CH27 Cells Research Articles

Fenofibrate induces human hepatoma Hep3B cells apoptosis and necroptosis through inhibition of thioesterase domain of fatty acid synthase

This study demonstrated that fenofibrate, a lipid-lowering drug, induced a significant time-dependent cytotoxicity of hepatoma Hep3B cells. Hep3B cells are significantly more sensitive to cell killing by fenofibrate than hepatoma HepG2, lung cancer CH27 and oral cancer HSC-3 cells. From the result of docking simulation, fenofibrate can bind excellently to the thioesterase domain of fatty acid synthase (FASN) binding site as orlistat, a FASN inhibitor, acts. The fenofibrate-induced cell cytotoxicity was protected by addition of palmitate, indicating that the cytotoxic effect of fenofibrate is due to starvation of Hep3B cells by inhibiting the formation of end product in the FASN reaction. Inhibition of lipid metabolism-related proteins expression, such as proteins containing thioesterase domain and fatty acid transport proteins, was involved in the fenofibrate-induced Hep3B cell death. Fenofibrate caused S and G2/M cell cycle arrest by inducing cyclin A/Cdk2 and reducing cyclin D1 and E protein levels in Hep3B cells. The anti-tumor roles of fenofibrate on Hep3B cells by inducing apoptosis and necroptosis were dependent on the expression of Bcl-2/caspase family members and RIP1/RIP3 proteins, respectively. These results suggest that fenofibrate has an anti-cancer effect in Hep3B cells and inhibition of lipid metabolism may be involved in fenofibrate-induced Hep3B cells apoptosis and necroptosis.

Development and utilization of a unique in vitro antigen presentation co-culture model for detection of immunomodulating substances

Current regulatory immunotoxicity studies require the use of animal models. However, evolving regulatory requirements, the need to evaluate large numbers of chemicals efficiently and societal pressures are driving the development and utilization of alternative in vitro methods for identifying potential immunotoxicants. In line with these efforts, we developed a novel in vitro cell-based assay to evaluate effects on antigen presentation – a key step in successful immunization. In this assay, Ch27 B cells acquire and present hen egg lysozyme peptides to antigen-restricted 3A9 T cells, causing them to produce and secrete IL-2. IL-2 levels in the culture medium may be monitored to identify effects of immunotoxicant exposure on antigen uptake, processing or presentation by the Ch27 cells and on antigen recognition and IL-2 production and secretion by the 3A9 cells. IL-2 production was reduced in response to treatment with well-known immunotoxicants cyclosporin A (CYA), dexamethasone (DEX), azathioprine (AZPR), methotrexate (MOT) and benzo(a)pyrene (BAP) but was not affected by treatment with cyclophosphamide (CYPH). A negative control compound mannitol (MANN) altered neither cell viability nor IL-2 levels whereas the lysosomotrophic compound ammonium chloride (AMCL) reduced IL-2 production. This novel in vitro assay of immune function may be suitable for integration into a tiered testing battery for screening and prioritization of potential immunosuppressants.

Mechanisms underlying lung resistance-related protein (LRP)-mediated doxorubicin resistance of non-small cell lung cancer cells.

Lung resistance-related protein (LRP) is a human major vault protein (MVP) implicated in drug resistance of cancer cells in a cell-type dependent manner. The primary goal of the study was to understand the role(s) of LRP in doxorubicin (DOX) resistance of non-small cell lung cancer (NSCLC) cells and the underlying working mechanisms. In the study, the roles of LRP in the regulation of DOX dynamics, nuclear import of minor vault proteins (vault poly (ADP-ribose) polymerase, vPARP and telomerase associated protein-1, TEP-1) and DOX-mediated cytotoxicity were examined in CH27 and H460 cells. Our results were the first to show that the CH27 cells with higher LRP expression levels were more resistant to DOX-induced cytotoxicity as shown in apoptosis experiments. LRP at the nuclear membrane could regulate DOX efflux from the nucleus to the cytosol, and also the reverse vPARP/TEP1 influx from the cytosol, to protect NSCLC cells from DOX-induced apoptosis. Cytosolic LRP could bind to DOX, vPARP and TEP1 to clear DOX away from the nucleus and promote the assembly of vaults for cell protection again. Based on the data obtained, the molecular mechanisms responsible for DOX resistance of NSCLC were delineated to demonstrate that LRP, vPARP and TEP1 were potential targets for NSCLC therapy. Inhibitors of these proteins, including small interfering LRP (siLRP), wheat-germ agglutenin (WGA) (WGA), 3-aminobenzamide (3-AB) and 3,6,9-trisubstituted acridine 9-[4-(N,N-dimethylamino) phenylamino]-3,6-bis(3-pyrrolodinopropionamido) acridine (BRACO-19), break down the DOX resistance of NSCLC cells, particularly in CH27 cells, and may have therapeutic values in the control of NSCLC.

Gene Therapy for Human Lung Adenocarcinoma Using a Suicide Gene Driven by a Lung-Specific Promoter Delivered by JC Virus-Like Particles.

Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter–driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter’s tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP’s gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter–driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk–carrying JCPyV VLPs. In mice injected with pSPB-tk–carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.

Hypoxia can impair doxorubicin resistance of non-small cell lung cancer cells by inhibiting MRP1 and P-gp expression and boosting the chemosensitizing effects of MRP1 and P-gp blockers.

Non-small cell lung cancers (NSCLCs) frequently exhibit resistance to therapeutic drugs, which seriously hampers their treatment. Here, we set out to assess the roles of the multidrug resistance protein 1 (MRP1) and P-glycoprotein (P-gp) in the doxorubicin (DOX) resistance of NSCLC cells, as well as the putative therapeutic efficacy of MRP1 and P-gp blockers on DOX-treated NSCLC cells. The impact of DOX on cell survival, DOX efflux and MRP1 and P-gp expression was assessed in 5 different NSCLC-derived cell lines (parental CH27, A549, H1299, H460, and DOX resistant CH27) in the absence or presence of MK571 (MRP1 inhibitor) or Verapamil (P-gp inhibitor), under both normoxic and hypoxic conditions. We found that in response to DOX treatment, NSCLC cells that express high levels of MRP1 and P-gp (such as CH27) showed a better DOX efflux and a higher DOX resistance. MK571 and Verapamil were found to abolish DOX resistance and to act as chemosensitizers for DOX therapy in all cell lines tested. We also found that hypoxia could inhibit MRP1 and P-gp expression in a HIF-1α-dependent manner, abolish DOX resistance and boost the chemosensitizer effect of MK571 and Verapamil on DOX treatment of all the NSCLC cells tested, except the DOX-resistant CH27 cells. From our data we conclude that MRP1 and P-gp play critical roles in the DOX resistance of the NSCLC cells tested. MRP1 and P-gp targeted therapy using MK571, Verapamil, CoCl2 or ambient hypoxia appeared to be promising in abolishing the DOX efflux and DOX resistance of the NSCLC cells. The putative therapeutic efficacies of MRP1 and/or P-gp blockers on NSCLC cells are worthy of note.

TGFβ can stimulate the p(38)/β-catenin/PPARγ signaling pathway to promote the EMT, invasion and migration of non-small cell lung cancer (H460 cells).

