β-elemene, a compound derived from Rhizoma zedoariae, is a promising new plant-derived drug with broad-spectrum anticancer activity. However, the underlying mechanism by which this agent inhibits human lung cancer cell growth has not been well elucidated. In this study, we showed that β-elemene inhibits human non-small cell lung carcinoma (NSCLC) cell growth, and increased phosphorylation of ERK1/2, Akt and AMPKα. Moreover, β-elemene inhibited expression of DNA methyltransferase 1 (DNMT1), which was not observed in the presence of the specific inhibitors of ERK (PD98059) or AMPK (compound C). Overexpression of DNMT1 reversed the effect of β-elemene on cell growth. Interestingly, metformin not only reversed the effect of β-elemene on phosphorylation of Akt but also strengthened the β-elemene-reduced DNMT1. In addition, β-elemene suppressed Sp1 protein expression, which was eliminated by either ERK1/2 or AMPK inhibitor. Conversely, overexpression of Sp1 antagonized the effect of β-elemene on DNMT1 protein expression and cell growth. Taken together, our results show that β-elemene inhibits NSCLC cell growth viaERK1/2- and AMPKα-mediated inhibition of transcription factor Sp1, followed by reduction in DNMT1 protein expression. Metformin augments the effect of β-elemene by blockade of Akt signalling and additively inhibition of DNMT1 protein expression. The reciprocal ERK1/2 and AMPKα signalling pathways contribute to the overall responses of β-elemene. This study reveals a potential novel mechanism by which β-elemene inhibits growth of NSCLC cells.

BackgroundBerberine (BBR), a component from traditional Chinese medicine, has been shown to possess anti-tumor activity against a wide spectrum of cancer cells including human lung cancer, but the detailed mechanism underlining this has not been well elucidated.MethodsIn this study, the effect of berberine on cell growth and apoptosis were assessed by MTT, flow cytometry and Hoechst 33258 staining assays. The phosphorylation of p38 MAPK and ERK1/2, and expressions of p38 MAPK isoforms α and β, total ERK1/2, p53, FOXO3a and p21 protein were evaluated by Western Blot analysis. Silencing of p38 MAPK isoform α and β, p53, FOXO3a and p21 were performed by siRNA methods. Exogenous expression of FOXO3a was carried out by electroporated transfection assays.ResultsWe showed that BBR significantly inhibited growth and induced cell cycle arrest of non small cell lung cancer (NSCLC) cells in the G0/G1 phase in a dose-dependent manner. Furthermore, we found that BBR increased phosphorylation of p38 MAPK and ERK1/2 in a time-dependent and induced protein expression of tumor suppressor p53 and transcription factor FOXO3a in a dose-dependent fashion. The specific inhibitor of p38 MAPK (SB203580), and silencing of p38α MAPK by small interfering RNAs (siRNAs), but not ERK1/2 inhibitor (PD98059) blocked the stimulatory effects of BBR on protein expression of p53 and FOXO3a. Interestingly, inhibition of p53 using one specific inhibitor (Pifithrin-α) and silencing of p53 using siRNAs overcome the inhibitory effect of BBR on cell growth. Silencing of FOXO3a appeared to attenuate the effect of BBR on p53 expression, cell proliferation and apoptosis. Furthermore, BBR induces the protein expression of cell cycle inhibitor p21 (CIP1/WAF1), which was not observed in cells silencing of p53 or FOXO3α gene. Intriguingly, exogenous expression of FOXO3a enhanced the expression of p21 (CIP1/WAF1) and strengthened BBR-induced apoptosis.ConclusionOur results show that BBR inhibits proliferation and induces apoptosis of NSCLC cells through activation of p38α MAPK signaling pathway, followed by induction of the protein expression of p53 and FOXO3a. The latter contribute to the BBR-increased p21 (CIP1/WAF1) protein expression. The exogenous FOXO3a, interaction and mutually exclusive events of p53 and FOXO3a augment the overall response of BBR.

