Modulation Of Cell Proliferation By Acetylbritannilactone And Analogues

Neointimal formation, predominantly consisting of vascular smooth muscle cell (VSMC) growth and migration, is a major pathogenic process of hyperproliferative vascular disorders, such as atherosclerosis and restenosis after balloon angioplasty and stent placement. Although the mechanisms implicated in the process of restenosis have not been completely resolved, the accumulating evidences suggest that inflammation plays a key role in neointimal growth after angioplasty. Recently, abnormally elevated expression of cyclooxygenase-2 (COX-2) has been frequently observed in cancer tissues and some researches supports the concept that COX-2 could provide an early target for cancer prevention. The epidemiological studies have reported the inverse association between the regular use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of many cancers. Therefore, using the anti-inflammatory drugs and agents could suppress the production of inflammatory mediators and in turn block the initiation and progression of inflammation-associated diseases, including hyperproliferative vascular disorders and cancers.Acetylbritannilactone (ABL), a new active extract isolated from a traditional Chinese medicinal herb Inula Britannica L, is a kind of sesquiterpenes and has been shown to possess anti-inflammatory and anticancer activities. In the previous work, it is demonstrated that that ABL inhibits the expression of inflammation-associated genes and it possesses anticancer properties. It is showed that the properties of ABL have been attributed, at least in part, to its ability to inhibit COX-2. Here, we evaluated the effects of ABL on VSMC and cancer cells and then investigated the intracellular signaling pathways as possible mechanisms.1 Acetylbritannilactone induce G1 arrest and apoptosis in vascular smooth muscle cellsNeointimal formation is a major pathogenic process of hyperproliferative vascular disorders, such as atherosclerosis and restenosis after balloon angioplasty and stent placement. In response to arterial injury, varieties of inflammatory vasoactive and mitogenic factors are released. Among them, platelet-derived growth factor (PDGF) is one of the most potent mitogenic and chemotactic agents for SMCs and plays a pivotal role in the onset and development of various vascular proliferative diseases. Therefore, inhibition of VSMC growth, either by targeting cellular mediators of the proliferative response or by interfering with the cell cycle machinery, represents a potentially effective therapeutic approach to prevent against restenosis after revascularization therapies. ABL has been receiving strong attention as a preventing agent against cancer and inflammatory diseases. However, the effects of ABL on VSMC proliferation and apoptosis have not yet been clarified. Therefore, in the present study, we evaluated the regulation of ABL on VSMC cycle and apoptosis and then investigated the intracellular signaling pathways as possible mechanisms in vitro and in vivo.1.1 ABL inhibits DNA synthesis and cell proliferation in VSMCsGrowth-arrested VSMCs were treated with PDGF (20 ng/ml) for 24 h in the presence of ABL, and DNA synthesis was measured by BrdU-incorporation assay. Pretreatment for 2 h with ABL efficiently inhibited PDGF-stimulated cell proliferation in a concentration-dependent manner. Similar results obtained under the same concentrations by cell counts.1.2 ABL induces G1 cell cycle arrestTo characterize the contribution of cell cycle arrest to the reduction in VSMC proliferation, flow cytometric analysis was performed for DNA content. ABL treatment arrested the cell cycle at G1 phase, resulting in a decrease in the fraction of cells in the S phase. These data suggested that inhibition of VSMC proliferation by ABL might be associated with the induction of G1 arrest.1.3 ABL up-regulates p21cip1 and inhibits cyclins and CDKs As it has been shown that cyclins, CDKs, and CDKIs play crucial roles in the regulation of cell cycle progression, we analyzed the effects of ABL on the expression of these cell cycle regulatory proteins. Cyclin D1, A, and E were up-regulated in response to PDGF at 24 h and the effects could be blocked by ABL treatment. Similarly, a significant reduction in the expression of CDK2, CDK4, and CDK6 was observed. We also examined the effect of ABL on the induction of p21cip1 and showed that the decrease in p21cip1 expression stimulated by PDGF was markedly attenuated