Ming-Yue Li

Apoptosis induced by troglitazone is both peroxisome proliferator‐activated receptor‐γ‐ and ERK‐dependent in human non‐small lung cancer cells

The role of the peroxisome proliferator‐activated receptor‐gamma (PPARγ) in cell differentiation, cell‐cycle arrest, and apoptosis has attracted increasing attention. We have recently demonstrated that PPARγ ligands‐troglitazone (TGZ) induced apoptosis in lung cancer cells. In this report, we further studied the role of ERK1/2 in lung cancer cells treated by TGZ. The result demonstrated that TGZ induced PPARγ and ERK1/2 accumulation in the nucleus, in which the co‐localization of both proteins was found. The activation of ERK1/2 resulted in apoptosis via a mitochondrial pathway. Both PPARγ siRNA and U0126, a specific inhibitor of ERK1/2, were able to block these effects of TGZ, suggesting that apoptosis induced by TGZ was PPARγ and ERK1/2 dependent. Inhibition of ERK1/2 by U0126 also led to a significant decrease in the level of PPARγ, indicating a positive cross‐talk between PPARγ and ERK1/2 or an auto‐regulatory feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and apoptosis. In addition to ERK1/2, TGZ also activated Akt. Interestingly, inhibition of ERK1/2 prevented the activation of Akt whereas the suppression of Akt had no effect on ERK1/2, suggesting that Akt was not necessary for TGZ‐PPARγ‐ERK pathway. However, the inhibition of Akt promoted the release of cytochrome c, suggesting the activation of Akt may have a negative effect on apoptosis induced by TGZ. In conclusion, our study has demonstrated that TGZ, a synthetic PPARγ ligand, induced apoptosis in NCI‐H23 lung cancer cells via a mitochondrial pathway and this pathway was PPARγ and ERK1/2 dependent. J. Cell. Physiol. 209: 428–438, 2006.

Activation of peroxisome proliferator‐activated receptor‐γ by troglitazone (TGZ) inhibits human lung cell growth

Peroxisome proliferator‐activated receptor‐gamma (PPAR‐γ) is a member of the nuclear hormone receptor superfamily of ligand‐activated transcription factors and a crucial regulator of cellular differentiation. PPAR‐γ ligands have been demonstrated to inhibit growth of several cancer cells. In this study, two human lung cancer cells (NCI‐H23 and CRL‐2066) and one human lung normal cell (CRL‐202) were used for the experiments. The results showed that in consistence with the loss of viability, troglitazone (TGZ) induced apoptosis of CRL‐2066 and NCI‐H23 cells but not CCL‐202 cells. TGZ upregulated PPAR‐γ expression in all the three lung cell lines, especially in the cancer cells. In association of the time‐dependent inhibition of the cell proliferation, TGZ downregulated the expression of Bcl‐w and Bcl‐2 but activated extracellular signal‐regulated kinase (ERK)1/2 and p38, suggesting that the growth‐inhibitory effect of TGZ is associated with the reduction of Bcl‐w and Bcl‐2 and the increase of ERK1/2 and p38 activation. SAPK/JNK activation assay showed a decreased activity in all the three cell lines tested after TGZ treatment. It was also demonstrated that TGZ could activate PPAR‐γ transcriptionally. We conclude that TGZ inhibits growth of human lung cancer cells via the induction of apoptosis and the inhibition of cell growth, at least in part, in a PPAR‐γ‐relevant manner. The mechanism of TGZ is associated with the activation of ERK and p38, the reduction of SAPK/JNK activity, and the alteration of Bcl‐w and Bcl‐2.

4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A 2 and its receptor

The role of thromboxane A2 (TxA2) in smoking-associated lung cancer is poorly understood. This study was conducted to study the role of TxA2 in smoking carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-promoted cell survival and growth in human lung cancer cells. We found that NNK increased TxA2 synthase (TxAS) expression and thromboxane B2 (TxB2) generation in cultured lung cancer cells, the result of which was supported by the increased level of TxAS in lung cancer tissues of smokers. Both TxAS-specific inhibitor furegrelate and TxA2 receptor antagonist SQ29548 completely blocked NNK-mediated cell survival and growth via inducting apoptosis. TxA2 receptor agonist U46619 reconstituted a near-full survival and growth response to NNK when TxAS was inhibited, affirming the role of TxA2 receptor in NNK-mediated cell survival and growth. Suppression of cyclic adenosine monophosphate response element binding protein (CREB) activity by its small interference RNA blocked the effect of NNK. Phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) also had a positive role. Altogether, our results have revealed that NNK stimulates TxA2 synthesis and activates its receptor in lung cancer cells. The increased TxA2 may then activate CREB through PI3K/Akt and extracellular ERK pathways, thereby contributing to the NNK-promoted survival and growth of lung cancer cells.

Roles of Peroxisome Proliferator–Activated Receptor–α and –γ in the Development of Non–Small Cell Lung Cancer

Peroxisome proliferator-activated receptor (PPAR)-α and PPARγ participate in cell proliferation and apoptosis. Few studies have simultaneously investigated both PPARα and PPARγ in lung cancers in vivo. The roles of PPARα and -γ were investigated in the development of pulmonary tumors induced in the adult A/J mouse by treatment with 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone (NNK). Compared with the normal lung tissues, PPARγ expression was much higher in the NNK-induced lung tumor tissues. However, PPARγ transcriptional activity, and the levels of two major endogenous PPARγ ligands, 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, were significantly lower in the NNK-treated lung tissues. The ligand changes in mice were confirmed in human lung cancer tissues. Along with the alteration of PPARγ and its endogenous ligands, the level of PPARα and its activity were increased in the NNK-induced mouse lung tumors. Treatment of mice with the synthetic PPARγ ligand, pioglitazone, significantly inhibited the formation of mouse lung tumors induced by NNK. Our study demonstrated that the reduction of endogenous PPARγ ligands and increased PPARα occurred before the formation of lung tumors, indicating that the molecular changes play a role in lung carcinogenesis. The results suggest that the enhancement of PPARγ activity with its ligands, and the suppression of PPARα with its inhibitors, may prevent the formation of lung tumors, as well as accelerate the therapy of lung cancer. Our findings may also reveal the possibility of using the level of endogenous PPARγ ligands and the activities of PPARγ or PPARα as tumor markers for lung cancer.

Function of Pparγ and Its Ligands in Lung Cancer

Peroxisome proliferator-activated receptors gamma (PPARγ) is a transcriptional factor belonging to the ligand-activated nuclear receptor superfamily. PPARγ is highly expressed in adipose tissue and has a dominant regulatory role in adipocyte differentiation. In humans, PPARγ is expressed in multiple tissues such as the breast, colon, lung, ovary, and placenta. In addition to adipogenic and anti-inflammatory effects, PPARγ activation has been shown to be anti-proliferative by virtue of its differentiation-promoting effect, suggesting that activation of PPARγ may be useful in slowing or arresting the proliferation of de-differentiated tumor cells. A number of PPARγ ligands, such as natural prostaglandins and synthetic anti-diabetic thiazolidinediones (TZDs), have been identified. The discovery of PPARγ agonists has enabled the elucidation of the mechanisms involved in the multiple effects of PPARγ on the inhibition of tumor cell growth. The importance of this transcription factor in physiology and pathophysiology has stimulated much research in this field. This review describes structural features of PPARγ, mechanisms of PPARγ gene transcription, and recent developments in the discovery of its biological functions on growth inhibition of lung tumors. Prospects for future research leading to new therapies for lung cancer are also discussed.

Haem oxygenase-1 plays a central role in NNK-mediated lung carcinogenesis

The tobacco-specific nitrosamine, 4-(N-methyl-N-nitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung cancer inducer. However, how NNK induces lung cancer is still largely unknown. Haem oxygenase (HO)-1 was evaluated in 30 pairs of lung cancer tumour samples and matched nontumour tissues from patients with a history of cigarette smoking. Expression of HO-1, p21Cip1/Waf1/Cid1 (p21), B-cell lymphoma (Bcl)-2 family members, mitogen-activated protein kinase and nuclear factor (NF)-κB was also studied in lung cancer cells treated with NNK. The levels of HO-1 and p21 were significantly increased in lung tumour tissues. There was a positive relationship between these two proteins in the tumour. NNK stimulated lung cell proliferation and elevated the levels of HO-1, p21, inhibitor of apoptosis protein (c-IAP)2 and Bcl-2, but downregulated Bad. These effects of NNK were blocked by zinc protoporphyrin-XII, an HO-1 inhibitor. The NNK-mediated expression of HO-1 was governed by NF-κB and extracellular signal-regulated kinase 1/2, since blocking either of these prevented the stimulatory effect of NNK on HO-1, as well as molecules downstream of HO-1, such as p21, c-IAP2, Bcl-2 and Bad. In conclusion, haem oxygenase-1 plays a central role in NNK-mediated cell proliferation by promoting the expression of p21Cip1/Waf1/Cid1, inhibitor of apoptosis protein 2 and B-cell lymphoma-2 but inhibiting the activity of Bad. Nuclear factor-κB and extracellular signal-regulated kinase 1/2 function upstream of haem oxygenase-1. Therefore, haem oxygenase-1 is likely to be a potential target in the treatment of smoking-related lung cancer.

Increased inducible nitric oxide synthase in lung carcinoma of smokers

Cigarette smoking is well known to play an important role in the development of lung cancer. Inducible nitric oxide synthase (iNOS) can either promote or inhibit cell proliferation and growth, which makes its role in the development of malignant tumors controversial. The relation between cigarette smoking and iNOS in human lung cancer is unknown.

Thromboxane A2 exerts promoting effects on cell proliferation through mediating cyclooxygenase-2 signal in lung adenocarcinoma cells

AbstractBackground Lung cancer concerns a worldwide health problem and the efficacy of available treatments is unsatisfactory. Recently, thromboxane A2 (TXA2) synthase (TXAS) and receptor (TXA2R) have been documented to play a role in lung cancer development. Therefore, dual TXA2R modulator (i.e., the dual blocker of TXAS and TXA2R) may be more efficacious to kill lung tumor cells than single TXAS inhibitor or TXA2R antagonism. The close relationship between cyclooxygenase (COX)-2 and TXAS also raises whether or how TXA2 contributes to the oncogenic activity of COX-2. This study is therefore conducted to answer these questions.MethodsVarious inhibitors and siRNA were used to evaluate the roles of TXA2 and COX-2 in the proliferation and apoptosis of lung adenocarcinoma cells. Cell proliferation was detected using both MTS ELISA and BrdU labeling ELISA. Cell cycle distribution and apoptosis were examined by flow cytometric analysis. TXB2 level, reflecting the biosynthesis of TXA2, was detected by peroxidase-labeled TXB2 conjugates using an enzyme immunoassay kit. Western blotting was performed to evaluate many biomarkers for cell cycles, apoptosis and proliferation. The levels of COXs were screened by reverse transcriptase and real-time quantitative PCR.ResultsWe found either single TXAS inhibitor/TXA2R antagonist or the dual TXA2 modulators offered a similar inhibition on cell proliferation. Moreover, inhibition of TXA2 arrested cells at the G2/M phase and induced apoptosis. It is further demonstrated that TXA2 was able to function as a critical mediator for tumor-promoting effects of COX-2 in lung adenocarcinoma cells.ConclusionThe present study has for the first shown that dual TXA2 modulators and the single blocker of TXAS or TXA2R offer a similar inhibitory role in lung adenocarcinoma cell proliferation and that the tumor-promoting effects of COX-2 can largely be relayed by TXA2. Thus, TXA2 should be regarded as a critical molecule in COX-2-mediated tumor growth and a valuable target against lung cancer.

Inhibition of thromboxane synthase induces lung cancer cell death via increasing the nuclear p27.

The role of thromboxane in lung carcinogenesis is not clearly known, though thromboxane B2 (TXB(2)) level is increased and antagonists of thromboxane receptors or TXA2 can induce apoptosis of lung cancer cells. p27, an atypical tumor suppressor, is normally sequestered in the nucleus. The increased nuclear p27 may result in apoptosis of tumor cells. We hypothesize that the inhibition of thromboxane synthase (TXS) induces the death of lung cancer cells and that such inhibition is associated with the nuclear p27 level. Our experiment showed that the inhibition of TXS significantly induced the death or apoptosis in lung cancer cells. The activity of TXS was increased in lung cancer. The nuclear p27 was remarkably reduced in lung cancer tissues. The inhibition of TXS caused the cell death and apoptosis of lung cancer cells, likely via the elevation of the nuclear p27 since the TXS inhibition promoted the nuclear p27 level and the inhibition of p27 by its siRNA recovered the cell death induced by TXS inhibition. Collectively, lung cancer cells produce high levels of TXB(2) but their nuclear p27 is markedly reduced. The inhibition of TXS results in the p27-related induction of cell death in lung cancer cells.

Ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis

ObjectiveTo examine the apoptotic effect of ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), a compound isolated from Pteris semipinnata L (PsL), in human lung cancer A549 cells.MethodsA549 cells were treated with 5F (0–80 μg/ml) for different time periods. Cytotoxicity was examined using a MTT method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay (ELISA) and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species (ROS) level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined.Results5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor (AIF), and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin.Conclusion5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis.