Preparation and characterization of 2-deacetyl-3-O-sulfo-heparosan and its antitumor effects via the fibroblast growth factor receptor pathway.

Heparosan, with a linear chain of disaccharide repeating units of â†’ 4) ß-D-glucuronic acid (GlcA) (1 â†’ 4)-α-D-N-acetylglucosamine (GlcNAc) (1→, is a potential starting chemical for heparin synthesis. However, the chemoenzymatic synthesis of single-site sulfated heparosan and its antitumor activity have not been studied. In this study, 2-deacetyl-3-O-sulfo-heparosan (DSH) was prepared successively by the N-deacetylation chemical reaction and enzymatic modification of human 3-O-sulfotransferase-1 (3-OST-1). Structural characterization of DSH was shown the success of the sulfation with the sulfation degree of 0.87. High performance gel permeation chromatography (HPGPC) analysis revealed that DSH had only one symmetrical sharp peak with a molecular weight of 9.6334 × 104 Da. Biological function studies showed that DSH could inhibit tumor cell (A549, HepG2 and HCT116) viability and induce the apoptosis of A549 cells. Further in vitro mechanistic studies showed that DSH may induce apoptosis via the JNK signaling pathway, and the upstream signal of this process may be fibroblast growth factor receptors. These results indicated that DSH could be developed as one of a potential chemical for tumor treatment.

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.

Identification of nagilactone E as a protein synthesis inhibitor with anticancer activity.

Norditerpenoids and dinorditerpenoids represent diterpenoids widely distributed in the genus Podocarpus with notable chemical structures and biological activities. We previously reported that nagilactone E (NLE), a dinorditerpenoid isolated from Podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. In this study we investigated the in vivo effect of NLE against lung cancer as well as the underlying mechanisms. We administered NLE (10 mg·kg-1·d-1, ip) to CB-17/SCID mice bearing human lung cancer cell line A549 xenograft for 3 weeks. We found that NLE administration significantly suppressed the tumor growth without obvious adverse effects. Thereafter, RNA sequencing (RNA-seq) analysis was performed to study the mechanisms of NLE. The effects of NLE on A549 cells have been illustrated by GO and pathway enrichment analyses. CMap dataset analysis supported NLE to be a potential protein synthesis inhibitor. The inhibitory effect of NLE on synthesis of total de novo protein was confirmed in Click-iT assay. Using the pcDNA3-RLUC-POLIRES-FLUC luciferase assay we further demonstrated that NLE inhibited both cap-dependent and cap-independent translation. Finally, molecular docking revealed the low-energy binding conformations of NLE and its potential target RIOK2. In conclusion, NLE is a protein synthesis inhibitor with anticancer activity.

Casticin Induces DNA Damage and Affects DNA Repair Associated Protein Expression in Human Lung Cancer A549 Cells (Running Title: Casticin Induces DNA Damage in Lung Cancer Cells).

Casticin was obtained from natural plants, and it has been shown to exert biological functions; however, no report concerns the induction of DNA damage and repair in human lung cancer cells. The objective of this study was to investigate the effects and molecular mechanism of casticin on DNA damage and repair in human lung cancer A549 cells. Cell viability was determined by flow cytometric assay. The DNA damage was evaluated by 4',6-diamidino-2-phenylindole (DAPI) staining and electrophoresis which included comet assay and DNA gel electrophoresis. The protein levels associated with DNA damage and repair were analyzed by western blotting. The expression and translocation of p-H2A.X were observed by confocal laser microscopy. Casticin reduced total viable cell number and induced DNA condensation, fragmentation, and damage in A549 cells. Furthermore, casticin increased p-ATM at 6 h and increased p-ATR and BRCA1 at 6-24 h treatment but decreased p-ATM at 24-48 h, as well as decreased p-ATR and BRCA1 at 48 h. Furthermore, casticin decreased p-p53 at 6-24 h but increased at 48 h. Casticin increased p-H2A.X and MDC1 at 6-48 h treatment. In addition, casticin increased PARP (cleavage) at 6, 24, and 48 h treatment, DNA-PKcs and MGMT at 48 h in A549 cells. Casticin induced the expressions and nuclear translocation of p-H2AX in A549 cells by confocal laser microscopy. Casticin reduced cell number through DNA damage and condensation in human lung cancer A549 cells.

Silencing carbonic anhydrase 1 inhibits the proliferation, invasion and migration of A549 cells in lung cancer / 中国临床药理学与治疗学

AIM: To investigate the effects of silencing carbonic anhydrase 1 (CA1) on proliferation, apoptosis, invasion and migration of human lung cancer A549 cells. METHODS: CA1-specific siRNA (si-CA1 group) and negative control (si-NC group) were transfected into lung cancer A549 cells by lipofection. The A549 cells transfected with empty liposome were used as blank control group. Real-time quantitative PCR (qPCR) and Western blot (Western blot) were used to detect the expression of CA1 mRNA and protein. Cell counting kit method (CCK-8), flow cytometry and Transwell assay were used to detect proliferation and apoptosis of A549 cells, invasion and migration capabilities. RESULTS:qPCR and Western blot showed that the expression levels of CA1 mRNA and protein in A549 cells transfected with CA1 siRNA were significantly down-regulated (P0.05). CONCLUSION: Silencing CA1 can inhibit the proliferation, invasion and migration of lung cancer A549 cells and promote cell apoptosis.

Sodium selenite induced human lung cancer A549 cells apoptosis through Keap1/Nrf2/ARE signaling pathway / 中国药理学通报

Aim: To study the induction of apoptotic effect of sodium selenite on human lung cancer A549 cells and its mechanisms. Methods: A549 cells were exposed to different concentrations of sodium selenite for 24 h. MTT assay was applied to determine A549 cell proliferation. Inverted fluorescence microscope was used to investigate the morphological changes in A549 cells. Flow cytometry analysis was applied to assess the apoptotic rates of A549 cells. Laser confocal microscope was employed to measure the reactive oxygen species (ROS) fluorescence intensity. A multi-detection reader was used to determine the antioxidant parameter. Western blot was utilized to detect the expression of Keapl, Nrf2, HO-1 and Nrf2 in cytoplasm and nucleus. Results: MTT results showed that sodium selenite inhibited the proliferation of A549 cells in a concentration-dependent manner. After treatment with sodium selenite for 24 h, the apoptotic rate of A549 cells was markedly increased through Hoechst 33342 staining and flow cytometry measurement. Sodium selenite significantly up-regulated ROS and malondialdehyde (MDA) content and down-regulated the levels of superoxide dismutase (SOD) and glutathione (GSH). Meanwhile, sodium selenite treatment also reduced the expressions of Keapl, Nrf2 and HO-1 at protein levels and inhibited Nrf2 protein nuclear translocation in A549 cells. Conclusions: Treatment with sodium selenite induces A549 cells apoptosis, which may contribute to the anti-proliferation activity, induction of apoptosis and regulation of oxidative stress reaction and Keapl/Nrf2/ARE antioxidative signaling pathway expression.

Delicaflavone induces autophagic cell death in lung cancer via Akt/mTOR/p70S6K signaling pathway.

Searching for potential anticancer agents from natural sources is an effective strategy for developing novel chemotherapeutic agents. In this study, data supporting the in vitro and in vivo anticancer effects of delicaflavone, a rarely occurring biflavonoid from Selaginella doederleinii, were reported. Delicaflavone exhibited favorable anticancer properties, as shown by the MTT assay and xenograft model of human non-small cell lung cancer in male BALB/c nude mice without observable adverse effect. By transmission electron microscopy with acridine orange and Cyto-ID®Autophagy detection dyes, Western blot analysis, and RT-PCR assay, we confirmed that delicaflavone induces autophagic cell death by increasing the ratio of LC3-II to LC3-I, which are autophagy-related proteins, and promoting the generation of acidic vesicular organelles and autolysosomes in the cytoplasm of human lung cancer A549 and PC-9 cells in a time- and dose-dependent manner. Delicaflavone downregulated the expression of phospho-Akt, phospho-mTOR, and phospho-p70S6K in a time- and dose-dependent manner, suggesting that it induced autophagy by inhibiting the Akt/mTOR/p70S6K pathway in A549 and PC-9 cells. Delicaflavone is a potential anticancer agent that can induce autophagic cell death in human non-small cell lung cancer via the Akt/mTOR/p70S6K signaling pathway. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer. KEY MESSAGES: Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer.

