Wangbing Chen

Simultaneous modulation of COX-2, p300, Akt, and Apaf-1 signaling by melatonin to inhibit proliferation and induce apoptosis in breast cancer cells.

Abstract:  Melatonin exhibits anti‐inflammatory and anticancer effects and could be a chemopreventive and chemotherapeutic agent against cancers, but the precise mechanisms involved remain largely unresolved. In this study, we evaluated the mechanism of action of melatonin in human MDA‐MB‐361 breast cancer cells. Melatonin at pharmacological concentrations (10−3 m) significantly suppressed cell proliferation and induced apoptosis in a dose‐dependent manner. The observed suppression of proliferation was accompanied by the melatonin‐mediated inhibition of COX‐2, p300, and NF‐κB signaling. Melatonin significantly inhibited COX‐2 expression and prostaglandin E(2) (PGE2) production, abrogated p300 histone acetyltransferase activity and p300‐mediated NF‐κB acetylation, thereby blocking NF‐κB binding and p300 recruitment to COX‐2 promoter. Pretreatment with a COX‐2‐ or p300‐selective inhibitor abrogated the melatonin‐induced inhibition of cell proliferation, whereas PGE2 treatment or COX‐2 transfection reversed the inhibition by melatonin. Moreover, melatonin markedly inhibited phosphorylation of PI3K, Akt, PRAS40, and GSK‐3 proteins, thereby inactivating the PI3K/Akt signaling pathway. Pretreatment with a PI3K‐ or an Akt‐selective inhibitor or an Akt‐specific siRNA blocked the melatonin‐mediated inhibition of cell proliferation. Conversely, gene delivery of a constitutively active Akt effectively reversed the inhibition by melatonin. Furthermore, melatonin induced Apaf‐1 expression, triggered cytochrome C release, and stimulated caspase‐3 and caspase‐9 activities and cleavage, leading to an activation of the Apaf‐1‐dependent apoptotic pathway. Pretreatment with an Apaf‐1‐specific siRNA effectively attenuated the melatonin‐induced apoptosis. These results therefore indicate that melatonin inhibits cell proliferation and induces apoptosis in MDA‐MB‐361 breast cancer cells in vitro by simultaneously suppressing the COX‐2/PGE2, p300/NF‐κB, and PI3K/Akt/signaling and activating the Apaf‐1/caspase‐dependent apoptotic pathway.

Melatonin suppresses proinflammatory mediators in lipopolysaccharide-stimulated CRL1999 cells via targeting MAPK, NF-κB, c/EBPβ, and p300 signaling.

Abstract:  Melatonin is an indoleamine secreted by the pineal gland as well as a plant‐derived product that exerts potential anti‐inflammatory properties, but the mechanisms of action remain unclear. Here, we investigated the roles of melatonin in regulation of proinflammatory mediators and identified the underlying mechanisms in human vascular smooth muscle (VSM) cell line CRL1999 stimulated by lipopolysaccharide (LPS). We found that treatment with melatonin significantly inhibited the production and expression of TNF‐α and interleukin (IL)‐1β, cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase, prostaglandin E(2) (PGE2), and nitric oxide (NO) in a dose‐dependent manner. Moreover, we also found that the suppression of proinflammatory mediators by melatonin was mediated through inhibition of MAPK, NF‐κB, c/EBPβ, and p300 signaling in LPS‐stimulated CRL1999 cells. Treatment with melatonin markedly inhibited phosphorylation of ERK1/2, JNK, p38 MAPK, IκB‐α, and c/EBPβ, blocked binding of NF‐κB and c/EBPβ to promoters, and suppressed p300 histone acetyltransferase (HAT) activity and p300 HAT‐mediated NF‐κB acetylation. Transfection with an ERK‐, IκB‐, or c/EBPβ‐specific siRNA or pretreatment with an ERK‐, p38 MAPK‐, or p300‐selective inhibitor considerably abrogated the melatonin‐mediated inhibition of proinflammatory mediators. Conversely, exogenous overexpression of a constitutively active p300, but not its HAT mutant, effectively reversed the melatonin‐mediated inhibitions. Collectively, these results indicate that melatonin suppresses proinflammatory mediators by simultaneously targeting the multiple signaling such as ERK/p38 MAPK, c/EBPβ, NF‐κB, and p300, in LPS‐stimulated VSM cell line CRL1999, and suggest that melatonin is a potential candidate compound for the treatment of proinflammatory disorders.

Ursolic acid simultaneously targets multiple signaling pathways to suppress proliferation and induce apoptosis in colon cancer cells.

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways.

Berberine Targets AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth

Berberine (BBR), an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC) cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2β expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2β by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.

Melatonin potentiates the antiproliferative and pro-apoptotic effects of ursolic acid in colon cancer cells by modulating multiple signaling pathways.

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is largely distributed in medical herbs and edible plants. Melatonin is an indoleamine compound produced in the pineal gland and also a plant‐derived product. Both UA and melatonin have been shown to inhibit cancer cell growth in numerous studies, but they have never been combined altogether as an anticolon cancer treatment. In this study, we investigated whether the association between UA and melatonin leads to an enhanced antiproliferative and pro‐apoptotic activities in colon cancer SW480 and LoVo cells. We found that combined treatment with UA and melatonin significantly enhanced inhibition of cell viability and migration, promoted changes in cell morphology and spreading, and increased induction of apoptosis, thereby potentiating the effects of UA alone in colon cancer cells. Moreover, we found that the enhanced effects of UA and melatonin combination are mediated through simultaneous modulation of cytochrome c/caspase, MMP9/COX‐2, and p300/NF‐κB signaling pathways. Combined treatment with UA and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, induced cleavage of caspase and PARP proteins, enhanced inhibition of MMP9 and COX‐2 expression, promoted p300 and NF‐κB translocation from cell nuclei to cytoplasm, and abrogated NF‐κB binding and p300 recruitment to COX‐2 promoter in colon cancer cells. These results, therefore, demonstrated that melatonin potentiated the antiproliferative and pro‐apoptotic effects of UA in colon cancer cells by modulating multiple signaling pathways and suggest that such a combinational treatment might potentially become an effective way in colon cancer therapy.

Small interfering RNA-based molecular therapy of cancers

RNA interference (RNAi) has become a gold standard for validating gene function in basic life science research and provides a promising therapeutic modality for cancer and other diseases. This mini-review focuses on the potential of small interfering RNAs (siRNAs) in anticancer treatment, including the establishment and screening of cancer-associated siRNA libraries and their applications in anticancer drug target discovery and cancer therapy. This article also describes the current delivery approaches of siRNAs using lipids, polymers, and, in particular, gold nanoparticles to induce significant gene silencing and tumor growth regression.

TFAP2A Regulates Nasopharyngeal Carcinoma Growth and Survival by Targeting HIF-1a Signaling Pathway

TFAP2A is a transcription factor that orchestrates a variety of cell processes, including cell growth and tissue differentiation. However, the regulation of TFAP2A in human nasopharyngeal carcinoma tumorigenesis and its precise mechanism of action remain largely unknown. In this study, we investigated the biologic role and clinical significance of TFAP2A in nasopharyngeal carcinoma growth and progression and identified the underlying molecular mechanisms. We found that TFAP2A was highly expressed in various nasopharyngeal carcinoma cell lines and tumor tissue specimens and was significantly correlated with hypoxia-inducible factor-1α (HIF-1α) expression. A positive correlation of TFAP2A overexpression with advanced tumor stage, local invasion, clinical progression, and poor prognosis of patients with nasopharyngeal carcinomas were also observed. Moreover, we found that knockdown of TFAP2A expression by siRNA significantly inhibited tumor cell growth in nasopharyngeal carcinoma cell lines and in a subcutaneous xenograft mouse model by targeting the HIF-1α–mediated VEGF/pigment epithelium–derived factor (PEDF) signaling pathway. Treatment of nasopharyngeal carcinoma cells with TFAP2A siRNA dramatically inhibited the expression and the release of VEGF protein but did not change the level of PEDF protein, resulting in a significant reduction of the ratio of VEGF/PEDF. Pretreatment with a HIF-1α siRNA did not significantly change the TFAP2A siRNA-mediated inhibition in cell viability. Our results indicate that TFAP2A regulates nasopharyngeal carcinoma growth and survival through the modulation of the HIF-1α–mediated VEGF/PEDF signaling pathway, and suggest that TFAP2A could be a potential prognostic biomarker and therapeutic target for nasopharyngeal carcinoma treatment. Cancer Prev Res; 7(2); 266–77. ©2013 AACR.

CPSF4 activates telomerase reverse transcriptase and predicts poor prognosis in human lung adenocarcinomas

The elevated expression and activation of human telomerase reverse transcriptase (hTERT) is associated with the unlimited proliferation of cancer cells. However, the excise mechanism of hTERT regulation during carcinogenesis is not well understood. In this study, we discovered cleavage and polyadenylation specific factor 4 (CPSF4) as a novel tumor‐specific hTERT promoter‐regulating protein in lung cancer cells and identified the roles of CPSF4 in regulating lung hTERT and lung cancer growth. The ectopic overexpression of CPSF4 upregulated the hTERT promoter‐driven report gene expression and activated the endogenous hTERT mRNA and protein expression and the telomerase activity in lung cancer cells and normal lung cells. In contrast, the knockdown of CPSF4 by siRNA had the opposite effects. CPSF4 knockdown also significantly inhibited tumor cell growth in lung cancer cells in vitro and in a xenograft mouse model in vivo, and this inhibitory effect was partially mediated by decreasing the expression of hTERT. High expression of both CPSF4 and hTERT proteins were detected in lung adenocarcinoma cells by comparison with the normal lung cells. Tissue microarray immunohistochemical analysis of lung adenocarcinomas also revealed a strong positive correlation between the expression of CPSF4 and hTERT proteins. Moreover, Kaplan‐Meier analysis showed that patients with high levels of CPSF4 and hTERT expression had a significantly shorter overall survival than those with low CPSF4 and hTERT expression levels. Collectively, these results demonstrate that CPSF4 plays a critical role in the regulation of hTERT expression and lung tumorigenesis and may be a new prognosis factor in lung adenocarcinomas.

Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation

Cleavage and polyadenylation specific factor 4 (CPSF4), a member of CPSF complex, plays a key role in mRNA polyadenylation and mRNA 3′ ends maturation. However, its possible role in lung cancer pathogenesis is unknown. In this study, we investigated the biological role and clinical significance of CPSF4 in lung cancer growth and survival and elucidated its underlying molecular mechanisms. We found that CPSF4 was highly expressed in lung adenocarcinoma cell lines and tumor tissue but was undetectable in 8 normal human tissues. We also found that CPSF4 overexpression was correlated with poor overall survival in patients with lung adenocarcinomas (P<0.001). Multivariate survival analyses revealed that higher CPSF4 expression was an independent prognostic factor for overall survival of the patients with lung adenocarcinomas. Suppression of CPSF4 by siRNA inhibited lung cancer cells proliferation, colony formation, and induced apoptosis. Mechanism studies revealed that these effects were achieved through simultaneous modulation of multiple signaling pathways. Knockdown of CPSF4 expression by siRNA markedly inhibited the phosphorylation of PI3K, AKT and ERK1/2 and JNK proteins. In contrast, the ectopic expression of CPSF4 had the opposite effects. Moreover, CPSF4 knockdown also induced the cleavage of caspase-3 and caspse-9 proteins. Collectively, these results demonstrate that CPSF4 plays a critical role in regulating lung cancer cell proliferation and survival and may be a potential prognostic biomarker and therapeutic target for lung adenocarcinoma.

TFAP2B overexpression contributes to tumor growth and a poor prognosis of human lung adenocarcinoma through modulation of ERK and VEGF/PEDF signaling

BackgroundTFAP2B is a member of the AP2 transcription factor family, which orchestrates a variety of cell processes. However, the roles of TFAP2B in regulating carcinogenesis remain largely unknown. Here, we investigated the regulatory effects of TFAP2B on lung adenocarcinomas growth and identified the underlying mechanisms of actions in non-small cell lung cancer (NSCLC) cells.MethodsWe first examined the expression of TFAP2B in lung cancer cell lines and tumor tissues. We also analyzed the prognostic predicting value of TFAP2B in lung adenocarcinomas. Then we investigated the molecular mechanisms by which TFAP2B knockdown or overexpression regulated lung cancer cell growth, angiogenesis and apoptosis, and further confirmed the role of TFAP2B in tumor growth in a lung cancer xenograft mouse model.ResultsTFAP2B was highly expressed in NSCLC cell lines and tumor tissues. Strong TFAP2B expression showed a positive correlation with the poor prognoses of patients with lung adenocarcinomas (P < 0.001). TFAP2B knockdown by siRNA significantly inhibited cell growth and induced apoptosis in NSCLC cells in vitro and in a lung cancer subcutaneous xenograft model, whereas TFAP2B overexpression promoted cell growth. The observed regulation of cell growth was accompanied by the TFAP2B-mediated modulation of the ERK/p38, caspase/cytochrome-c and VEGF/PEDF-dependent signaling pathways in NSCLC cells.ConclusionsThese results indicate that TFAP2B plays a critical role in regulating lung adenocarcinomas growth and could serve as a promising therapeutic target for lung cancer treatment.