were independently associated with overall survival (P < 0.05). CONCLUSIONS: Our results provided the first evidence that down-regulation of miR-32 was correlated with NSCLC progression, and miR-32 might be a potential molecular biomarker for predicting the prognosis of patients.

Involvement of microRNA-198 overexpression in the poor prognosis of esophageal cancer.

OBJECTIVE: This study aimed to investigate whether the miR-198 expression level is related to clinicopathological factors and prognosis of esophageal cancer. METHODS: MicroRNA was extracted from esophageal cancer patients who underwent surgery for assessment using the Taqman@ MicroRNA assay. The correlation between miR-198 expression and clinicopathological features was analyzed, and the significance of miR-198 as a prognostic factor and its relationship with survival was determined. RESULTS: MicroRNA-198 (miR-198) expression was higher in patients with poor prognosis than those with good prognosis (P < 0.05). Kaplan-Meier analysis results showed that the miR-198 expression level had a significant correlation with survival time (P = 0.030) and that patients with a higher expression of miR-198 had a shorter survival time. Cox multi-factor model analysis showed that patient prognosis (P = 0.014), tumor length (P = 0.040) and expression (P = 0.012), and survival time had a significant correlation; the corresponding risks were 7.268, 1.246, and 3.524, respectively. CONCLUSION: miR- 198 overexpression is involved in the poor prognosis of esophageal cancer and can be used as a biomarker for selection of cases requiring especial attention.

Differential expression of miRNAs in esophageal cancer tissue.

The aim of this study was to explore the association of specific microRNAs (miRNAs) with the development of esophageal cancer (EC) and to identify new molecular markers for EC by analyzing the expression profiles of miRNAs in EC tissues. The expression profiles of miRNAs in paired EC and paracancerous normal tissues were detected and bioinformatically analyzed using miRNA assays. The outcomes were validated using real-time polymerase chain reaction. The miRNA assays revealed a total of 60 differentially expressed miRNAs in the EC tissues compared with those in the paracancerous normal tissues. Among them, 51 had doubled or more than doubled their expression levels and 9 had halved their expression levels. The most markedly upregulated miRNAs were hsa-miR-15a, hsa-miR-28-3p, hsa-miR-31, hsa-miR-99b, hsa-miR-101, hsa-miR-130a, hsa-miR-143, hsa-miR-196b, hsa-miR-200a, hsa-miR-210, hsa-miR-452 and hsa-miR-27a, whereas the most markedly downregulated miRNAs included hsa-miR-30b, hsa-miR-223, hsa-miR-454, hsa-miR-486, hsa-miR-574-3p and hsa-miR-126. Specific miRNA expression profiles exist in EC tissues and may serve as novel EC molecular markers.

miR-19a promotes cell growth and tumorigenesis through targeting SOCS1 in gastric cancer.

Accumulating evidence has shown that microRNAs are involved in cancer development and progression. However, it remains unknown about the potential role of miR-19a in the pathogenesis of gastric cancer. Here, we report that suppressor of cytokine signaling 1 (SOCS1) is a novel target of miR-19a in gastric cancer cells and that miR-19a expression is inversely correlated with SOCS1 expression in gastric cancer cells and a subset of gastric cancer tissues. Ectopic expression of miR-19a dramatically promoted proliferation and tumorigenicity of gastric cancer cells both in vitro and in vivo. Moreover, we showed that silencing of SOCS1 promoted cell growth and colony formation resembling that of miR-19a overexpression, whereas re-introduction of SOCS1 (without the 3'-UTR) attenuated the pro-tumorigenic functions. Taken together, our findings suggest that the SOCS1 gene is a direct target of miR-19a, which functions as an oncogenic miRNA in gastric cancer by repressing the expression of tumor suppressor SOCS1.