by ABL. Furthermore, we immunoprecipitated p21cip1 from total cell lysates, and studied it binding with CDK2, CDK4 and CDK6, which showed an increase in the bound levels of the proteins after ABL treatment. Thus, these results revealed that the ABL-induced enhancement of the p21cip1 played an important role in the ABL-induced G1 arrest of cell cycle progression in VSMCs, possibly through their inhibition of CDK kinase activity.1.4 ABL induces apoptosis of VSMCsTo determine whether the ABL-induced loss of the proliferation in VSMCs was associated with the induction of apoptosis, nucleosome fragmentation in the cytoplasm was determined. The data showed that ABL dramatically enhanced apoptosis in VSMCs in a concentration-dependent manner. Moreover, cell lysates were prepared from VSMCs stimulated with PDGF and following treatment for 24 h with ABL and were subjected to western bolt for Bcl-xL, Bcl-2, and Bax. This revealed that ABL treatment induced a concentration-dependent reduction in the levels of the anti-apoptotic proteins Bcl-xL and Bcl-2 with a concomitant increase in the levels of pro-apoptotic protein Bax compared with the cells that were not treated with ABL. Based on the above results showing induction of apoptosis by ABL, we analyzed the levels of cleaved caspase-9 and caspase-3 treated with ABL for 24 h. Our data showed that ABL treatment significantly increased the cleaved caspase-9 and caspase-3 in VSMCs induced by PDGF.1.5 ABL suppresses VSMC migrationMigration of VSMCs is regarded as the essential step leading to neointimal thickening in atherosclerosis and restenosis. The wounding assay showed that PDGF (20 ng/ml) enhanced the basal migration of VSMCs by≈5-fold. Pretreatment with ABL potently suppressed chemoattractant induced migration of VSMCs in a concentration-dependent manner. Similar results were obtained in the Boyden chamber assay. The results also showed that ABL inhibited the chemotaxis of VSMCs stimulated with chemoattractant in the lower chamber. Moreover, ABL also reduced the random motion induced by PDGF (20 ng/ml) in both the upper and lower chambers.1.6 ABL inhibits PDGF-stimulated phosphorylation of ERK1/2To investigate the molecular mechanisms of the antiproliferative and pro-apoptotic effects exerted by ABL, the phosphorylation of each mitogen-activated protein kinase (MAPK) pathways were examined. ABL treatment significantly inhibited the ERK1/2 activation stimulated with PDGF in a concentration-dependent manner. Likewise, ABL also effectively suppressed the capacity of PDGF to stimulate the phosphorylation of MEK1/2. In addition, we examined the influence of ABL on the ligand-induced tyrosine phosphorylation of theβPDGFR. The data showed that ABL significantly inhibited theβPDGFR phosphorylation stimulated with PDGF. Therefore, it is very likely that ABL interferes with the pathway from theβPDGFR, via MEK1/2, to ERK1/2.1.7 ABL suppresses VSMC proliferation and induces apoptosis in vivoFurthermore, we showed that ABL also resulted in a significantly reduction of neointimal formation in carotid arteries (neointimal area, 0.15±0.04 vs. 0.05±0.02 mm2, P < 0.01). To evaluate the effects of ABL on VSMC apoptosis in vivo, we performed TUNEL assay. At 14 days after injury, the ABL-treated group showed higher levels of apoptosis than the vehicle-treated group (ABL vs. vehicle-treated, 15.6±2.9% vs. 4.3±1.1%), confirming that the induction of apoptosis was one of mechanisms of ABL inhibiting neointimal formation.In summary, the present study provides some important new insights into the molecular mechanisms of action of ABL in VSMCs. Our results suggest that ABL is capable of suppressing the abnormal VSMC proliferation, accompanied by the induction of apoptosis in vivo and in vitro. In this regard, it may be of interest to investigate the feasibility of ABL administration in patients with vascular restenosis.2 Acetylbritannilactone synergistically potentiates the growth inhibitory effect of celecoxib on human breast cancer cells through suppressing cyclooxygenase-2 expressionRecently, the epidemiological studies have reported the inverse association between the regular use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of breast cancer. However, the long-term use of traditional NSAIDs may be limited due to undesired side effects among the users. Unlike traditional NSAIDs, several studies have indicated that celecoxib may provide effective approaches to prevent and treat breast cancer. ABL is a kind of