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This study investigated B16F10 melanoma cell death induced by melatonin combined with endoplasmic reticulum (ER) stress through the PI3K/Akt/mTOR pathway. Cell viability was significantly decreased after treatment with melatonin combined with ER stress from thapsigargin or tunicamycin compared to no treatment or treatment with melatonin only. Combined melatonin and ER stress also significantly reduced expression of p85β, p-Akt (Ser473, Thr308), and p-mTOR (Ser2448, Ser2481) compared to treatment with melatonin only. The ER stress protein p-PERK and p-eIF2α were significantly increased under combined melatonin and ER stress treatment compared to no treatment or treatment with melatonin only. Combined melatonin and ER stress significantly reduced Bcl-2 protein and augmented Bax protein compared to melatonin-only treatment. Also, the combined treatment significantly lowered expression of catalase, Cu/Zn-SOD, and Mn-SOD proteins compared to melatonin only. Expression of p85β was significantly more decreased under treatment with melatonin and thapsigargin or tunicamycin plus the PI3K inhibitors LY294002 or wortmannin than under treatment with only melatonin or a PI3K inhibitor. The PI3K downstream target p-Akt (Ser473, Thr308) showed significantly decreased expression under treatment with melatonin and thapsigargin or tunicamycin plus PI3K inhibitors than under treatment with melatonin or PI3K inhibitors only. These results indicate that survival of B16F10 melanoma cells after combined treatment with melatonin and ER stress inducers is suppressed through regulation of the PI3K/Akt/mTOR pathway. Melatonin combined with thapsigargin or tunicamycin appears to be a promising strategy for effective melanoma treatment.

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The extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) signaling cascade mediates the transduction of growth promoting signals such as growth factors, cytokines and proto-oncogenes through the activation of RAS, which leads to the activation of RAF (mitogen activated protein kinase kinase kinase, MAP3K) and then to activation of MEK (mitogen activated protein kinase kinase, MAP2K). MEK then phosphorylates and activates ERK (MAP kinase), and finally ERK activates the transcription factors FOS and JUN for the biological outcome.1) The ERK/MAPK signaling pathway not only promotes cell proliferation but also mediates cell survival, and ERK/MAPK signaling is up-regulated in cancer cells. Hence, ERK/MAPK signaling is a good target for cancer therapy, and several ERK/MAPK kinase signaling inhibitors have been developed for cancer therapy. 1,2)The nuclear factor-kB (NF-kB) signaling pathway plays major roles in inflammation, cell adhesion, differentiation and apoptosis.3) NF-kB signaling is tightly regulated by the cellular localization of NF-kB. Before activation, NF-kB is associated with the inhibitory kappa B (IkB) protein and localized in the cytoplasm. During activation, IkB kinase (IKK) phosphorylates IkB protein and ubiquitinates IkB for degradation. This process releases NF-kB protein, allowing the NF-kB to be translocated into the nucleus. The nuclear NF-kB binds to a specific sequence (kB element) and activates the transcription of target genes.3) Constitutive NF-kB activation has been observed in a wide variety of cancers, indicating that NF-kB signaling is critical for the initiation and progression of human cancers. 4,5) Therefore, the NF-kB signaling pathway and the ERK/MAPK kinase pathway are good targets for cancer therapies. 4,5) Plants from the genus Erythrina have a significant history of medicinal use for the treatment of diseases such as female fertility, stomach pain and gonorrhea, and Erythrina species are well known for their alkaloid and flavonoid contents. 6)Erythrina lysistemon is native to Africa and contains many prenylated flavonoids. 6) Alpinumisoflavone (AIF) is isolated from Erythrina lysistemon and little is known about its biological functions. Alpinumisoflavone is reported to inhibit mouse brain monoamine oxidase in vitro, 7) and it has a weak inhibitory effect on human protein tyrosine phosphatase 1B. 8)Recently, AIF and its derivative, methylalpinumisoflavone, have been shown to inhibit hypoxia inducible factor-1 (HIF-1) activation.9) Here we report that AIF induces the apoptosis of lung tumor cells and the apoptosis induced by AIF may be via deregulating both the ERK/MAPK and NF-kB pathways. MATERIALS AND METHODS Plant MaterialThe stem bark of Erythrina lysistemon was collected in Buea, Cameroon, in July 2003 and identified by Zacharias Tanee Fomum at the University of Yaoundé I (Yaoundé, Cameroon). A voucher specimen (No. 0005) was deposited at the Department of Botany, Yaoundé I University, Cameroon.Extraction and Isolation The dried and ground stem...

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