Signaling pathway(s) responsible for transforming growth factor β (TGFβ)-induced epithelial mesenchymal transition (EMT), invasion and migration of H460 cells (non-small cell lung cancer/NSCLC) was identified in the study. The results showed that TGFβ-induced p(38)/β-catenin/PPARγ signaling pathway played a critical role in the promotion of EMT, invasion and migration of H460 cells. All these pathological outcomes attributed to PPARγ-increased expression of p-EGFR, p-c-MET and Vimentin and the decrease of E-cadherin. Transforming growth factor β and p(38)-induced β-catenin not only stimulated the expression of PPARγ but also physically interacted with it. Blocking the ligand binding domain of PPARγ (with GW9662) could significantly interfere the binding between PPARγ and β-catenin, and interrupt the nuclear infiltration of both factors. These findings suggested that β-catenin was an upstream regulator and a ligand of PPARγ, and the binding between these two molecules was critical for their nuclear infiltration. Transforming growth factor β-induced tumor invasion and migration was also seen in U373 cells (brain glioma, with high inducible PPARγ) in a PPARγ-dependent manner, but not in CH27 cells (squamous NSCLC, with low PPARγ). PPARγ shRNA, GW9662, JW67 and 2,4-diaminoquinazoline were all revealed to have important values in the control of the intrinsic and TGFβ-induced EMT, tumor invasion and migration of H460 cells. The results further suggested that PPARγ and β-catenin may be the potential markers for the early diagnosis and/or treatment of metastatic tumors.

Application of proteomics to identify the target molecules involved in Lonicera japonica-induced photokilling in human lung cancer CH27 cells

BackgroundThe Lonicera japonica has been used as natural and healthy drink for its anti-inflammatory effect and pleasant odor in China and Taiwan.Methods2D electrophoresis was used to analyze the proteins involved in photoactivated Lonicera japonica-induced CH27 cell apoptosis. The fluorescent dyes MitoTracker Red CMXRos, calcein AM and JC-1 were used to elucidate mitochondrial function. The protein expression was performed by Western blotting. Fluorescent image of endoplasmic reticulum was accomplished by using ER-Tracker Green. This study used fluorescent dye CM-H2DCFDA to detect intracellular generation of reactive oxygen species.ResultsThe identified proteins can be classified into three major groups, which include proteins involved in mitochondrial function, cytoskeleton-related proteins and proteins associated with endoplasmic reticulum (ER) stress. Photoactivated Lonicera japonica caused a significant effect on the mitochondrial function and ER stress in CH27 cells. The reactive oxygen species producing was found to be involved in photoactivated Lonicera japonica-induced CH27 cell apoptosis.ConclusionMitochondria and endoplasmic reticulum are the integral targets in photoactivated Lonicera japonica-induced CH27 cell apoptosis. We also demonstrated that ethyl acetate fraction of Lonicera japonica extracts caused photocytotoxicity in a dose-dependent manner in CH27 cells. This could explain the fact that the ethyl acetate fraction of Lonicera japonica extracts may contain compounds which exhibit the photosensitizing activity in CH27 cells.

Molecular modelling, synthesis, cytotoxicity and anti-tumour mechanisms of 2-aryl-6-substituted quinazolinones as dual-targeted anti-cancer agents

Our previous study demonstrated that 6-(pyrrolidin-1-yl)-2-(3-methoxyphenyl)quinazolin-4-one (HMJ38) was a potent anti-tubulin agent. Here, HMJ38 was used as a lead compound to develop more potent anti-cancer agents and to examine the anti-cancer mechanisms. Using computer-aided drug design, 2-aryl-6-substituted quinazolinones (MJ compounds) were designed and synthesized by introducing substituents at C-2 and C-6 positions of HMJ38. The cytotoxicity of MJ compounds towards human cancer cells was examined by Trypan blue exclusion assay. Microtubule distribution was visualized using TubulinTracker(TM) Green reagent. Protein expression of cell cycle regulators and JNK was assessed by Western blot analysis. Compounds MJ65-70 exhibited strong anti-proliferative effects towards melanoma M21, lung squamous carcinoma CH27, lung non-small carcinoma H460, hepatoma Hep3B and oral cancer HSC-3 cells, with one compund MJ66 (6-(pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one) highly active against M21 cells (IC50 about 0.033 μM). Treatment of CH27 or HSC-3 cells with MJ65-70 resulted in significant mitotic arrest accompanied by increasing multiple asters of microtubules. JNK protein expression was involved in the MJ65-70-induced CH27 and M21 cell death. Consistent with the cell cycle arrest at G2/M phase, marked increases in cyclin B1 and Bcl-2 phosphorylation were also observed, after treatment with MJ65-70. MJ65-70 are dual-targeted, tubulin- and JNK-binding, anti-cancer agents and induce cancer cell death through up-regulation of JNK and interfering in the dynamics of tubulin. Our work provides a new strategy and mechanism for developing dual-targeted anti-cancer drugs, contributing to clinical anti-cancer drug discovery and application.

Abstract 3028: Peroxisome proliferator-activated receptor ≤ (PPARδ) reveals a critical role in promoting the TGFβ-induced EMT and metastasis of non-small cell lung cancer cell (H460)

Abstract Although transforming growth factor ≤ (TGFβ) is anti-cancer identified, it was also known to promote tumor malignancy by enhancing the growth, epithelial to mesenchymal transition (EMT), tissue invasion and metastasis in a variety of cancer cells, including non-small cell lung cancers (NSCLCs). The aim of this study was to know the contribution of peroxisome proliferator-activated receptor ≤ (PPARδ) to TGFβ-promoted EMT and metastasis in NSCLCs. The degree of EMT, cell scattering, migration and invasion, and protein expression of PPARδ were first analyzed and compared between H460 and CH27 cells with or without TGFβ treatment (both are NSCLC cell lines). Chemical or shRNA blocking assay was also utilized to further confirm the importance of PPARδ in promoting TGFβ-induced tumor metastasis. Finally, signaling pathway(s) responsible for TGFβ/PPARδ-mediated metastasis was evaluated. The results demonstrated that TGFβ can effectively triggered the EMT, cell scattering, migration and invasion in H460 cell, but only a slight EMT was seen in TGFβ-treated CH27 cell. Meanwhile, PPARδ expression was induced by TGFβ in H460 cells but remained unchanged in CH27 cell. This indicated that PPARδ may involve in TGFβ-mediated metastasis. Co-treatment of TGFβ with GW9662 (a specific PPARδ inhibitor) significantly blocked TGFβ-promoted metastasis of H460 cell indicating the presence of PPARδ was indeed critical for TGFβ-attributed tumor metastasis. TGFβ appeared to act through P38 signaling pathway to trigger the expression of PPARδ and the subsequent activation of epidermal growth factor receptor (EGFR)/c-mesenchymal-epithelial transition factor (c-MET) signaling pathway and the reduction of E-cadherin that eventually led to the EMT and metastasis of H460 cell. Additionally, TGFβ also act through P38 to trigger the expression of β-catenin which could bind to and up-regulate the expression of PPARδ. Blocking of β-catenin activity, however, did not interfere with the metastasis indicating β-catenin may not play a direct role in promoting the metastasis of H460 cell. Nevertheless, we can not preclude the possibility that β-catenin may still act through PPARδ to regulate the metastasis of H460 cell, indirectly. In summary, TGFβ-induced EMT and metastasis in H460 cell was PPARδ-dependent and GW9662 revealed a therapeutical value in the control of NSCLCs (H460). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3028. doi:1538-7445.AM2012-3028

Diffusion of Single B Cell Receptors in Resting and Stimulated B Lymphocytes using Super-Resolution Microscopy

The B cell antigen receptor (BCR) is an integral part of our immune systems that communicates binding of antigen in the extracellular environment through the plasma membrane to the cytoplasm. Antigen binding to the BCR results in phosphorylation of intracellular tyrosine activating motifs (ITAMs), which act as binding partners for SH2 domain containing adapters and Src family kinases. To examine the spatio-temporal dynamics of the BCR during stimulation, we utilize stochastic optical reconstruction microscopy (STORM) in live CH27 cells. Using Alexa-647 labeled anti-IgM Fab fragments, we resolve the BCR down to tens of nanometers with acquisition rates of 40 frames per second. We then examine BCR diffusion by tracking the localized receptors and found that the BCRs in resting cells exhibit a lognormal distribution of diffusion constants centered at 10-1 μm2/s. Upon stimulation, this population is quickly shifted to one centered at 10-2 μm2/s. In separate experiments, we examined the calcium response of CH27 B cells and found that the buffers used for STORM experiments do not alter calcium responses after receptor crosslinking. We also perform an auto correlation analysis of the localized receptors, which shows an increase in BCR clustering on the same timescale as the reduction in diffusion. In addition, we are investigating the dynamics of other proteins involved in BCR signaling, namely the Src family kinase Lyn and Ezrin. We use two color simultaneous emission of a photoswitchable fluorescent protein mEos2 in conjunction with Alexa-647 in fixed and live cells to resolve the behavior of these interaction partners with respect to the BCR.

Peroxisome proliferator-activated receptor γ (PPARγ) plays a critical role in the development of TGFβ resistance of H460 cell

The primary goal of the study was to investigate how peroxisome proliferator-activated receptor γ (PPARγ) played a critical role in the protection of H460 cell, one of the non-small cell lung cancer (NSCLC) cells with multidrug resistance, from transforming growth factor β (TGFβ)-mediated mitoinhibition. In the study, TGFβ resistance of H460 cell was first confirmed by analyses of PPARγ expression, its interaction with TGFβ-induced Smad3 and phospho-Smad3 (p-Smad3) and survival of H460. Results showed that enable to escape from G2/M phase arrest, H460 cell had higher resistance to TGFβ-mediated mitoinhibition than CH27 (a drug sensitive control). TGFβ significantly increased PPARγ expression of H460 but not of CH27 cell whereas nuclear accumulation of p-Smad3 was only limited to CH27, the latter was believed to contribute to the induction of P 21 waf1/cip1 and cyclin B1, cell cycle arrest at G2/M phase and TGFβ-mediated mitoinhibition of CH27 cell. TGFβ-induced PPARγ of H460 cell was further demonstrated to bind to Smad3 and p-Smad3, and GW9662 (PPARγ inhibitor) or PPARγ-specific shRNA could disrupt the binding. GW9662 also increased the nuclear accumulation of p-Smad3 that eventually led to the reduction of TGFβ resistance of H460. A transient knockdown of PPARγ with shRNA revealed a similar effect as GW9662. In addition, activation of P 38 instead of ERK played a critical role in TGFβ-induced expression of PPARγ, which subsequently activated RhoA in H460 cell.

Rottlerin Inhibits Lonicera japonica‐Induced Photokilling in Human Lung Cancer Cells through Cytoskeleton‐Related Signaling Cascade

This study demonstrated that many apoptotic signaling pathways, such as Rho family, PKC family, MAP kinase family, and mitochondria-mediated apoptotic pathway, were triggered by Lonicera japonica extracts and irradiation in CH27 cells. Rottlerin, a PKCδ -selective inhibitor, reversed the photoactivated Lonicera japonica extract-induced decrease in PKCδ protein expression and change in cell morphology in this study. In addition, rottlerin inhibited the photoactivated Lonicera japonica-induced decrease in protein expression of Ras, ERK, p38, PKCα, and PKCε, which are the kinases of prosurvival signaling pathway. We also demonstrated that pretreatment with rottlerin prevented actin microfilaments and microtubules from damage during the photoactivated Lonicera japonica-induced CH27 cell death. Furthermore, the promotion of the cytoskeleton-related signaling cascade following rottlerin by upregulation of cytoskeleton-related mediators (p38, HSP27, FAK, paxillin, and tubulin) and molecules of downstream of F-actin (mitochondria-mediated apoptosis pathway) reduces CH27 cell death, indicating that cytoskeleton is the potential target in the photoactivated Lonicera japonicaextract-induced photokilling of CH27 cells.

Proteomic analysis reveals ATP-dependent steps and chaperones involvement in luteolin-induced lung cancer CH27 cell apoptosis

The present study applied 2D electrophoresis to analyze the proteins involved in luteolin (50 μM)-induced CH27 cell apoptosis. We found 7 proteins to be markedly changed. According to the data of analysis of these protein spots, we hypothesized that ATP synthetic pathway and heat shock proteins were involved in luteolin-induced CH27 cell apoptosis. In this study, luteolin induced a significant change in intracellular ATP levels and mitochondrial activity of CH27 cells. Further experiments demonstrated that pretreatment with forskolin blocked the luteolin-induced cell death. P38 and heat shock protein 27 may be important participants in the luteolin-induced changes in organization of actin microfilaments in this study. In addition, endoplasmic reticulum stress is also important in the luteolin-induced CH27 cell apoptosis. Our findings suggested that the function of mitochondria and endoplasmic reticulum is the integral factor in luteolin-induced CH27 cell apoptosis.

Anticancer Effects of Alpinia pricei Hayata Roots

The leaves and roots of Alpinia pricei Hayata are used as a traditional wrapping for food and as a cooking substitute for fresh ginger. Our previous study showed that ethanol extracts from the roots of A. pricei Hayata (EEAP) and its phenolic compounds have anti-inflammatory effects. The aims of this work were to further study the in vitro anticancer activity of EEAP and its active compounds with respect to various cancer cells. The results from an MTT assay demonstrated that EEAP decreased the cell population growth of CH27, HL-60, and A549 cells. Flow cytometric analysis of HL-60 cells exposed to EEAP showed that the number of apoptotic cells increased in a time- and dose-dependent manner. Western blot data revealed that EEAP stimulated an increase in the level of protein expression of Fas, FasL, caspase-8, and tBid. Moreover, the ratio of the expression levels of pro- and anti-apoptotic Bcl-2 family members was changed after treatment with EEAP. EEAP-induced apoptosis involved the release of mitochondrial cytochrome c and subsequently induced the activation of caspase-9 and caspase-3, which were followed by the cleavage of poly(ADP-ribose) polymerase (PARP). The results also demonstrated that phenolic compounds (caffeic acid, apigenin, curcumin, and pinocembrin) from EEAP decreased the rate of population growth of HL-60 cells. Treatment of HL-60 cells with these phenolic compounds caused the loss of mitochondrial membrane potential. Our finding could provide critical information regarding the chemopreventive potential of ethanol extracts from A. pricei Hayata. These results also demonstrate that the EEAP-induced apoptotic ability in HL-60 cells might be related to the phenolic compounds.

XIAP-mediated protection of H460 lung cancer cells against cisplatin

Molecular mechanism(s) responsible for drug resistance of non-small cell lung cancer (NSCLC) cells to cisplatin was investigated. Results showed that cisplatin (50muM)-induced cell death (apoptosis) was more significant in CH27 and A549 cell lines than in H460. The high protein levels of X-linked inhibitor-of-apoptosis protein (XIAP) observed in H460 cells appeared to play a key role in the regulation of cisplatin resistance of H460 cells. XIAP can bind to and suppress the activities of caspase 3 in H460 cells and lead to apoptosis inhibition of these cells. Blockade of XIAP activity by Embelin (XIAP inhibitor) or siRNA has increased caspase 3 activities and promoted cisplatin-induced cell death of H460 cells. The results indicate a therapeutic value of Embelin and/or XIAP siRNA in the control of cisplatin-resistant NSCLC cells (H460).

Mechanisms of Apoptotic Effects Induced by Resveratrol, Dibenzoylmethane, and Their Analogues on Human Lung Carcinoma Cells

While lung cancer accounts for approximately 20% of cancer diagnoses, it is the leading cause of tumor-related deaths. The apoptotic effects of 3,5,4'-trihydroxystilbene (resveratrol), dibenzoylmethane (DBM), and their analogues on human lung cancer cells are generally unclear. The aims of this study were to evaluate the apoptotic effects and molecular mechanisms of resveratrol, DBM, and their analogues on human lung cancer cells. The results of the MTT and lactate dehydrogenase (LDH) leakage assays indicated that resveratrol, 3,5,4'-trimethoxy-trans-stilbene (MR-3), and 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB) could inhibit cell population growth and induce cell injury in A549 and CH27 cell lines. Resveratrol and HMDB could induce apoptotic cell death in the A549 and CH27 cell lines. Moreover, cellular growth of the A549 and CH27 cell lines might be inhibited by MR-3 through induction of apoptosis and regulation of the cell cycle. The A549 and CH27 cell lines treated with resveratrol, MR-3, and HMDB showed a time-dependent reduction of mitochondrial membrane potential, and the Bax/Bcl-2 ratio increased gradually with a higher concentration of polyphenols. The resveratrol-, MR-3-, and HMDB-induced apoptosis in the A549 and CH27 cell lines were controlled through activation of caspase-9 and caspase-3 and subsequent cleavage of PARP. In conclusion, we have demonstrated that resveratrol, DBM, and their analogues could be effective candidates for chemoprevention of lung cancer and HMDB might have the strongest ability for inducing apoptosis.

P38-associated pathway involvement in apoptosis induced by photodynamic therapy with Lonicera japonica in human lung squamous carcinoma CH27 cells

Photodynamic therapy (PDT) is an effective therapy for local malignant tumors. Lonicera japonica was found to have the anti-tumor effect. The aim of this study is to explore the mechanisms of apoptosis induced by PDT in lung CH27 carcinoma cells with alcohol extract from Lonicera japonica as photosensitizer. Our study indicated that Lonicera japonica extracts exhibited significant photocytotoxicity in CH27 cells at a concentration range of 50–150 μg/ml, with 0.4–1.2 J/cm 2 light dose. PDT with Lonicera japonica extracts-induced cell death is a typical apoptosis that was accompanied by DNA condensation, externalization of phosphatidylserine and formation of apoptotic bodies. PDT with Lonicera japonica extracts was shown to be caspase-3-independent apoptosis via activation of AIF in this study. P38-associated pathway may be involved in apoptosis induced by PDT with Lonicera japonica extracts in CH27 cells. We also have demonstrated that PDT with Lonicera japonica extracts-induced CH27 cells apoptosis was probably related to its ability to change the protein expression and distribution of heat shock protein 27.

Antioxidant enzymes activity involvement in luteolin-induced human lung squamous carcinoma CH27 cell apoptosis

Luteolin (3′,4′,5,7-tetrahydroxyflavone) is an active constituent of Lonicera japonica (Caprifoliaceae), and has been reported to produce anti-tumor activities. However, the apoptosis-inducing activity of luteolin still remains unknown. Flavonoids have been found to posses prooxidant and antioxidant action. The biological and pharmacological effect of flavonoid may depend upon its behavior as either an antioxidant or a prooxidant. Our experiments found that luteolin-induced CH27 cell apoptosis was accompanied by activation of antioxidant enzymes, such as superoxide dismutase and catalase, but not through the production of reactive oxygen species and disruption of mitochondrial membrane potential. Therefore, the effects of luteolin on CH27 cell apoptosis were suspected to result from the antioxidant rather than the prooxidant action of luteolin.

Luteolin induced DNA damage leading to human lung squamous carcinoma CH27 cell apoptosis

Luteolin is an active compound from the Lonicera japonica (Caprifoliaceae). Luteolin (50 μM)-induced human lung carcinoma CH27 cell apoptosis is a typical apoptosis that was accompanied by a significant DNA condensation and apoptotic body formation. Luteolin-induced apoptosis is related to its ability to change the expression of apoptotic markers, such as caspase-3 (caspase-dependent) and apoptosis-inducing factor (caspase-independent) protein expression. The alkaline microgel electrophoresis technique (comet assay), which is the most sensitive, was used for estimation of the luteolin-induced DNA single strand breaks in this study. DNA-damaging effects of luteolin on DNA single strand breaks have been demonstrated in our study. In this study, luteolin induced S-phase cell cycle arrest and increased the mRNA of DNA repair enzymes such as human MutT homologue, 8-oxoguanine-glycosylase and apurinic endonuclease. Our data suggested that luteolin induces CH27 cell apoptosis by caspase-dependent and -independent pathway and the effect of luteolin on apoptosis of CH27 cells is associated with DNA damage and the expression of DNA repair enzymes.

Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs.

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.