No 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, a molecule produced throughout the animal and plant kingdoms, and berberine, a plant derived agent, both exhibit antitumor and multiple biological and pharmacological effects, but they have never been combined altogether for the inhibition of human lung cancers. In this study, we investigated the role and underlying mechanisms of melatonin in the regulation of antitumor activity of berberine in lung cancer cells. Treatment with melatonin effectively increased the berberine-mediated inhibitions of cell proliferation, colony formation and cell migration, thereby enhancing the sensitivities of lung cancer cells to berberine. Melatonin also markedly increased apoptosis induced by berberine. Further mechanism study showed that melatonin promoted the cleavage of caspse-9 and PARP, enhanced the inhibition of Bcl2, and triggered the releasing of cytochrome C (Cyto C), thereby increasing the berberine-induced apoptosis. Melatonin also enhanced the berberine-mediated inhibition of telomerase reverses transcriptase (hTERT) by down-regulating the expression of AP-2β and its binding on hTERT promoter. Moreover, melatonin enhanced the berberine-mediated inhibition of cyclooxygenase 2 (COX-2) by inhibiting the nuclear translocation of NF-κB and its binding on COX-2 promoter. Melatonin also increased the berberine-mediated inhibition of the phosphorylated Akt and ERK. Collectively, our results demonstrated that melatonin enhanced the antitumor activity of berberine by activating caspase/Cyto C and inhibiting AP-2β/hTERT, NF-κB/COX-2 and Akt/ERK signaling pathways. Our findings provide new insights in exploring the potential therapeutic strategies and novel targets for lung cancer treatment.

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 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.