Abstract. Recent studies have revealed that 3,3-diindolylmethane (DIM) has antitumor effects in both in vivo and in vitro tumor models. However, the biological function of DIM in human gastric cancer cells is unknown. Genetic and biological studies have confirmed the importance of the novel Hippo tumor-suppressor pathway in regulating cell proliferation, apoptosis, organ size and tumorigenesis in mammals. Thus, the purpose of this study was to investigate the cytotoxic effects of DIM in human gastric cancer cells and to elucidate whether DIM induces cell death by activating the Hippo signaling pathway. Two human gastric cancer cell lines (SNU-1 and SNU-484) were used to investigate the DIM response. DIM significantly inhibited the proliferation of human gastric cancer cells in a dose-dependent manner. The percentage of G1 phase cells increased 24 h following DIM treatment. DIM reduced CDK2, CDK4, CDK6 and cyclin D1 protein levels, while increasing p53 protein levels. DIM induced the levels of cleaved poly(ADP-ribose) polymerase, cleaved-caspase-9, and diminished pro-caspase-3 protein production. In addition, DIM increased pLATS1, Mob1, pMob1, pYAP and Ras association domain family 1 (RASSF1) protein levels and reduced Yap protein production levels. DIM stimulated the binding of RASSF1 with the Mst1/2-LATS1-Mob1 complex, promoting an active Hippo signaling pathway and favoring YAP phosphorylation (pYAP) that inactivates cell proliferation. Furthermore, DIM inhibited the growth of human gastric tumors in a xenograft mouse model. These results indicate that DIM suppresses the growth of gastric cancer cells by activating the Hippo signaling pathway.

Cancer development is a complex process governed by the interaction of several signaling pathways. The Hippo and PI3K/Akt pathways have been shown to play a critical role in controlling tissue growth involved in the regulation of cell proliferation. 3, 3'-diindolylmethane (DIM) is a natural compound that selectively kills cancer cells without causing toxicity to normal cells. This study aims to investigate whether DIM has an effect on the Hippo signaling pathway mediated via the PI3K/Akt signaling pathway in colon cancer cells. Our study provides new insights into the mechanisms of crosstalk between Hippo signaling and the Akt pathway controlling cell proliferation by PI3K inhibitor and DIM treatment in colon cancer cells. DIM strongly potentiates the lethality of LY294002 in HCT116 cells and inhibits proliferation of colon cancer cells via inactivation of Akt and YAP. Thus, DIM has dramatic therapeutic effects when it is combined with the PI3K inhibitor in the treatment of colon cancer cells. These findings highlight the potential usefulness of DIM and can help develop therapeutic strategies for the prevention and treatment of colon cancer.

No abstract

Abstract. Gastric cancer is the fourth most common cancer and is one of the leading causes of cancer-related mortality worldwide. Forkhead box M1 (FOXM1) is overexpressed in gastric cancer, suggesting that it is important in gastric cancer oncogenesis. However, no studies have investigated the role of 3,3'-diindolylmethane (DIM), a component of cruciferous vegetables, in the regulation of FOXM1 and its signaling pathway in gastric cancer. Here, we report for the first time that DIM effectively downregulated Akt/FOXM1 in gastric cancer cells. Combination treatment with DIM and paclitaxel significantly and dose-dependently inhibited the proliferation of SNU638 cells when compared to treatment with DIM or paclitaxel alone. Colony formation of SNU638 cells was significantly attenuated by treatment with DIM and paclitaxel, and DIM potentiated the inhibition of colony formation in SNU638 cells by paclitaxel when compared to treatment with a single agent. Treatment with DIM plus paclitaxel substantially increased apoptosis as indicated by increased levels of cleaved polyADP-ribose polymerase (PARP) and cleaved caspase-9 protein. DIM dose-dependently sensitized gastric cancer cells through downregulation of FOXM1 and potentiated the effects of paclitaxel. FOXM1 effector genes such as CDK4, p53 and cyclin D1 were downregulated in gastric cancer cells by combination treatment with DIM and paclitaxel. In addition, DIM significantly and dose-dependently inhibited phosphorylation of Akt and potentiated paclitaxel-induced inhibition of Akt function in gastric cancer cells. Therefore, our results indicate that DIM effectively potentiates the efficacy of chemotherapeutic agents such as paclitaxel by downregulation of the Akt/ FOXM1 signaling cascade in gastric cancer cells. Our findings suggest that DIM enhances the therapeutic efficacy of paclitaxel in gastric cancer and is a potential clinical anticancer agent for the prevention and/or treatment of gastric cancer.

