• Asian Pacific Journal of Cancer Prevention
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• v.17 no.11
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• pp.4893-4898
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• 2016

Objective: To investigate the impact of a Croton tiglium extract on cellular proliferation and apoptosis in a non-small cell lung cancer cell line (A549) in vitro. Methods: A Croton tiglium seed methanol extract was prepare and assessed for effects on A549 cells regarding cellular proliferation, apoptotic rates, and expression of apoptosis related genes and proteins using real-time PCR and immunofluorescence. Results: The tested Croton tiglium extract inhibited A549 cell proliferation in a dose- and time-dependent manner, with significant elevation of apoptotic indexes at various concentrations after 24 h. In addition, rates in both early and late stages were higher in treated than untreated groups, the $100{\mu}g/ml$ dose causing the highest levels of apoptosis. RT-PCR showed that A549 cells treated with $100{\mu}g/ml$ Croton tiglium extract for 24 h has markedly higher Bax mRNA expression levels and obviously lower Bcl-2 expression levels than controls, equivalent results being observed for proteins by immunofluorescence. However, the mRNA expression levels of Fas and caspase-8 were not significantly altered. Conclusion: A Croton tiglium extract can inhibit proliferation of A549 cells and promote apoptosis though Bax/Bcl-2 pathways.

• CELLMED
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• v.3 no.1
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• pp.9.1-9.10
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• 2013

In traditional systems of medicine including homeopathy, the Condurango extract (Con) is often used to cure stomach cancer mainly, without having any scientific validation of its anti-cancer ability. Con has therefore been tested against non-small-cell lung cancer cells (NSCLC) A549 and NCI-H522 (H522) known to contain the KRAS mutation, making them resistant to most chemotherapeutic agents. As cancer cells generally defy cytotoxicity developed by chemopreventive agents and escape cell death, any drug showing the capability of preferentially killing cancer cells through apoptosis is worth consideration for judicious application. A549 and H522 cells were exposed to $0.35{\mu}g/{\mu}l$ and $0.25{\mu}g/{\mu}l$ of Con, respectively, for 48 h and analysed based on various protocols associated with apoptosis and DNA damage, such as MTT assay to determine cell viability, LDH assay, DNA fragmentation assay, comet assay, and microscopical examinations of DNA binding fluorescence stains like DAPI, Hoechst 33258 and acridine orange/ethidium bromide to determine the extent of DNA damage made in drug-treated and untreated cells and the results compared. Changes in mitochondrial membrane potential and the generation of reactive oxygen species were also documented through standard techniques. Con killed almost 50% of the cancer cells but spared normal cells significantly. Fluorescence studies revealed increased DNA nick formation and depolarized membrane potentials after drug treatment in both cell types. Caspase-3 expression levels confirmed the apoptosis-inducing potential of Con in both the NSCLC lines. Thus, overall results suggest considerable anticancer potential of Con against NSCLC in vitro, validating its use against lung cancer by practitioners of traditional medicine including homeopathy.

• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.343-352
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• 2015

H9 is an ethanol extract prepared from nine traditional/medicinal herbs. This study was focused on the anticancer effect of H9 in non-small-cell lung cancer cells. The effects of H9 on cell viability, apoptosis, mitochondrial membrane potential (MMP; ${\Delta}\psi_{m}$), and apoptosisrelated protein expression were investigated in A549 human lung cancer cells. In this study, H9-induced apoptosis was confirmed by propidium iodide staining, expression levels of mRNA were determined by reverse transcriptase polymerase chain reaction, protein expression levels were checked by western blot analysis, and MMP (${\Delta}\psi_{m}$) was measured by JC-1 staining. Our results indicated that H9 decreased the viability of A549 cells and induced cell morphological changes in a dose-dependent manner. H9 also altered expression levels of molecules involved in the intrinsic signaling pathway. H9 inhibited Bcl-xL expression, whereas Bax expression was enhanced and cytochrome C was released. Furthermore, H9 treatment led to the activation of caspase-3/caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase; the MMP was collapsed by H9. However, the expression levels of extrinsic pathway molecules such as Fas/FasL, TRAIL/TRAIL-R, DR5, and Fas-associated death receptor were downregulated by H9. These results indicated that H9 inhibited proliferation and induced apoptosis by activating intrinsic pathways but not extrinsic pathways in human lung cancer cells. Our results suggest that H9 can be used as an alternative remedy for human non-small-cell lung cancer.