Study on Inhibitory Mechanism of Timosaponin B-Ⅱ on the Proliferation and Migration of Human Lung Cancer A549 Cells / 中国药房

OBJECTIVE:To study the inhibitory mechanism of timosaponin B-Ⅱ(TB-Ⅱ) on the proliferation and migration of human lung cancer A549 cells. METHODS:A549 cells were treated with TB-Ⅱ [0(blank control),1,10 and 100 μg/ml] for 48 h,and total RNA and total protein were extracted respectively. Real time fluorescence quantitative-PCR and Western blot were used to detect mRNA and protein levels of IL-18. IL-18 in A549 cells was silenced by transfection;the expression of IL-18 mRNA and protein were compared among untransfection group,negative control group and transfection group;and then human lung can-cer A549 cells with silenced gene were treated with 10 μg/ml TB-Ⅱ for 24,48 and 72 h. The activity of cell proliferation was de-tected with CCK-8,and the change of cell migration ability was observed by streak method. RESULTS:Compared with blank con-trol,the expression of IL-18 mRNA and protein in A549 cells all increased after treated with TB-Ⅱ(P<0.05 or P<0.01),and were positively correlated with concentration. Compared with untransfection group,the expression of IL-18 mRNA and protein de-creased in transfection group(P<0.01). Compared with untransfected cell treated with TB-Ⅱ,the viability and migration ability of A549 cells with transfection gene increased after treated with TB-Ⅱ for 72 h(P<0.01). CONCLUSIONS:TB-Ⅱ can inhibit the proliferation and migration of A549 cells by up-regulating IL-18 gene expression.

Effects of Thymalfasin for Injection on the Apoptosis of Humun Lung Cancer A549 Cells / 中国药房

OBJECTIVE:To study the effects of Thymalfasin for injection on the apoptosis of human lung cancer A549 cells. METHODS:After treated with 0(blank control),25,50,100,200 and 400 mg/L Thymalfasin for injection for 24,48 and 72 h, the cell proliferation inhibitory rate was analyzed with MTT and calculated. After treated with 0(blank control),50 and 100 mg/L Thymalfasin for injection for 48 h,cell apoptosis was detected by flow cytometry,and the expression of Caspase-3,Bcl-2 and Bax and the phosphorylation level of Akt were deteced by Western blot. RESULTS:Compared with blank control group,proliferation in-hibitory rate of A549 cells increased after treated with Thymalfasin for injection,in concentration and time-dependent manner(P<0.05). The apoptotic rate of A549 cells increased after treated with Thymalfasin for injection 50,100 mg/L for 48 h (P<0.05). The expression of Caspase-3 increased while the Bcl-2/Bax and phosphorylation level of Akt decreased in A549 cells after treated with Thymalfasin for injection 100 mg/L (P<0.05). CONCLUSIONS:Thymalfasin for injection can inhibit the proliferation of A549 cells by activating Caspase-3,decreasing Bcl-2/Bax ratio,inhibiting Akt signal pathway and induce the apoptosis of A549 cells.

Cross-talk between endothelial and tumor cells via basic fibroblast growth factor and vascular endothelial growth factor signaling promotes lung cancer growth and angiogenesis.

The present study aimed to investigate the origin and potential mechanisms of angiogenesis in lung cancer cells. Normal endothelial cells (ECs) were isolated from human umbilical vein ECs (HUVECs) and cultured. The human lung cancer A549 cell line was also used. The cross-talk model between the HUVECs and the A549 cell line was constructed in vitro using a Millicell co-culture system. Cluster of differentiation (CD)31 and CD146 were selected as markers of the HUVECs. CD105 was used as a marker of activated blood vessel ECs in the tumor microenvironment and glucose-regulated protein-78 (GRP-78) was used as a biomarker of the A549 cells. The four markers were detected by immunofluorescence, and the mean optical density was calculated. The growth curves were constructed using the cell proliferation reagent, WST-1. The expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the media was measured using an ELISA. The average proliferation rates of the co-cultured HUVECs and A549 cells were significantly higher than those observed in the control groups. The fluorescence intensity of CD105 expression in the co-cultured HUVECs was higher than that in the control group. The fluorescence intensity of GRP-78 in the co-cultured A549 cells was higher than that in the A549 cells cultured alone. The average expression levels of VEGF and bFGF in the co-cultured model were higher than in the control groups. Therefore, it was hypothesized that cancer cells may induce the differentiation of normal ECs into vascular ECs via the secretion of VEGF and bFGF. Furthermore, vascular ECs can affect the proliferation and differentiation of cancer cells.

Chrysophanol-induced cell death (necrosis) in human lung cancer A549 cells is mediated through increasing reactive oxygen species and decreasing the level of mitochondrial membrane potential.

Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is one of the anthraquinone compounds, and it has been shown to induce cell death in different types of cancer cells. The effects of chrysophanol on human lung cancer cell death have not been well studied. The purpose of this study is to examine chrysophanol-induced cytotoxic effects and also to investigate such influences that involved apoptosis or necrosis in A549 human lung cancer cells in vitro. Our results indicated that chrysophanol decreased the viable A549 cells in a dose- and time-dependent manner. Chrysophanol also promoted the release of reactive oxygen species (ROS) and Ca(2+) and decreased the levels of mitochondria membrane potential (ΔΨm ) and adenosine triphosphate in A549 cells. Furthermore, chrysophanol triggered DNA damage by using Comet assay and DAPI staining. Importantly, chrysophanol only stimulated the cytocheome c release, but it did not activate other apoptosis-associated protein levels including caspase-3, caspase-8, Apaf-1, and AIF. In conclusion, human lung cancer A549 cells treated with chrysophanol exhibited a cellular pattern associated with necrotic cell death and not apoptosis in vitro. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 740-749, 2014.

Effect of combination valdecoxib and pirarubicin on promoting apoptosis in human lung cancer cell / 中国药学杂志

OBJECTIVE: To study the effect of valdecoxib and pirarubicina combination on cell cycle and apoptosis of human lung cancer A549 cell line in vitro. METHODS: MTT assay was used to analyze the effect of valdecoxib and pirarubicina on the growth of human lung cancer A549 cell line. The median-effect principle was applied to determine the combination effect of valdecoxib and pirarubicina. Flow cytometry (FCM) was used to observe the cell cycle and apoptosis. The expressions of Bcl-2, Bax, and Caspase-3 were detected by western blotting. RESULTS: The inhibition ratio of A549 cell in valdecoxib and pirarubicina combination group was increased compared with their respective application, and CI<1. In valdecoxib and pirarubicina combination group, the apoptosis rate and the expression of Bax and Caspase-3 was increased, and the expression of Bcl-2 was decresed. CONCLUSION: Valdecoxib and pirarubicina combination has synergistic effect, which is partly due to the increase of the apoptosis rate.

Study of Kanglaite Injection Combined with Chemotherapeutic Drugs in Inhibiting the Growth of Transplanted Human Lung Cancer A549 in Nude Mice / 中药新药与临床药理

Objective To observe the attenuated effect of Kanglaite Injecti on (KI) combined with chemotherapeutic drugs on transplanted human lung cancer A54 9 in nude mice. Methods Ninety- six nude mice were transplanted subcutaneousl y with human lung cancer A549; 7 days later, the mice were randomized into 16 gr oups(n=6) and were given antitumor drugs according the regimen. Other 12 normal mice were used as controls. KI groups were given KI 1.25, 2.5 and 5.0 g/kg respe ctively and were administered (iv) continuously for 10 times on 7th day of trans plantation; cisplatin (DDP, 1.5 mg/mg or 3 mg/kg) and Gemzar (30 mg/kg or 60 mg/ kg) were given one dose by intraperitoneal injection on 10th day of transplantat ion; Gemzar was given again 4h after medication of DDP. Taxotere (25 mg/kg) was given one dose by intraperitoneal injection on 7th day of transplantation. The m ice were killed to examine the body weight, tumor weight and tumor- inhibiting rate. Results KI 1.25, 2.5 and 5.0 g/kg respectively combined with DDP 3mg/kg and Gemzar 60 mg/kg had a better inhibitory effect on the growth of human lung c ancer A549 than KI, DDP and Gemzar alone. KI in three doses respectively combine d with DDP 1.5 mg/kg and Gemzar 30 mg/kg not only had a better inhibitory effect than those of KI, DDP and Gemzar alone, but also promoted the antitumor actions . The tumor- inhibitory rate of KI in three doses respectively combined with Ta xotere (25 mg/kg) was higher than KI and Taxotere alone. Conclusion KI combine d with DDP and Gemzar or with Taxotere can shorten the tumor mass and has tumor - inhibitory and attenuated effect.