Modulatory effects of bombyxin on ecdysteroidogenesis in Bombyx mori prothoracic glands.

In the present study, we investigated the modulatory effects of ecdysteroidogenesis of prothoracic glands (PGs) by bombyxin, an endogenous insulin-like peptide in the silkworm, Bombyx mori. The results showed that bombyxin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. Moreover, the injection of bombyxin into day 4-last instar larvae increased ecdysteroidogenesis 24h after the injection, indicating its possible in vivo function. Phosphorylation of the insulin receptor and Akt, and the target of rapamycin (TOR) signaling were stimulated by bombyxin, and stimulation of Akt phosphorylation and TOR signaling appeared to be dependent on phosphatidylinositol 3-kinase (PI3K). Bombyxin inhibited the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and the inhibition appeared to be PI3K-independent. Bombyxin-stimulated ecdysteroidogenesis was blocked by either an inhibitor of PI3K (LY294002) or a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, AICAR), indicating involvement of the PI3K/Akt and AMPK signaling pathway. Bombyxin did not stimulate extracellular signal-regulated kinase (ERK) signaling of PGs. Bombyxin, but not prothoracicotropic hormone (PTTH) stimulated cell viability of PGs. In addition, bombyxin treatment also affected mRNA expression levels of insulin receptor, Akt, AMPKα, -ß, and -γ in time-dependent manners. These results suggest that bombyxin modulates ecdysteroidogenesis in B. mori PGs during development.

Involvement of phosphorylation of adenosine 5'-monophosphate-activated protein kinase in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

In this study, we investigated inhibition of the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) by prothoracicotropic hormone (PTTH) in prothoracic glands of the silkworm, Bombyx mori. We found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPKα phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside, AICAR) increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. Examination of gene expression levels of AMPKα, ß, and γ by quantitative real-time PCR (qRT-PCR) showed that PTTH did not affect AMPK transcription. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, may play a role in PTTH stimulation of ecdysteroidogenesis.

Involvement of PI3K/Akt signaling in PTTH-stimulated ecdysteroidogenesis by prothoracic glands of the silkworm, Bombyx mori.

The prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by prothoracic gland in larval insects. Previous studies showed that Ca(2+), cAMP, extracellular signal-regulated kinase (ERK), and tyrosine kinase are involved in PTTH-stimulated ecdysteroidogenesis by the prothoracic glands of both Bombyx mori and Manduca sexta. In the present study, the involvement of phosphoinositide 3-kinase (PI3K)/Akt signaling in PTTH-stimulated ecdysteroidogenesis by B. mori prothoracic glands was further investigated. The results showed that PTTH-stimulated ecdysteroidogenesis was partially blocked by LY294002 and wortmannin, indicating that PI3K is involved in PTTH-stimulated ecdysteroidogenesis. Akt phosphorylation in the prothoracic glands appeared to be moderately stimulated by PTTH in vitro. PTTH-stimulated Akt phosphorylation was inhibited by LY294002. An in vivo PTTH injection into day 6 last instar larvae also increased Akt phosphorylation of the prothoracic glands. In addition, PTTH-stimulated ERK phosphorylation of the prothoracic glands was not inhibited by either LY294002 or wortmannin, indicating that PI3K is not involved in PTTH-stimulated ERK signaling. A23187 and thapsigargin, which stimulated B. mori prothoracic gland ERK phosphorylation and ecdysteroidogenesis, could not activate Akt phosphorylation. PTTH-stimulated ecdysteroidogenesis was not further activated by insulin, indicating the absence of an additive action of insulin and PTTH on the prothoracic glands. The present study, together with the previous demonstration that insulin stimulates B. mori ecdysteroidogenesis through PI3K/Akt signaling, suggests that crosstalk exists in B. mori prothoracic glands between insulin and PTTH signaling, which may play a critical role in precisely regulated ecdysteroidogenesis during development.

DNA damages induced by trans, trans-2,4-decadienal (tt-DDE), a component of cooking oil fume, in human bronchial epithelial cells.

