Gadd45-α and Gadd45-γ utilize p38 and JNK signaling pathways to induce cell cycle G2/M arrest in Hep-G2 hepatoma cells

The Gadd45 family of proteins, which includes α, β, and γ isoforms, has recently been shown to play a role in the G2/M cell cycle checkpoint in response to DNA damage; however, the mechanisms by which Gadd45 proteins inhibit cell cycle control are not fully understood. Using immunohistochemical analysis, we found that protein expression of Gadd45γ, but not Gadd45α, was down-regulated in hepatocellular carcinoma. We thus investigated possible mechanisms by which Gadd45α and Gadd45γ might differentially induce G2/M arrest in the human hepatoma Hep-G2 cell line. Flow cytometric analysis revealed significant G2/M arrest in cells transfected with either Gadd45α or Gadd45γ. Importantly, we found that expression of either Gadd45α or Gadd45γ activated the P38 and JNK kinase pathways to induce G2/M arrest. Taken together, these findings suggest that the induction of G2/M arrest by Gadd45α or Gadd45γ involves activation of two distinct signaling pathways in Hep-G2 hepatoma cell lines.     

Therapeutic ROS targeting of GADD45γ in the induction of G2/M arrest in primary human colorectal cancer cell lines by cucurbitacin E

Cucurbitacin E (CuE) or α-elaterin is a natural compound previously shown to be an antifeedant as well as a potent chemopreventive agent against several types of cancer. The present study investigated the anticancer effects of CuE on colorectal cancer (CRC) using primary cell lines isolated from five CRC patients in Taiwan, Specifically, we explored the anti-proliferation and cell cycle G2/M arrest induced by CuE in CRC cells. MPM-2 flow cytometry tests show that CuE-treated cells accumulated in metaphase (CuE 2.5–7.5 μM). Results further indicate that CuE produced G₂/M arrest as well as the downregulation of CDC2 and cyclin B1 expression and dissociation. Both effects increased proportionally with the dose of CuE; however, the inhibition of proliferation, arrest of mitosis, production of reactive oxygen species (ROS), and loss of mitochondrial membrane potential (ΔΨm) were found to be dependent on the quantity of CuE used to treat the cancer cells. In addition, cell cycle arrest in treated cells coincided with the activation of the gene GADD45(α, β, γ). Incubation with CuE resulted in the binding of GADD45γ to CDC2, which suggests that the delay in CuE-induced mitosis is regulated by the overexpression of GADD45γ. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activities in established CRC.     

Activation of PPAR-α induces cell cycle arrest and inhibits transforming growth factor-β1 induction of smooth muscle cell phenotype in 10T1/2 mesenchymal cells

Transforming growth factor-β1 (TGF-β1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-β1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G₀/G₁ phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G₀/G₁ phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells.     

The dietary phytochemical 3,3'-diindolylmethane induces G2/M arrest and apoptosis in oral squamous cell carcinoma by modulating Akt-NF-κB, MAPK, and p53 signaling

In light of the growing incidence of oral cancer in Taiwan, this study is aimed at investigating the antitumor activity of 3,3'-diindolylmethane (DIM), an active metabolite of the phytochemical indole-3-carbinol (I3C), in oral squamous cell carcinoma (OSCC). DIM exhibited substantially higher antiproliferative potency than I3C in three OSCC cell lines with IC(50) values in SCC2095, SCC9, and SCC15 cells, respectively, of 22 versus 168μM, 25 versus 176μM, and 29versus 300μM. Flow cytometric analysis and Comet assay indicated that DIM suppressed the viability of SCC2095 cells by inducing apoptosis and G2/M arrest. Western blot analysis of various signaling markers revealed the ability of DIM to target pathways mediated by Akt, mitogen-activated protein (MAP) kinases, nuclear factor (NF)-κB, and p53, of which the concerted action underlined its antitumor efficacy. The concomitant inactivation of Akt and MAP kinases in response to DIM facilitated the dephosphorylation of the proapoptotic protein Bad at Ser-136 and Ser-112, respectively. Through endoplasmic reticulum (ER) stress, DIM stimulated the activation of p53 via Ser-15 phosphorylation, leading to increased expression of the BH3-only proapoptotic Bcl-2 members Puma and Noxa. Together, these changes decreased the mitochondrial threshold for apoptosis. G2/M arrest might be attributable to the suppressive effect of DIM on the expression of cyclin B1 and cdc25c. As many downstream effectors of the Akt-NF-κB pathway, including glycogen synthase kinase 3β, IκB kinase α, and cyclooxygenase-2, have been shown to promote oral tumorigenesis, the ability of DIM to inhibit this signaling axis underscores its chemopreventive potential in oral cancer.