BackgroundBaicalein, a natural flavonoid obtained from the Scutellaria baicalensis root, has been reported to inhibit growth of human lung cancer. However, the detailed mechanism underlying this has not been well elucidated.MethodsCell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Apoptosis was detected by flow cytometry analysis and caspase 3/7 assays. The expression of RUNX3 and FOXO3a mRNA were measured by real time RT-PCR methods. Western blot analysis was performed to measure the phosphorylation and protein expression of AMP-activated protein kinase alpha (AMPKα) and extracellular signal-regulated kinase 1/2 (ERK1/2), runt-related transcription factor 3 (RUNX3) and forkhead box O3a (FOXO3a). Silencing of FOXO3a and RUNX3 were performed by small interfering RNA (siRNA) methods. Exogenous expression of FOXO3a or RUNX3 was carried out by electroporated transfection assays.ResultsWe showed that baicalein significantly inhibited growth and induced apoptosis of non-small cell lung cancer (NSCLC) cells in a time- and dose-dependent manner. Baicalein induced RUNX3 and FOXO3a protein expression, and increased phosphorylation of AMPKα and ERK1/2. Moreover, the inhibitors of AMPK and MEK/ERK1/2 reversed the effect of baicalein on RUNX3 and FOXO3a protein expression. Interestingly, while compound C had little effect on blockade of baicalein-induced phosphorylation of ERK1/2, PD98059 significantly abrogated baicalein-induced phosphorylation of AMPKα. Intriguingly, while silencing of RUNX3 abolished the effect of baicalein on expression of FOXO3a and apoptosis, silencing of FOXO3a significantly attenuated baicalein-reduced cell proliferation. On the contrary, overexpression of FOXO3a restored the effect of baicalein on cell growth inhibition in cells silencing of endogenous FOXO3a gene and enhanced the effect of baicalein on RUNX3 protein expression. Finally, exogenous expression of RUNX3 increased FOXO3a protein and strengthened baicalein-induced phosphorylation of ERK1/2.ConclusionCollectively, our results show that baicalein inhibits growth and induces apoptosis of NSCLC cells through AMPKα- and MEK/ERK1/2-mediated increase and interaction of FOXO3a and RUNX3 protein. The crosstalk between AMPKα and MEK/ERK1/2 signaling pathways, and the reciprocal interplay of FOXO3a and RUNX3 converge on the overall response of baicalein. This study reveals a novel mechanism for regulating FOXO3a and RUNX3 signaling axis in response to baicalein and suggests a new strategy for NSCLC associated targeted therapy.

Solamargine (SM), a major steroidal alkaloid glycoside extracted from a traditional Chinese medicinal herb, Solanum nigrum L. (SNL), has been shown to inhibit growth and induce apoptosis of various cancer cells. However, the molecular mechanisms underlying this are poorly understood. In this study, we showed that SM inhibited growth and induced apoptosis of non-small-cell lung cancer (NSCLC) cells in a time- and dose-dependent manner. To further explore this, we found that SM increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in a time-dependent fashion. SM also inhibited phosphorylation and protein expression of signal transducer and activator of transcription 3 (Stat3), a transcription factor, which was abrogated by the SB203580, a specific inhibitor of p38 MAPK. In addition, SM induced protein expression of p21, one of cyclin-dependent kinase inhibitors, and this was not observed in cell overexpression of Stat3 or cells treated with SB203580. Finally, while silencing of Stat3 had no further effect, exogenous expression of Stat3 overcame the effect of SM on cell proliferation. Collectively, our results show that SM inhibits proliferation and induces apoptosis in lung cancer cells through p38 MAPK-mediated suppression of phosphorylation and protein expression of Stat3, followed by inducing Stat3 downstream effector p21. This unveils a potential new mechanism by which SM inhibits growth of human lung cancer cells.

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Hepatocellular carcinoma (HCC), the major histological subtype of primary liver cancer, remains one of the most common malignancies worldwide. Due to the complicated pathogenesis of this malignancy, the outcome for comprehensive treatment is limited. Chinese herbal medicine (CHM) is emerging as a promising choice for its multi-targets and coordinated intervention effects against HCC. Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid found in CHM, exerts anti-tumor effects and is emerging as an effective compound for cancer prevention and therapy. However, the molecular mechanisms underlying the action of UA remain largely unknown. In this study, we showed that UA inhibited the growth of HCC cells and induced apoptosis in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of AMP-activated protein kinase alpha (AMPKα) and suppressed the protein expression of DNA methyltransferase 1 (DNMT1) in the dose-dependent manner. The inhibitor of AMPK, compound C blocked, while an activator of AMPK, metformin augmented the effect of UA on DNMT1 expression. In addition, UA suppressed the expression of transcription factor Sp1. Conversely, overexpression of Sp1 reversed the effect of UA on DNMT1 expression and cell growth. Collectively, our results show for the first time that UA inhibits growth of HCC through AMPKα-mediated inhibition of Sp1; this in turn results in inhibition of DNMT1. This study reveals a potential novel mechanism by which UA controls growth of HCC cells and suggests that DNMT1 could be novel target for HCC chemoprevention and treatment.

BackgroundUrsolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown.MethodsCell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)-methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays.ResultsWe showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth.ConclusionOur results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells.

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