Epidemiological studies have demonstrated that cooking oil fumes (COF) are an environmental risk factor for the development of lung adenocarcinoma among nonsmoking females in Taiwan. Aside from polycyclic aromatic hydrocarbons, aldehydes, especially trans, trans-2,4-decadienal (tt-DDE) are found to be abundant in COF. Although there is indication that tt-DDE induces DNA damage, the precise role of tt-DDE in the induction of DNA damage in lung cells is still not clear. When we assessed DNA breaks with the Comet assay, we found that the DNA breaks induced by 1 muM tt-DDE in human bronchial epithelial cells (BEAS-2B) could be significantly reduced by antioxidants, suggesting that oxidative stress was involved. Indeed, when tt-DDE-treated cells were coincubated with endonuclease III/formamidopyrimidine-DNA glycosylase or with nuclear extract (NE), an enhancement of DNA breaks was observed at 1 hr after tt-DDE exposure. Furthermore, when NE was incubated with an antibody against 8-oxoguanine DNA glycosylase (anti-OGG1), a reduction in tt-DDE/NE-induced DNA breaks could be demonstrated. Since OGG1 is a specific repair enzyme for 8-oxo-deoxyguanosine (8-oxo-dG), these findings indicated that 8-oxo-dG was involved. On the other hand, when NE was incubated with antibodies against nucleotide excision repair enzymes, there was a significant reduction in tt-DDE/NE-induced DNA breaks at 4 hr after tt-DDE treatment. These observations indicate that, in addition to early oxidative DNA damage, nonoxidative DNA damage such as bulky adduct formation, was also induced by tt-DDE. Our study further affirms that tt-DDE is genotoxic to human lung cells and can increase carcinogenic risk.

Increased sensitivity of Hep G2 cells toward the cytotoxicity of cisplatin by the treatment of piper betel leaf extract.

Piper betel leaves (PBL) are used in Chinese folk medicine for the treatment of various disorders. PBL has the biological capabilities of de-toxication, anti-oxidation and anti-mutation. In this study we first examined the effect of PBL extract on the activity of Glutathione S-transferase (GST) isoforms, and found that it inhibited total GST and the alpha class of GST (GSTA), but not the pi class of GST (GSTP), and the mu class of GST (GSTM), activity in Hep G2 cells. RT-PCR results verified a reduction in the expression of GSTA1. Next, we examined whether PBL extract could increase the sensitivity of Hep G2 cells to anti-cancer drugs. The data showed that the cytotoxicity of cisplatin was significantly enhanced by the presence of PBL extract, accompanied by a reduction in the expression of multidrug resistance protein 2 (MRP2). These effects of PBL extract were compared to its major constitute, eugenol. Although eugenol decreased MRP2 level more effectively than PBL extract, it exhibited less sensitizing effect. In conclusion, we demonstrated that PBL extract was able to increase the sensitivity of Hep G2 cells to cisplatin via at least two mechanisms, reducing the expression of MRP2 and inhibiting the activity of total GST and the expression of GSTA. The data of this study support an application of PBL as an additive to reduce drug resistance.

Geniposide activates GSH S-transferase by the induction of GST M1 and GST M2 subunits involving the transcription and phosphorylation of MEK-1 signaling in rat hepatocytes.

Geniposide, an iridoid glycoside isolated from the fruit of Gardenia jasminoides Ellis, has biological capabilities of detoxication, antioxidation, and anticarcinogenesis. We have recently found that geniposide possesses a potential for detoxication by inducing GST activity and the expression of GST M1 and GST M2 subunits. In this study, the signaling pathway of geniposide leading to the activation of GSH S-transferase (GST) was investigated. Primary cultured rat hepatocytes were treated with geniposide in the presence or absence of mitogen-activated protein kinase (MAPK) inhibitors and examined for GST activity, expression of GST M1 and M2 subunits, and protein levels of MAPK signaling proteins. Western blotting data demonstrated that geniposide induced increased protein levels of GST M1 and GST M2 (approximately 1.76- and 1.50-fold of control, respectively). The effect of geniposide on the increased protein levels of GST M1 and GST M2 was inhibited by the MEK-1 inhibitor PD98059, but not by other MAPK inhibitors. The GST M1 and GST M2 transcripts as determined by RT-PCR and GST activity were also inhibited concurrently by the MEK-1 inhibitor PD98059. The protein levels of up- and down-stream effectors of the MEK-1, including Ras, Raf, and Erk1/2, and the phosphorylation state of Erk1/2 were found to be induced by geniposide, indicating a two-phase influence of geniposide. The results suggest that geniposide induced GST activity and the expression of GST M1 and GST M2 acting through MEK-1 pathway by activating and increasing expression of Ras/Raf/MEK-1 signaling mediators.

Differential induction of the expression of GST subunits by geniposide in rat hepatocytes.

Geniposide, an iridoid glycoside isolated from the fruit of Gardenia jasminoides Ellis, has the biological capabilities of detoxication, antioxidation, and anticarcinogenesis. In this study, the mechanism of geniposide affecting the GST (glutathione S-transferase) system was investigated. Primary cultured rat hepatocytes were treated with geniposide and examined for total GST activity and expression of GST subunits. The results showed that the geniposide-induced GST activity was dose and time dependent. Western blotting data demonstrated that geniposide induced increased protein levels of GSTM1 and GSTM2 (approximately 1.7- and 1.8-fold of control, respectively), but did not increase those of GSTA1. The corresponding transcripts levels were confirmed by RT-PCR. Using PD98059, the effect of geniposide was verified to be via the MEK pathway. The results suggest that geniposide possesses a potential for detoxication by inducing GST activity via increasing the transcription of GSTM1 and GSTM2.