Isokotomolide A, a new butanolide extracted from the leaves of Cinnamomum kotoense, arrests cell cycle progression and induces apoptosis through the induction of p53/p21 and the initiation of mitochondrial system in human non-small cell lung cancer A549 cells

This study is the first to investigate isokotomolide A (IKA), a butanolide compound isolated from the leaves of Cinnamomum kotoense Kanehira & Sasaki (Lauraceaee), which exhibits an anti-proliferative activity in human non-small cell lung cancer A549 cells. The results show that IKA inhibits the proliferation of A549 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels and reduced amounts of cyclin D1, cyclin E, Cdk2, Cdk4, and Cdk6 in a p53-mediated manner. IKA treatment also increased p53 phosphorylation (Ser15) and decreased the interaction of p53-MDM2. IKA treatment triggered the mitochondrial apoptotic pathway, indicated by changing Bax/Bcl-2 ratios, cytochrome c release and caspase-9 activation. In addition, pre-treatment of cells with caspase-9 inhibitor inhibited IKA-induced apoptosis, indicating that caspase-9 activation was involved in A549 cells' apoptosis induced by IKA. Our study reports here for the first time that the induction of p53/p21 and the initiation of the mitochondrial apoptotic system may participate in the anti-proliferative activity of IKA in human non-small cell lung cancer cells.     

Putranjivain A from Euphorbia jolkini inhibits proliferation of human breast adenocarcinoma MCF-7 cells via blocking cell cycle progression and inducing apoptosis

Putranjivain A, isolated from the whole plant of Euphorbia jolkini Bioss (Euphorbiaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. The results showed that putranjivain A inhibited the proliferation of MCF-7 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. Enzyme-linked immunosorbent assay showed that putranjivain A increased the expression of p21/WAF1 concomitantly as MCF-7 cell underwent G0/G1 arrest. An enhancement in Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by putranjivain A. Our study reports here for the first time that the induction of p21/WAF1 and the activity of Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of putranjivain A in MCF-7 cells.     

Chalcone inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis.

Chalcones are discussed to represent cancer preventive food components in a human diet that is rich in fruits and vegetables. In this study, we examined chalcone (1,3-diphenyl-2-propenone) for its effect on proliferation in human breast cancer cell lines, MCF-7 and MDA-MB-231. The results showed that chalcone inhibited the proliferation of MCF-7 and MDA-MB-231 by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Immunoblot assay showed that chalcone significantly decreased the expression of cyclin B1, cyclin A and Cdc2 protein, as well as increased the expression of p21 and p27 in a p53-independent manner, contributing to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), was responsible for the apoptotic effect induced by chalcone. In addition, chalcone also triggered the mitochondrial apoptotic signaling by increasing the amount of Bax and Bak and reducing the level of Bcl-2 and Bcl-X(L), and subsequently activated caspase-9 in MCF-7 and MDA-MB-231 cells. Taken together, our study suggests that the blockade of cell cycle progression and initiation of cell apoptotic system may participate in the antiproliferative activity of chalcone in human breast cancer cells.     

Casuarinin from the bark of Terminalia arjuna induces apoptosis and cell cycle arrest in human breast adenocarcinoma MCF-7 cells

Casuarinin, a hydrolyzable tannin isolated from the bark of Terminalia arjuna L. (Combretaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. The results showed that casuarinin inhibited the proliferation of MCF-7 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. An enzyme-linked immunosorbent assay showed that casuarinin increased the expression of p21/WAF1 concomitantly as the MCF-7 cells underwent G0/G1 arrest. An enhancement in Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by casuarinin. Our study reports here for the first time that the induction of p21/WAF1 and the activity of Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of casuarinin in MCF-7 cells.     

Upregulation of Fas/Fas ligand-mediated apoptosis by gossypol in an immortalized human alveolar lung cancer cell line

Gossypol has wide antineoplastic effects in vitro, but its effects on human lung cancer have not been explored. To evaluate the activity of gossypol against alveolar cell lung cancer and to provide information on the mechanism of action, we examined the effects of gossypol on the proliferation of A549 cells indirectly using an XTT assay and on the distribution of cells within the phases of the cell cycle using flow cytometry. We also examined several factors that may affect apoptosis, including p53, p21/WAF1, Fas receptor, Fas ligand (FasL) and caspase 8 activity. The results showed that gossypol inhibited proliferation of A549 cells at a concentration of 0.5 micromol/L after 12 h treatment. The effect was both dose- and time-dependent by the induction of apoptosis without the effect of p53 and p21/WAF1. Upregulation of Fas/FasL, in association with the activation of downstream caspase 8 activity, was observed following treatment with gossypol. The Fas/FasL pathway accounted for 75% of gossypol-mediated apoptosis. We suggest that the Fas/FasL apoptotic system is the major pathway for gossypol-mediated apoptosis of A549 cells. Gossypol had no effect on the distribution of A549 cells within the phases of the cell cycle. In conclusion, gossypol inhibited A549 cells mainly by induction of the Fas/FasL apoptotic pathway, but not the p53 and p21/WAF1 pathway.     

Isoliquiritigenin inhibits the proliferation and induces the apoptosis of human non-small cell lung cancer a549 cells

1. Isoliquiritigenin (ISL) is a natural pigment with the simple chalcone structure 4,2',4'-trihydroxychalcone. In the present study, we report, for the first time, ISL-induced inhibition of the proliferation of the human non-small cell lung cancer A549 cell line. 2. The results showed that ISL not only inhibited A549 cell proliferation, but also induced apoptosis and blocked cell cycle progression in the G1 phase. An ELISA assay demonstrated that ISL significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. 3. An enhancement in Fas and its two ligands, namely membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), may be responsible for the apoptotic effect induced by ISL. 4. Taken together, the results indicate that the p53 and Fas/FasL apoptotic system may participate in the antiproliferative activity of ISL in A549 cells.     

Induction of cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells by casuarinin from the bark of Terminalia arjuna Linn

Casuarinin, a hydrolyzable tannin isolated from the bark of Terminalia arjuna Linn. (Combretaceae), inhibits human non-small cell lung cancer A549 cells by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. Enzyme-linked immunosorbent assay showed that the G0/G1 phase arrest is due to p53-dependent induction of p21/WAF1. An enhancement in Fas/APO-1 and the two forms of Fas ligand (FasL), membrane-bound FasL and soluble FasL, might be responsible for the apoptotic effect induced by casuarinin. Our study reports here for the first time that the induction of p53 and the activity of the Fas/FasL apoptotic system may participate in the antiproliferative activity of casuarinin in A549 cells.     

Norsolorinic acid from Aspergillus nidulans inhibits the proliferation of human breast adenocarcinoma MCF-7 cells via Fas-mediated pathway

Norsolorinic acid, isolated from the Aspergillus nidulans, was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. To identity the anticancer mechanism of norsolorinic acid, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that norsolorinic acid induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that Fas/Fas ligand apoptotic system is the main pathway of norsolorinic acid-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of norsolorinic acid in MCF-7 cells.     

Involvement of reactive oxygen species/c-Jun NH(2)-terminal kinase pathway in kotomolide A induces apoptosis in human breast cancer cells

The anticancer effects of kotomolide A (KTA), a new butanolide constituent isolated from the leaves of Cinnamomum kotoense (Lauraceae), on the two human breast cancer cell lines MCF-7 and MDA-MB-231, were first investigated in our study. KTA exhibited selectively antiproliferative effects in cancer cell lines without showing any toxicity in normal mammary epithelial cells. Treatment of cancer cells with KTA to trigger G2/M phase arrest was associated with increased p21/WAF1 levels and reduced amounts of cyclin A, cyclin B1, cdc2 and cdc25C. KTA induced cancer cell death treatment by triggering mitochondrial and death receptor 5 (DR5) apoptotic pathways, but did not act on the Fas receptor. Exposure of MCF-7 and MDA-MB-231 cells to KTA resulted in cellular glutathione reduction and ROS generation, accompanied by JNK activation and apoptosis. Both antioxidants, NAC and catalase, significantly decreased apoptosis by inhibiting the phosphorylation of JNK and subsequently triggering DR5 cell death pathways. The reduction of JNK expression by siRNA decreased KTA-mediated Bim cleavage, DR5 upregulation and apoptosis. Furthermore, daily KTA i.p. injections in nude mice with MDA-MB-231 s.c. tumors resulted in a 50% decrease of mean tumor volume, compared with vehicle-treated controls. Taken together, the data show that cell death of breast cancer cells in response to KTA is dependent upon ROS generation and JNK activation, triggering intrinsic and extrinsic apoptotic pathways. The ROS/JNK pathway could be a useful target for novel approaches in breast cancer chemotherapy.     

Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by pterocarnin A from the bark of Pterocarya stenoptera via the Fas-mediated pathway

Pterocarnin A, isolated from the bark of Pterocarya stenoptera (Juylandaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. To identify the anticancer mechanism of pterocarnin A, we assayed its effects on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that pterocarnin A induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that the Fas/Fas ligand apoptotic system is the main pathway of pterocarnin A-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of pterocarnin A in MCF-7 cells.     

A phenylacetate derivative,SCK6, inhibits cell proliferation via G1 cell cycle arrest and apoptosis

Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G(1) arrest in CH27 cells. Western blot analysis of G(1) phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21(CIP1/WAF1). Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G(1)-associated Cdks and cyclins and upregulation of p53 and p21(CIP1/WAF1) may contribute to SCK6-mediated G(1)-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.     

Indole-3-carbinol induces apoptosis through p53 and activation of caspase-8 pathway in lung cancer A549 cells

Indole-3-carbinol (I3C) has anti-tumor effects in various cancer cell lines. However, the anti-tumor effect of I3C on human lung cancers has been rarely reported. We investigated the anti-tumor effects and its mechanism of I3C on human lung carcinoma A549 cell line. Treatment of the A549 cells with I3C significantly reduced cell proliferation, increased formations of fragmented DNA and apoptotic body, and induced cell cycle arrest at G0/G1 phase. I3C increased not only the protein levels of cyclin D1, phosphorylated p53, and p21 but also the expression of Fas mRNA. Cleavage of caspase-9, -8, -3 and PARP also was increased by I3C. Treatment with wortmannin significantly suppressed both I3C-induced Ser15 phosphorylation and accumulation of p53 protein. The inhibition of caspase-8 by z-IETD-FMK significantly decreased cleavage of procaspase-8,-3 and PARP in I3C-treated A549 cells. Taken together, these results demonstrate that I3C induces cell cycle arrest at G0/G1 through the activation of p-p53 at Ser 15 and induces caspase-8 mediated apoptosis via the Fas death receptor. This molecular mechanism for apoptotic effect of I3C on A549 lung carcinoma cells may be a first report and suggest that I3C may be a preventive and therapeutic agent against lung cancer.     

Eupatilin, a dietary flavonoid, induces G2/M cell cycle arrest in human endometrial cancer cells

This study is the first to investigate the antiproliferative effect of eupatilin in human endometrial cancer cells. Eupatilin, a naturally occurring flavonoid isolated from Artemisia princeps, has anti-inflammatory, anti-oxidative, and anti-tumor activities. In the present study, we investigated the potential effect of eupatilin on cell growth and its molecular mechanism of action in human endometrial cancer cells. Eupatilin was more potent than cisplatin in inhibiting cell viability in the human endometrial cancer cell lines Hec1A and KLE. Eupatilin showed relatively low cytotoxicity in normal human endometrial cells HES and HESC cells when compared to cisplatin. Eupatilin induced G2/M phase cell cycle arrest in a time- and dose-dependent manner, as indicated by flow cytometry analysis. In addition, treatment of Hec1A cells with eupatilin resulted in a significant increase in the expression of p21^WAF1/CIP1 and in the phosphorylation of Cdc25C and Cdc2. Knockdown of p21 using specific siRNAs significantly compromised eupatilin-induced cell growth inhibition. Interestingly, levels of mutant p53 in Hec1A cells decreased markedly upon treatment with eupatilin, and p53 siRNA significantly increased p21 expression. Moreover, eupatilin modulated the phosphorylation of protein kinases ERK1/2, Akt, ATM, and Chk2. These results suggest that eupatilin inhibits the growth of human endometrial cancer cells via G2/M phase cell cycle arrest through the up-regulation of p21 by the inhibition of mutant p53 and the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway.     