sesquiterpenes and has been shown to possess anti-inflammatory and anticancer activities. It is showed that the properties of ABL have been attributed, at least in part, to its ability to inhibit COX-2. With the goal of enhancing the chemopreventive effects in breast cancer, we tested the effects of the celecoxib and ABL combination in vitro and in vivo.2.1 Celecoxib and ABL synergistically inhibited human breast cancer cell growthThe growth-inhibitory effects of celecoxib (2.5-40μM) and ABL (5-150μM) on cell viability were first assessed by MTT assay and calculated as the percentage of viable cells relative to untreated cells, respectively. Celecoxib and ABL inhibited growth of these three cancer cell lines (MDA-MB-231, MDA-MB-468 and MCF-7) in a concentration dependent manner. Although single ABL (50μM) induced only weak growth inhibition, the addition of the dose of ABL to celecoxib (5μM) that was almost no effect on the growth of the breast cancer cells, induced a pronounced decrease in cell viability. Furthermore, MDA-MB-231 cells were treated with both celecoxib and ABL simultaneously at fixed 1: 5 and 1:10 dose ratio for 48 h. CI values were determined using the commercial software package Calcusyn. The CI values of the celecoxib and ABL were less than 1. These results showed a synergistic inhibition between celecoxib and ABL in the growth of MDA-MB-231 cells, suggesting that celecoxib and ABL may be an effective combination for the inhibition of MDA-MB-231 cell growth due to their synergistic efficacy. Because the combination of celecoxib and ABL was more effective at inhibiting cultured cancer cells, effect on tumor development was examined next in vivo. Treatment with single celecoxib (5 mg/kg) or ABL (15 mg/kg) had weaker inhibitory effect on MDA-MB-231 tumor growth, while the combination treatment significantly reduced tumor volume by 50% (P < 0.05) after 30 days of treatment.2.2 Celecoxib and ABL synergistically induced apoptosisTo examine whether the synergistic growth inhibition by combined treatment with celecoxib and ABL may be explained by the induction of apoptosis, the extent of apoptosis was assessed following 48 h exposure of cells to the agents. The combination of celecoxib (5μM) and ABL (50μM) significantly increased the percentage of apoptotic cells compared with single agents in MDA-MB-231 cells. However, celecoxib, ABL, or combined agents showed weaker induction of apoptosis in MCF-7 and MDA-MB-468 cells that express low levels of COX-2. To confirm the induction of apoptosis by the combination, expression of apoptosis markers also analyzed in MDA-MB-231 cells by Western blot after treatments with the agents. The combination treatment, but not individual agents, resulted in marked increased degradation of caspase-3 and caspase-9 in MDA-MB-231 cells. These events were associated with increased apoptosis proportion of cancer cells.2.3 Celecoxib and ABL synergistically suppressed COX-2 expression and activityTo determine the mechanisms in MDA-MB-231 cell apoptosis induced by the combination treatment of celecoxib and ABL, the expression of COX-2 mRNA and protein was examined. COX-2 was overexpressed at mRNA and protein levels in MDA-MB-231 cells, slightly decreased by the single treatments with celecoxib (5μM) or ABL (50μM), but markedly decreased by the combination of the two agents. Consistent with the Western blot results, in immunofluorescence staining analyses, the combination treatment of celecoxib (5μM) and ABL (50μM) on MDA-MB-231cells for 48 h led to a drastic decrease in cytoplasmic COX-2 expression was not affected by the two agents treatment. Moreover, the combination of celecoxib (5μM) and ABL (50μM) also markedly suppressed TPA (0.1μM)-induced COX-2 expression in MCF-7 cells. PGE2 is derived by COX-2 catalysis, which represents COX-2 activity. The combination of celecoxib and ABL almost totally (>90%) diminished PGE2 production.2.4 Celecoxib and ABL synergistically suppressed COX-2 gene transcriptionTo investigate the mechanisms by which the agent combination inhibited COX-2 expression, the luciferase-reporter construct containing a fragment of the COX-2 promoter (?1432/+59) was transfected into MCF-7 cells that was induced by TPA to express COX-2. The combination treatment of celecoxib and ABL significantly reduced TPA-induced COX-2 promoter activity (P < 0.05). The inhibitory effect of the combination of the two agents was significantly stronger than the effects produced by either celecoxib or ABL at their low doses. Furthermore, the recruitments of transcription factors to the CRE and AP-1 elements were evaluated by ChIP assay. TPA increased the recruitment of the transcription factors ATF-2, CREB-1, c-Fos and c-Jun to the COX-2 promoter region containing CRE and AP-1 sites, respectively. However, the combination of celecoxib and ABL significantly reduced the binding of ATF-2, CREB-1 and c-Fos binding activity than each agent alone. The similar results were observed in DAPA experiments using the probe containing the CRE and AP-1 binding sites.2.5 Synergistic inhibition of the transcription factors by celecoxib and ABL is associated with suppression of Akt and p38 signalingThe activation of specific transcription factors, which triggers COX-2 expression, is regulated by Akt and MAPKs cascades. For this, the roles of these intracellular signaling pathways in inhibition of ATF-2, CREB-1 and c-Fos activation by single and combination of celecoxib and ABL were examined using antibodies that identify the active (phosphorylated) forms of these kinases. Western blot analysis showed that exposure of MCF-7 cells to celecoxib or ABL alone for 2 h resulted in a slight decrease in Akt and p38 phosphorylation induced by TPA. However, the combination of agents reduced levels of active Akt and p38 to a greater degree versus either agent alone. To further determine whether the observed changes in signaling are responsible for reduction of transcription activation of COX-2 gene, MCF-7 cells were transfected with the COX-2-Luc along with a constitutively active Akt expression (Ca-Akt) or p38 expression (MKK6b) vectors. The results showed that transfection of cells with Ca-Akt or MKK6b significantly abolished the inhibition of TPA-induced COX-2 gene promoter activity by the combination of celecoxib and ABL. In addition, the Ca-Akt or MKK6b also protected cells from agents-induced apoptosis and supported the idea that the attenuation of Akt and p38 activation contributed to the enhanced induction of apoptosis by the combination of celecoxib and ABL.In summary, celecoxib and ABL combination synergistically inhibit the growth of breast cancer cells in vitro and in vivo. In particular, ABL may synergistically enhance the activity of celecoxib on breast cancer growth inhibition via suppressing COX-2 transcriptional activation and expression.3 ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activationIn the previous work, it is demonstrated that that ABL inhibits the expression of inflammation-associated genes and it possesses anticancer properties. In the course of our continuing search for cytotoxic ABL analogues, we synthesized the compound 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N), which in preliminary studies showed exceptional anti-proliferative activity against several human cancer cell types. Here, we showed that ABL-N was more potent than ABL in the ability to induce apoptosis, at a low concentration, of human breast cancer cells and investigated the therapeutic potential of the ABL-N and its underlying mechanism of action.3.1 ABL-N reduces the viability of various carcinoma cell linesThe inhibitory effects of ABL and ABL-N on various carcinoma cell lines were estimated using the MTT cellular survival assay. The results showed that ABL-N treatment inhibited cell growth with similar IC50 (approximately 12-20μM) after a 24 h treatment. It indicated that ABL-N was a broad-spectrum inhibitory agent of the human carcinoma cells.3.2 ABL-N arrests cells in G2/M phase of the cell cycleBecause ABL-N can effectively inhibit cell viability, we reasoned that this effect might be attributable to its ability to interfere with the cell cycle. MDA-MB-231 cells were incubated with 20μM ABL-N for 6, 12 and 24 h, and the cell cycle analysis was done by PI uptake. The results showed that the ratio of cells in G2/M phase and the cells with hypodiploid DNA contents (sub-G1) were significantly accumulated over the treatment periods. Moreover, we also analyzed cell cycle in MDA-MB-468 and MCF-7 cells and found the G2/M arrest induced by ABL-N as well.3.3 ABL-N induces apoptosis in breast cancer cellsWe next analyzed whether the ABL-N-induced cell viability reduction in human breast cancer cells involved apoptosis. MDA-MB-231 cells were treated with 20μM ABL-N and apoptosis was assayed by two different methods. DAPI staining showed that the condensed and fragmented nuclei increased with ABL-N treatment. Nucleosome fragmentation further determined by Cell Death Detection ELISAPLUS confirmed that cells treated for 6 h with 20μM ABL-N underwent apoptosis. Furthermore, caspase activities were measured with Caspase-Glo assays. Treatment with ABL-N induced the activation of caspases-3/7, -8 and -9 in MDA-MB-231 cells. However, we found that caspase-8 activity is significantly higher in MCF-7 cells treated with ABL-N. To define the role of caspase activation in ABL-N-induced apoptosis, we treated MDA-MB-231 cells with pan-caspase inhibitor z-VAD-fmk (50μM) and caspase-3-specific inhibitor (z-DEVD-fmk) (50μM) before challenge with ABL-N (20μM). z-VAD-fmk pretreatment for 1 h abrogated ABL-N-induced apoptosis as measured by the nucleosome fragmentation and the appearance of sub-G1 cells. The caspase-3-specific inhibitor z-DEVD-fmk also reduced ABL-N-induced apoptosis in MDA-MB-231 cells. These results suggested that activation of caspase cascade was essential for ABL-N-induced apoptosis in breast cancer cells.3.4 ABL-N modulates the expression of Bcl-2 family proteins in breast cancer cellsThe effects of ABL-N on the expression of anti-apoptotic protein Bcl-2 and the pro-apoptotic proteins Bax and Bad in breast cancer cells were evaluated. The data showed a marked increase in the level of Bax and Bad, which started at 6 h and peaked at 24 h of treatment with ABL-N in MDA-MB-231 cells. In contrast, reduced Bcl-2 protein appeared later at 12 h. Then, the ratio of Bax and Bcl-2 was measured by a densitometric analysis of the bands. The data showed that ABL-N treatment resulted in a time-dependent increase in Bax/Bcl-2 ratio in MDA-MB-231 cells that favors apoptosis.3.5 ABL-N induces the activation of JNK and p38 signaling in breast cancer cellsActivation of MAPK is involved in many aspects of the control of cellular proliferation and apoptosis in response to a variety of extracellular stimulus. We therefore examined the effects of ABL-N on the activation of several MAPK pathways. The data showed that ABL-N treatment induced activation of JNK and p38 in a time-dependent manner. The activation of JNK by ABL-N was further confirmed by the analysis of phosphorylation of its downstream substrate c-Jun. It showed that c-Jun was phosphorylated following ABL-N treatment, which occurred over the same sustained period as JNK activation. To gain further insight into the mechanism by which ABL-N treatment affects JNK, we assayed JNK activity in ABL-N-treated cells as well as the direct effect of ABL-N on GST-JNK1 fusion proteins activity. The data showed that JNK activity began to increase after treatment with 20μM ABL-N for 3 h and maximum activation was achieved 12 h after treatment. In addition, using GST-JNK1 fusion proteins, we found that the JNK activity was unaffected by the presence of ABL-N, indicating ABL-N activated JNK indirectly by activating signaling molecules located upstream in the JNK cascades. To determine the role of the activation of JNK and p38 in ABL-N-induced apoptosis, MDA-MB-231 cells were treated with the JNK inhibitor SP600125 or the p38 inhibitor SB203580 and their effects on cell apoptosis were examined. The data showed that reduction of cell viability by ABL-N was effectively abolished by SP600125, but SB203580 only had a slight effect on the decreased viability by ABL-N. These results suggested that the activation of JNK signaling is responsible for the ABL-N-induced apoptosis.3.6 ABL-N inhibits the growth of human breast cancer xenograftsBecause ABL-N treatment showed the effective growth inhibition in cultured breast cancer cells, we subsequently carried out in vivo study using MDA-MB-231-derived cancer xenografts in nude mice. The data showed that the i.p. treatment with ABL-N (15 mg/kg) caused a significant inhibition of tumor growth as early as 20 days after treatment and persisted after 34 days. In summary, our studies suggest that ABL-N significantly induces apoptosis in breast cancer cells. This induction is associated with the activation of caspases and JNK signaling pathways. Moreover, ABL-N treatment caused a significant inhibition of tumor growth in vivo. Therefore, it is thought that ABL-N might be a potential drug for use in breast cancer prevention and intervention.CONCLUSIONS1 Acetylbritannilactone induce G1 arrest and apoptosis in vascular smooth muscle cells.2 Acetylbritannilactone synergistically potentiates the growth inhibitory effect of celecoxib on human breast cancer cells through suppressing cyclooxygenase-2 expression. 3 ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation.