Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0–15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line

We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, down-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.     

An anthraquinone derivative from Luffa acutangula induces apoptosis in human lung cancer cell line NCI-H460 through p53-dependent pathway

The current study was designed to evaluate the in vitro antiproliferative activity of 1,8-dihydroxy-4-methylanthracene-9,10-dione (DHMA) isolated from the Luffa acutangula against human non-small cell lung cancer cell line (NCI-H460). Induction of apoptosis and reactive oxygen species (ROS) generation was determined through fluorescence microscopic technique. Quantitative real-time PCR and western blotting analysis was carried out to detect the expression of pro-apoptotic (p53, p21, caspase-3, Bax, GADD45A, and ATM) and anti-apoptotic (NF-κB) proteins in NCI-H460 cell line. In silico studies also performed to predict the binding mechanism of DHMA with MDM2-p53 protein. The DHMA inhibited the cell viability of NCI-H460 cells in a dose-dependent manner with an IC₅₀ of about 50?µg/ml. It significantly reduced cell viability correlated with induction of apoptosis, which was associated with ROS generation. The apoptotic cell death was further confirmed through dual staining and DNA fragmentation assay. DHMA significantly increased the expression of anti-apoptotic protein such as p53, p21, Bax, and caspase-3 but downregulated the expression of NF-κB in NCI-H460 cell line. In silico studies demonstrate that DHMA formed hydrogen bond interaction with key residues Trp26, Phe55 and Lys24 by which it disrupt the binding of p53 with MDM2 receptor. These findings suggested that DHMA induces apoptosis in NCI-H460 via a p53-dependent pathway. This the first study on cytotoxic and apoptosis inducing activity of DHMA from L. acutangula against NCI-H460 cell line. Therefore, DHMA has therapeutic potential for lung cancer treatment.     

重楼皂苷Ⅶ抑制肺癌H460细胞增殖和迁移能力研究

为研究重楼皂苷Ⅶ(polyphyllin Ⅶ)抑制人肺癌H460细胞增殖、迁移能力和诱导凋亡的作用和机制。本实验采用MTT法检测重楼皂苷Ⅶ处理后H460细胞生长抑制率,Hoechst 33258染色观察细胞形态,细胞集落形成实验考察细胞的增殖能力,划痕实验和Transwell小室实验研究H460细胞迁移和侵袭能力的改变,并通过western blot法检测在重楼皂苷Ⅶ处理前后细胞蛋白表达变化情况。结果发现,重楼皂苷Ⅶ可显著抑制H460细胞增殖,影响其集落形成,并使细胞形态发生变化,抑制细胞体外的迁移和侵袭能力,并可诱导凋亡的发生。重楼皂苷Ⅶ处理后,H460细胞中基质金属蛋白酶MMP-2和MMP-9显著降低,凋亡相关蛋白剪切型caspase-3、Bax表达增加,ICAD、Bcl-2表达降低,提示重楼皂苷Ⅶ体外可能通过调节基质金属蛋白酶抑制H460细胞迁移和侵袭,同时诱导凋亡的发生。     

Metformin promotes survivin degradation through AMPK/PKA/GSK-3β-axis in non–small cell lung cancer

Metformin, a first-line antidiabetic drug, has been reported with anticancer activities in many types of cancer. However, its molecular mechanisms remain largely unknown. As a member of inhibitor of apoptosis proteins, survivin plays an important role in the regulation of cell death. In the present study, we investigated the role of survivin in metformin-induced anticancer activity in non–small cell lung cancer in vitro. Metformin mainly induced apoptotic cell death in A549 and H460 cell lines. It remarkably suppressed the expression of survivin, decreased the stability of this protein, then promoted its proteasomal degradation. Moreover, metformin greatly suppressed protein kinase A (PKA) activity and induced its downstream glycogen synthase kinase 3β (GSK-3β) activation. PKA activators, both 8-Br-cAMP and forskolin, significantly increased the expression of survivin. Consistently both GSK-3β inhibitor LiCl and siRNA restored the expression of survivin in lung cancer cells. Furthermore, metformin induced adenosine 5′-monophosphate-activated protein kinase (AMPK) activation. Suppression of the activity of AMPK with Compound C reversed the degradation of survivin induced by metformin, and meanwhile, restored the activity of PKA and GSK-3β. These results suggest that metformin kills lung cancer cells through AMPK/PKA/GSK-3β-axis–mediated survivin degradation, providing novel insights into the anticancer effects of metformin.     

Antioxidant and anticancer activity of extract from Betula platyphylla var. japonica

The antioxidant and anticancer properties of a medicinal plant, Betula platyphylla var. japonica were investigated. The total methanol extract of B. platyphylla var. japonica had protective effects against hydrogen peroxide (H2O2) in the Chinese hamster lung fibroblast (V79-4) cell line and induced apoptotic cell death in human promyelocytic leukemia (HL-60) cells, a cancer cell line. B. platyphylla var. japonica extract significantly increased cell viability against H2O2. The extract also showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 2.4 microg/ml) and lipid peroxidation inhibitory activity (IC50 below 4.0 microg/ml). Furthermore, B. platyphylla var. japonica extract reduced the number of V79-4 cells arrested in G2/M in response to H2O2 treatment and increased the activities of several cellular antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. Treatment with B. platyphylla var. japonica extract induced cytotoxicity and apoptosis in HL-60 cells, as shown by nucleosomal DNA fragmentation, increases in the subdiploid cell population, and fluorescence microscopy. B. platyphylla var. japonica extract gradually increased the expression of pro-apoptotic Bax and led to the activation of caspase-3 and cleavage of PARP. These findings suggest that B. platyphylla var. japonica exhibits potential antioxidant and anticancer properties.     

Inhibition of fibroblast growth factor 2-induced apoptosis involves survivin expression, protein kinase C alpha activation and subcellular translocation of Smac in human small cell lung cancer cells

To investigate the mechanism by which fibroblast growth factor 2 (FGF-2) inhibits apoptosis in the human small cell lung cancer cell line H446 subjected to serum starvation, apoptosis was evaluated by flow cytometry, Hoechst 33258 staining, caspase-3 activity, and DNA fragmentation. Survivin expression induced by FGF-2 and protein kinase Cα (PKCα) translocation was detected by subcellular frac-tionation and Western blot analysis. In addition, FGF-2-in-duced release of Smac from mitochondria to the cytoplasm was analyzed by Western blotting and immunofluorescence. FGF-2 reduced apoptosis induced by serum starvation and up-regulated survivin expression in H446 cells in a dose-dependent andtime-dependentmanner, andinhibitedcaspase-3 activity. FGF-2 also inhibited the release of Smac from mitochondria to the cytoplasm induced by serum starvation and increased PKCα translocation from the cytoplasm to the cell membrane. In addition, PKC inhibitor inhibited the expression of survivin. FGF-2 up-regulates the expression of survivin protein in H446 cells and blocks the release of Smac from mitochondria to the cytoplasm. PKCα regulated FGF-2-induced survivin expression. Thus, survivin, Smac, and PKCα might play important roles in the inhibition of apoptosis by FGF-2 in human small cell lung cancer cells.     

TIPE2 Inhibits Lung Cancer Growth Attributing to Promotion of Apoptosis by Regulating Some Apoptotic Molecules Expression

Recent studies found that TIPE2 was involved in cancer development. However, little is known about TIPE2 in lung cancer. Our study aims to clarify the role of TIPE2 in lung carcinogenesis. We examined the expression of TIPE2 in lung squamous cancer (LSC), small cell lung cancer and lung adenocarcinoma (AdC) tissues and found that TIPE2 expression was lost in small cell lung cancer, compared with adjacent non-tumor tissues. Overexpression of TIPE2 significantly inhibited the growth of lung cancer cell H446 in vitro and even suppressed tumor formation in vivo. Flow cytometry analysis found TIPE2 overexpression promoted apoptosis of H446. In TIPE2 over-expression cells, caspase-3, caspase-9, and Bax were significantly up-regulated while Bcl-2 was down-regulated. Moreover, coincident results were shown by immunohistochemistry in tumors from nude mice. TIPE2 inhibited the phosphorylation of Akt, while promoting the phosphorylation of P38, but had no effect on IκBα and ERK pathway. Taken together, TIPE2 promoted lung cancer cell apoptosis through affecting apoptosis-related molecules caspase-3, caspase-9, Bcl-2 and Bax, possibly via regulating P38 and Akt pathways, indicating that TIPE2 might be a novel marker for lung cancer diagnosis and therapy.     

Arctigenin Enhances Chemosensitivity to Cisplatin in Human Nonsmall Lung Cancer H460 Cells through Downregulation of Survivin Expression

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin‐mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin‐treated non‐small‐cell lung cancer (NSCLC) H460 cells. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and annexin‐V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose‐dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase‐3 and poly(ADP‐ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin‐induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell‐cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina‐tion with chemotherapeutic agents for NSLC.     

Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.     

ATP sensitizes H460 lung carcinoma cells to cisplatin-induced apoptosis

Platinum resistance of cancer cells may evolve due to a decrease in intracellular drug accumulation, decreased cell permeability or by an increased deactivation of the drug by glutathione (GSH). The aim of this study was (1) to investigate the effect of adenosine 5′-triphosphate (ATP) on the cytotoxicity of cisplatin in a large cell lung carcinoma cell line (H460), and (2) to examine the potential involvement of increased cisplatin uptake, GSH depletion and pyrimidine starvation by ATP in this effect. H460 cells were harvested and seeded (5% CO₂; 37 °C). Subsequently, cells were incubated with medium or ATP followed by an incubation with cisplatin. Cytotoxicity screening was analyzed by the sulforhodamine B (SRB) colorimetric assay, lactate dehydrogenase and caspase-3/7 activity. Pre-incubation for 72 h with 0.3 and 3 mM ATP strongly enhanced the anti-proliferative potency of cisplatin 2.9- and 7.6-fold, respectively. Moreover, after incubation of H460 cells with 0.3 mM ATP the intracellular platinum concentration increased, indicating increased cisplatin uptake by ATP. ATP, despite lowering the LD₅₀ of cisplatin, did not modulate GSH levels in H460 cells. ATP itself showed a biphasic effect on H460 cell growth: 0.3 mM inhibited H460 cell growth via the pyrimidine starvation effect, activation of caspase-3/7 and LDH leakage, while 3 mM ATP showed no effect on cell growth. In conclusion, ATP sensitizes the H460 cells to cisplatin-induced apoptosis. The effect of 0.3 mM ATP is not due to GSH depletion but involves increased cisplatin uptake and pyrimidine starvation due to ATP conversion to adenosine followed by cellular uptake.     

BCI induces apoptosis via generation of reactive oxygen species and activation of intrinsic mitochondrial pathway in H1299 lung cancer cells

The compound(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1 H-inden-1-one(BCI) is known as an inhibitor of dual specific phosphatase 1/6 and mitogen-activated protein kinase. However, its precise anti-lung cancer mechanism remains unknown. In this study, the effects of BCI on the viability of non-small cell lung cancer cell lines NCI-H1299, A549, and NCI-H460 were evaluated. We confirmed that BCI significantly inhibited the viability of p53(-) NCI-H1299 cells as compared to NCI-H460 and A549 cells, which express wild-type p53. Furthermore, BCI treatment increased the level of cellular reactive oxygen species and pre-treatment of cells with N-acetylcysteine markedly attenuated BCI-mediated apoptosis of NCI-H1299 cells. BCI induced cellular morphological changes, inhibited viability, and produced reactive oxygen species in NCI-H1299 cells in a dose-dependent manner. BCI induced processing of caspase-9, caspase-3, and poly ADP-ribose polymerase as well as the release of cytochrome c from the mitochondria into the cytosol. In addition, BCI downregulated Bcl-2 expression and enhanced Bax expression in a dose-dependent manner in NCI-H1299 cells. However, BCI failed to modulate the expression of the death receptor and extrinsic factor caspase-8 and Bid, a linker between the intrinsic and extrinsic apoptotic pathways in NCI-H1299 cells. Thus, BCI induces apoptosis via generation of reactive oxygen species and activation of the intrinsic pathway in NCI-H1299 cells.     

Oleanolic acid initiates apoptosis in non-small cell lung cancer cell lines and reduces metastasis of a B16F10 melanoma model in vivo

Background

Drug resistance, a process mediated by multiple mechanisms, is a critical determinant for treating lung cancer. The aim of this study is to determine if oleanolic acid (OA), a pentacyclic triterpene present in several plants, is able to circumvent the mechanisms of drug resistance present in non-small cell lung cancer (NSCLC) cell lines and to induce their death.

Principal Findings

OA decreased the cell viability of the NSCLC cell lines A459 and H460 despite the presence of active, multidrug-resistant (MDR) MRP1/ABCC1 proteins and the anti-apoptotic proteins Bcl-2 and survivin. These effects are due to apoptosis, as evidenced by the capacity of OA to induce fragmentation of DNA and activate caspase 3. Induction of NSCLC cell death by OA cannot be explained by inhibition of the MDR proteins, since treatment with triterpene had little or no effect on the activity or expression of MRP1. Moreover, treatment with OA had no effect on the expression of the anti-apoptotic protein Bcl-2, but increased the expression of the pro-apoptotic protein Bax, altering the Bcl-2/Bax balance towards a pro-apoptotic profile. OA also decreased the expression of the anti-apoptotic protein survivin. Furthermore, OA decreased the expression of the angiogenic vascular endothelial growth factor (VEGF) and decreased the development of melanoma-induced lung metastasis.

Conclusion

Our data provide a significant insight into the antitumoral and antimetastatic activity of OA in NSCLC and suggest that including OA in the NSCLC regimens may help to decrease the number of relapses and reduce the development of metastases.     

p53 Regulates Cellular Responses to Environmental Carcinogen Benzo[a]pyrene-7,8-diol-9,10-epoxide in Human Lung Cancer Cells

The p53 tumor suppressor is a mutational target of environmental carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). We now demonstrate that p53 plays an important role in regulation of cellular responses to BPDE. Exposure of p53-null H1299 human lung cancer cells to BPDE resulted in S and G2 phase cell cycle arrest, but not mitotic block, which correlated with induction of cyclin B1 protein expression, down-modulation of cell division cycle 25C (Cdc25C) and Cdc25B protein levels, and hyperphosphorylation of Cdc25C (S216), cyclin-dependent kinase 1 (Cdk1; Y15), checkpoint kinase 1 (Chk1; S317 and S345) and Chk2 (T68). The BPDE-induced S phase block, but not the G2/M phase arrest, was significantly attenuated by knockdown of Chk1 protein level. The BPDE-mediated accumulation of sub-diploid fraction (apoptotic cells) was significantly decreased in H1299 cells transiently transfected with both Chk1 and Chk2 specific siRNAs. The H460 human lung cancer cell line (wild-type p53) was relatively more sensitive to BPDE-mediated growth inhibition and enrichment of sub-diploid apoptotic fraction compared with H1299 cells. The BPDE exposure failed to activate either S or G2 phase checkpoint in H460 cells. Instead, the BPDE-treated H460 cells exhibited a nearly 8-fold increase in sub-diploid apoptotic cells that was accompanied by phosphorylation of p53 at multiple sites. Knockdown of p53 protein level in H460 cells attenuated BPDE-induced apoptosis but enforced activation of S and G2 phase checkpoints. In conclusion, the present study points towards an important role of p53 in regulation of cellular responses to BPDE in human lung cancer cells.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Influence of gastrin on the expression of cyclooxygenase-2, hepatocyte growth factor and apoptosis-related proteins in gastric epithelial cells.

BACKGROUND: Several studies have shown a link between gastrin and gastric cancer, both in humans and animals, especially infected with Helicobacter pylori (H. pylori). However, the exact role of hypergastrinemia in gastric carcinogenesis remains still undetermined. The aim of the present study was to evaluate the interaction between gastrin, cyclooxygenase-2 (COX-2), hepatocyte growth factor (HGF) and apoptosis-related proteins (Bax, Bcl-2, caspase-3, survivin) in cultured gastric epithelial cancer cells. MATERIAL AND METHODS: In the present study, gastric cultured cancer cells (KATO III cells) were exposed to increasing concentrations of gastrin (1-1000 nM). Cells incubated with culture medium alone, without added gastrin, served as controls. Using RT-PCR and Western blot, we examined the mRNA and protein expression for COX-2, HGF and apoptosis-related proteins (Bax, Bcl-2, caspase-3 and survivin). In addition, the gene expression of gastrin and gastrin receptor (CCK-2) as well as the release of gastrin in culture medium in the unstimulated cells were examined by RT-PCR and RIA, respectively. The apoptosis rate in cells was measured by flow cytometric analysis. RESULTS: The present study shows that the gastric cultured epithelial cells exhibit the expression of gastrin and CCK-2 receptors and release of gastrin into the culture medium. The epithelial gastric cancer cells incubated with gastrin showed a concentration-dependent increase of COX-2 and HGF expression. Although no significant changes in apoptosis rate were observed, the exposure of these cells was associated with a dose-dependent increase in the expression of antiapoptotic proteins Bcl-2 and survivin. CONCLUSIONS: This study demonstrates that 1) gastrin stimulates the gene and protein expression of COX-2 and HGF in human cultured gastric cancer cells and 2) gastrin shows antiapoptotic activity through the upregulation of Bcl-2 and survivin.     

Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network formation ability of cells was significantly inhibited by survivin silencing and completely by combination of survivin silencing and EGCG treatment. Collectively, survivin silencing potentiated anti-cancer effects of EGCG in human malignant neuroblastoma cells having survivin overexpression.     

Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection

This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.     

CCL21/CCR7 prevents apoptosis via the ERK pathway in human non-small cell lung cancer cells

Previously, we confirmed that C-C chemokine receptor 7 (CCR7) promotes cell proliferation via the extracellular signal-regulated kinase (ERK) pathway, but its role in apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. A549 and H460 cells of NSCLC were used to examine the effect of CCL21/CCR7 on apoptosis using flow cytometry. The results showed that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant decline in the percent of apoptosis. Western blot and real-time PCR assays indicated that activation of CCR7 significantly caused upregulation of anti-apoptotic bcl-2 and downregulation of pro-apoptotic bax and caspase-3, but not p53, at both protein and mRNA levels. CCR7 small interfering RNA significantly attenuated these effects of exogenous CCL21. Besides, PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished these effects of CCL21/CCR7. Coimmunoprecipitation further confirmed that there was an interaction between p-ERK and bcl-2, bax, or caspase-3, particularly in the presence of CCL21. These results strongly suggest that CCL21/CCR7 prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax and caspase-3 potentially via the ERK pathway in A549 and H460 cells of NSCLC.