Acacetin inhibits the proliferation of Hep G2 by blocking cell cycle progression and inducing apoptosis

Flavonoids are a broadly distributed class of plant pigments, universally present in vascular plants and responsible for much of the coloring in nature. They are strong antioxidants that occur naturally in foods and can inhibit carcinogenesis in rodents. In this study, we examined acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound, for its effect on proliferation in a human liver cancer cell line, Hep G2. The results showed that acacetin inhibited the proliferation of Hep G2 by inducing apoptosis and blocking cell cycle progression in the G1 phase. Enzyme-linked immunosorbent assay showed that acacetin significantly increased the expression of p53 and p21/WAF1 protein, contributing to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand and soluble Fas ligand, as well as Bax protein, was responsible for the apoptotic effect induced by acacetin. Taken together, our study suggests that the induction of p53 and activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of acacetin in Hep G2 cells.     

c-Jun N-terminal kinase 1 is required for cordycepin-mediated induction of G2/M cell-cycle arrest via p21WAF1 expression in human colon cancer cells

Cordycepin (3′-deoxyadenosine) has many anti-cancer properties. However, neither its molecular mechanism nor its molecular targets are well understood. In the present study, we investigated novel molecular mechanisms for the anti-tumor effects of cordycepin in human colon cancer HCT116 cells. After treatment of cells with cordycepin, dose-dependent cell growth inhibition was observed at an IC₅₀ value of 200 μM. Cordycepin treatment resulted in G2/M-phase cell-cycle arrest, which was associated with increased p21WAF1 levels and reduced amounts of cyclin B1, Cdc2, and Cdc25c in a p53-independent pathway. Moreover, cordycepin treatment induced activation of JNK (c-Jun N-terminal kinases). Pretreatment with SP600125, a JNK-specific inhibitor, abrogated cordycepin-mediated p21WAF1 expression, cell growth inhibition, and reduced cell-cycle proteins. Furthermore, JNK1 inhibition by small interfering RNA (siRNA) produced similar results: suppression of cordycepin-induced p21WAF1 expression, decreased cell growth, and reduced cell-cycle proteins. Together, these results suggest a critical role for JNK1 activation in cordycepin-induced inhibition of cell growth and G2/M-phase arrest in human colon cancer cells.     

25-Methoxyhispidol A, a novel triterpenoid of Poncirus trifoliata, inhibits cell growth via the modulation of EGFR/c-Src signaling pathway in MDA-MB-231 human breast cancer cells

The fruit of Poncirus trifoliata (Rutaceae) has been used a medicinal food and traditional medicine. Recently we reported the isolation of 25-methoxyhispidol A (25-MHA) as a novel triterpenoid from the immature fruit of P. trifoliata with the potential growth inhibition of cancer cells. However, the molecular mechanisms on the anti-proliferative activity in cancer cells remain to be elucidated. In the present study, we investigated the anti-proliferative activity and mechanisms of actions mediated by 25-MHA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells. 25-MHA exhibited the growth inhibitory activity against MDA-MB-231 cells with the cell cycle arrest in the G0/G1 phase. The cell cycle arrest in the G0/G1 by 25-MHA was well correlated with the downregulation of cyclin D1, cyclin dependent kinase (CDK4), CDK2, cyclin A, phosphorylated retinoblastoma protein (pRb), and induction of cdk inhibitor p21^WAF1/Cip1 protein. 25-MHA also suppressed the activation of c-Src/epidermal growth factor receptor (EGFR)/Akt signaling, and consequently led to the inactivation of mTOR and its downstream signal molecules including 4E-binding protein (4E-BP) and p70 S6 kinase. These findings suggest that 25-MHA-mediated inhibitory activity of human breast cancer cell growth might be related with the cell cycle arrest and modulation of signal transduction pathways.     

Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by prodelphinidin B-2 3,3'-di-O-gallate from Myrica rubra via Fas-mediated pathway

Myrica rubra Sieb et Zucc. (Myricaceae) is well known as a rich source of tannins. Prodelphinidin B-2 3,3'-di-O-gallate (PB233'OG) is a proanthocyanidin gallate that has been reported to exhibit antioxidant and antiviral activity. In this study, we evaluated the anti-proliferative activity of PB233'OG isolated from the bark of M. rubra in human breast adenocarcinoma MCF-7 cells. To identity the anti-cancer mechanism of PB233'OG, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that PB233'OG induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that Fas/Fas ligand apoptotic system is the main pathway of PB233'OG-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the anti-proliferative activity of PB233'OG in MCF-7 cells.     

The antiproliferative activity of prodelphinidin B-2 3'-O-gallate from green tea leaf is through cell cycle arrest and Fas-mediated apoptotic pathway in A549 cells.

Prodelphinidin B-2 3'-O-gallate, a proanthocyanidin gallate isolated from green tea leaf, was investigated for its anti-proliferative activity in human non-small cell lung cancer A549 cells. The results showed that prodelphinidin B-2 3'-O-gallate inhibited the proliferation of A549 cells with no detectable toxic effects on normal WI-38 cells as measured by the XTT assay. Flow cytometric analysis showed that prodelphinidin B-2 3'-O-gallate blocked cell cycle progression in the G0/G1 phase. In addition, prodelphinidin B-2 3'-O-gallate effectively induced A549 cell apoptosis as determined by assessing the nucleosome level in cytoplasm. Enzyme-linked immunosorbent assay showed that the G0/G1 phase arrest is due to p53-independent induction of p21/WAF1. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by prodelphinidin B-2 3'-O-gallate. We suggested that prodelphinidin B-2 3'-O-gallate's activities might be potentially contribute to its overall chemopreventive effects against lung cancer, and can possibly be considered for future therapeutic application.     

Antiproliferative activity of paeoniflorin is through cell cycle arrest and the Fas/Fas ligand-mediated apoptotic pathway in human non-small cell lung cancer A549 cells

1. Paeoniflorin (PF), isolated from the paeony root, is reported to have immunoregulatory, neuromuscular blocking, anticonvulsant, antihyperglycaemic and antihypotensive effects. 2. The present study investigated the antiproliferative activity of PF. The results showed that PF inhibited the proliferation of A549 by blocking cell cycle progression in the G(0)/G(1) phase and inducing apoptosis. 3. An ELISA showed that G(0)/G(1) phase arrest may be due to p53-independent induction of p21/wild-type p53-activated fragment 1 (WAF1). Increased protein expression of Fas/apoptosis-1 (APO-1) and its two ligands, membrane-bound Fas ligand and soluble Fas ligand, may be responsible for the PF-induced apoptosis. 4. This is the first study to show that the induction of p21/WAF1 and the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of PF in A549 cells.     

Chalcone arrests cell cycle progression and induces apoptosis through induction of mitochondrial pathway and inhibition of nuclear factor kappa B signalling in human bladder cancer cells

Chalcones (1,3-diphenyl-2-propenone) are cancer preventive food components found in a human diet rich in fruits and vegetables. In this study, we first report the chemopreventive effect of chalcone in two human bladder cancer cell lines: T24 and HT-1376. The results show that chalcone inhibits the proliferation of T24 and HT-1376 cells by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Western blot assay showed that chalcone significantly increases the expression of p21 and p27 proteins, and decreases the levels of cyclin B1, cyclin A and Cdc2, thereby contributing to cell cycle arrest. In addition, chalcone increased the expression of Bax and Bak, but decreased the levels of Bcl-2 and Bcl-X(L) and subsequently triggered mitochondrial apoptotic pathway (release of cytochrome c and activation of caspase-9 and caspase-3). Our study suggests that the induction of mitochondrial pathway and inhibition of the nuclear factor kappa B survival system may play important roles in the antiproliferative activity of chalcone in T24 and HT-1376 cells.