p63 is a member of the p53 protein family and plays a crucial role in epithelial development. p63 is expressed in many types of tumors including esophageal cancer; however, its function in cancer is controversial and its role in esophageal cancer has not been clearly established. In the present study, we aimed to identify the mechanisms by which p63 promotes proliferation of esophageal squamous carcinoma cells. Four human esophageal cancer cell lines (TE-8, TE-12, BE3 and OE33) were used in this study. We found that ΔNp63 was the predominantly expressed p63 isoform in esophageal squamous cancer cells. Silencing of p63 mRNA in the esopha-geal cancer cell lines TE-8 and TE-12 resulted in significant inhibition of cell proliferation in a dose-dependent manner. A colony formation assay also showed that colony formation by TE-8 and TE-12 cells was significantly inhibited by p63 siRNA. Furthermore, p63 siRNA significantly suppressed p-Akt and induced Akt expression in esophageal squamous carcinoma cell lines. On the other hand, overexpression of p63 in the esophageal cell lines BE3 and OE33 increased p-Akt expression. Silencing of p63 in TE-8 and TE-12 cell lines induced p53 and p27 expression and suppressed cyclin D1 and cyclin E1 expression, whereas overexpression of p63 in BE3 and OE33 cell lines resulted in decreased levels of p53 and p27 and increased levels of cyclin D1 and cyclin E1. Taken together, our results suggest that p63 may play a pivotal role in the progression of esophageal squamous carcinoma cells through regulation of the cell cycle via the Akt signaling pathway.

Despite the fact that paclitaxel and doxorubicin are widely used as chemotherapy agents against several types of cancer, their combined effects on esophageal squamous cell carcinoma (ESCC) have never been fully elucidated. The present study was designed to investigate the biological effects of paclitaxel and doxorubicin in ESCC cells. Combination treatment with paclitaxel and doxorubicin significantly inhibited the proliferation of TE-12 cells in a dose-and time-dependent manner compared to treatment with paclitaxel or doxorubicin alone. FACS analysis showed that the percentage of cells in the G2/M phase was significantly increased at 12 h after treatment with the combination. Increased p-cdc2, p-Wee1 and p53 protein levels were observed, while Akt activation was suppressed by combination treatment with paclitaxel and doxorubicin. In addition, treatment with paclitaxel plus doxorubicin significantly increased apoptosis as indicated by increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-7 and -9 levels. These results suggest that combination treatment with paclitaxel and doxorubicin induced G2/M cell cycle arrest and apoptosis in human ESCC cells by suppressing Akt activity. These findings highlight the potent apoptotic effect of combination therapy with paclitaxel and doxorubicin in ESCC cells and the potential clinical benefits of these two drugs in esophageal cancer.

Multiple genetic and signaling pathway alterations underlie the development of colon cancer. We utilized genome-wide transcriptome analysis to identify important gene expression patterns following treatment with 3,3'-diindolylmethane (DIM), a natural compound derived from cruciferous vegetables, on colon cancer cells. Statistical analyses of gene expression data from DIM treated cells revealed that 692 genes were significantly upregulated, while 731 genes were down-regulated. Putative gene networks showed that several oncogenes (β-catenin, Myc and FOS) were significantly suppressed by DIM treatment. Using clinical data from colon cancer patients, activation of β-catenin was found to be significantly associated with patient prognosis by Kaplan-Meir plot analysis. We validated the mRNA and protein expression levels of c-Myc, β-catenin, and cyclin D1, all of which were significantly suppressed after DIM treatment in DLD-1 and HCT116 cells. System level characterization of our findings suggests for the first time that β-catenin and c-Myc, which are major genes involved in colon carcinogenesis, were significantly downregulated by DIM treatment in colon cancer cells. Therefore, targeting Wnt/β-catenin signaling by DIM may be an attractive strategy for the prevention and treatment of colon cancer.

Although 3,3'-diindolylmethane (DIM) has been suggested to reduce the risk of colorectal cancer, the underlying biological mechanism is not clearly understood. In the present study, we investigated the effect of DIM on the migratory and invasive activities of the human colorectal cancer cell lines DLD-1 and HCT116. DIM significantly inhibited the migration and invasion of colorectal cancer cells as assessed by wound healing and Matrigel invasion assays. The migratory ability of the DLD-1 and HCT116 cells was significantly reduced by DIM at 24 and 48 h. DIM also significantly inhibited the invasion rate of the DLD-1 and HCT116 cells in a dose-dependent manner. The mRNA expression levels of urokinase type plasminogen activator (uPA) and matrix metalloprotease 9 (MMP9) were significantly attenuated, whereas expression of E-cadherin mRNA was significantly enhanced, following DIM treatment. DIM also decreased the protein levels of uPA and MMP9, yet significantly increased E-cadherin protein expression. In addition, DIM significantly reduced the mRNA and protein levels of FOXM1 in the DLD-1 and HCT116 cells. Our results suggest that DIM can influence the cell migratory and invasive properties of human colorectal cancer cells and may decrease the invasive capacity of colorectal cancer through downregulation of uPA and MMP9 mediated by suppression of the transcription factor FOXM1.