• Journal of Pharmacopuncture
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• v.18 no.1
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• pp.19-26
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• 2015

Objectives: Quercetin, a flavonoid compound, has been reported to induce apoptosis in cancer cells, but its anti-inflammatory effects, which are also closely linked with apoptosis, if any, on non-small-cell lung cancer (NSCLC) have not so far been critically examined. In this study, we tried to determine if quercetin had any demonstrable anti-inflammatory potential, which also could significantly contribute to inducing apoptosis in a NSCLC cell line, A549. Methods: In this context, several assays, including cytotoxicity, flow cytometry and fluorimetry, were done. Gene expression was analyzed by using a western blot analysis. Results: Results revealed that quercetin could induce apoptosis in A549 cells through mitochondrial depolarization by causing an imbalance in B-cell lymphoma 2/Bcl2 Antagonist X (Bcl2/Bax) ratio and by down-regulating the interleukine-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway. An analysis of the data revealed that quercetin could block nuclear factor kappa-light-chain-enhancer of activated B cells (NF-${\kappa}B$) activity at early hours, which might cause a down-regulation of the IL-6 titer, and the IL-6 expression, in turn, could inhibit p-STAT3 expression. Down-regulation of both the STAT3 and the NF-${\kappa}B$ expressions might, therefore, cause down-regulation of Bcl2 activity because both are major upstream effectors of Bcl2. Alteration in Bcl2 responses might result in an imbalance in the Bcl2/Bax ratio, which could ultimately bring about mitochondria mediated apoptosis in A549 cells. Conclusion: Overall, the finding of this study indicates that a quercetin induced anti-inflammatory pathway in A549 cells appeared to make a significant contribution towards induction of apoptosis in NSCLC and, thus, may have a therapeutic use such as a strong apoptosis inducer in cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.3001-3003
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• 2013

Objective: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shown to have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector for Syk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. Methods: A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vector pLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant Syk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. After selection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive ability were examined. Results: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stably in the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulated significantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05). Conclusions: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibit their invasive ability in vitro.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3119-3121
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• 2012

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer deaths worldwide. Crizotinib has been approved by the U.S. Food and Drug Administration for the treatment of patients with advanced NSCLC. However, understanding of mechanisms of action is still limited. In our studies, we confirmed crizotinib-induced apoptosis in A549 lung cancer cells. In order to assess mechanisms, small molecular docking technology was used as a preliminary simulation of signaling pathways. Interesting, our results of experiments were consistent with the results of computer simulation. This indicates that small molecular docking technology should find wide use for its reliability and convenience.

• Biomedical Science Letters
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• v.25 no.4
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• pp.293-301
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• 2019

Combination chemotherapy is more effective than mono-chemotherapy and is widely used in clinical practice for enhanced cancer treatment. In this study, we investigated the potential synergistic effects of acetaminophen, a common component in many cold medicines, and romidepsin, a histone deacetylase (HDAC) inhibitor, in the A549 non-small cell lung cancer (NSCLC) cell line. The combination of acetaminophen and romidepsin also exerted significant cytotoxicity and apoptosis induced by activation of caspase-3 on tumor cells in vitro. Moreover, combination therapy significantly induced increased production of chemokines that stimulate migration of activated T-cells into tumor cells. This mechanism can lead to active T-cell mediated anti-tumor immunity in addition to the direct cytotoxic chemotherapeutic effect. Activated T-cells led to enhanced cytotoxicity in drug-treated A549 cells through interaction with tumor cells. These results suggested that the interaction between the two drugs is synergistic and significant. In conclusion, our data showed that the use of romidepsin and low concentrations acetaminophen could induce effective anti-tumor effects via enhanced tumor immune and direct cytotoxic chemotherapeutic responses. The combination of acetaminophen with romidepsin should be considered as a promising strategy for the treatment of lung cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.1
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• pp.161-166
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• 2014

Lung cancer is the most common causes of cancer-related deaths worldwide, and a lack of effective methods for early diagnosis has greatly impacted the prognosis and survival rates of the affected patients. Tumor-initiating cells (TICs) are considered to be largely responsible for tumor genesis, resistance to tumor therapy, metastasis, and recurrence. In addition to representing a good potential treatment target, TICs can provide clues for the early diagnosis of cancer. MicroRNA (miRNA) alterations are known to be involved in the initiation and progression of human cancer, and the detection of related miRNAs in TICs is an important strategy for lung cancer early diagnosis. As Hsa-miR-155 (miR-155) can be used as a diagnostic marker for non-small cell lung cancer (NSCLC), a smart molecular beacon of miR-155 was designed to image the expression of miR-155 in NSCLC cases. TICs expressing CD133 and CD338 were obtained from A549 cells by applying an immune magnetic bead isolation system, and miR-155 was detected using laser-scanning confocal microscopy. We found that intracellular miR-155 could be successfully detected using smart miR-155 molecular beacons. Expression was higher in TICs than in A549 cells, indicating that miR-155 may play an important role in regulating bio-behavior of TICs. As a non-invasive approach, molecular beacons could be implemented with molecular imaging to diagnose lung cancer at early stages.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.9
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• pp.4039-4044
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• 2014

Background: The aim of this study was to investigate the anti-cancer effects and mechanisms of immunochemotherapy of 5-fluorouracil (5-FU) and interleukin-2 (IL-2) on non-small cell lung cancer (NSCLC) A549 cells. Materials and Methods: In order to detect whether 5-FU+IL-2 could effectively inhibit tumor growth in vivo, we established an A549-bearing nude mouse model. The cytotoxicity of natural killer (NK) cells was evaluated using a standard chromium release assay. To evaluate the relevance of NK cells in 5-FU+IL-2-mediated tumor inhibitory effects, we depleted NK cells in A549-bearing mice by injecting anti-asialo-GM-1 antibodies. Effects of 5-FU+IL-2 on the expression and promoter activity of NKG2D ligands (MICA/MICB) in A549 cells in vitro were also assessed. Results: In A549-bearing nude mice, combination therapy significantly inhibited tumor growth in comparison with monotherapy with 5-FU or IL-2 and enhanced the recognition and lysis of tumor cells by NK cells. Further study of mechanisms showed that NK cells played a vital role in the anticancer immune response of 5-FU+IL-2 immunochemotherapy. In addition, the combination therapy synergistically stimulated the expression and promoter activity of MICA/MICB. Conclusions: 5-FU and IL-2 immunochemotherapy significantly inhibited tumor growth and activated NK cytotoxicity in vivo, and these effects were partly impaired after depleting NK cells in tumor-bearing mice. Combination treatment of 5-FU and IL-2 upregulated the expression and the promoter activity of MICA/MICB in A549 cells, which enhanced the recognition of A549 cells by NK cells. All of the data indicated that immunochemotherapy of 5-FU and IL-2 may provide a new treatment option for patients with lung cancer.

• Tuberculosis and Respiratory Diseases
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• v.67 no.4
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• pp.303-310
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• 2009

Background: Elevated expression of cyclooxygenase-2 (COX-2) and Polo-like kinase-1 (PLK-1) is observed in a wide variety of cancers. Augmented expression of COX-2 and enhanced production of prostaglandin $E_2(PGE_2)$ are associated with increased tumor cell survival and malignancy; COX-2 has been implicated in the control of human non-small cell lung carcinoma (NSCLC) cell growth. PLK-1 siRNA induced the cell death of lung cancer cells and the systemic administration of PLK-1 siRNA/atelocollagen complex inhibited the growth of lung cancer in a liver metastatic murine model. COX-2 and PLK-1 are involved in proliferation and in cell cycle regulation, and there is a significant correlation between their interaction in prostate carcinoma. Methods: In this study, we investigated the pattern of COX-2 and PLK-1 expression in NSCLC, after treatment with IL-1$\beta$, COX-2 inhibitor and PLK-1 siRNA. Results: Expression of PLK-1 was decreased in A549 COX-2 sense cells, and was increased in A549 COX-2 anti-sense cells. Knock out of PLK-1 expression by PLK-1 siRNA augmented COX-2 expression in A549 and NCl-H157 cells. When A549 and NCI-H157 cells were treated with COX-2 inhibitor on a dose-dependent basis, PLK-1 and COX-2 were reduced. However, when the expression of COX-2 was induced by IL-1$\beta$, the production of PLK-1 decreased. Conclusion: These results demonstrate that COX-2 and PLK-1 are regulated and inhibited by each other in NSCLC, and suggest that these proteins have a reverse relationship in NSCLC.