The Hippo pathway is often dysregulated in many carcinomas, which results in various stages of tumor progression. Ursolic acid (UA), a natural compound that exists in many herbal plants, is known to obstruct cancer progression and exerts anti-carcinogenic effect on a number of human cancers. In this study, we aimed to examine the biological mechanisms of action of UA through the Hippo pathway in gastric cancer cells. MTT assay showed a decreased viability of gastric cancer cells after treatment with UA. Following treatment with UA, colony numbers and the sizes of gastric cancer cells were significantly diminished and apoptosis was observed in SNU484 and SNU638 cells. The invasion and migration rates of gastric cancer cells were suppressed by UA in a dose-dependent manner. To further determine the gene expression patterns that are related to the effects of UA, a microarray analysis was performed. Gene ontology analysis revealed that several genes, such as the Hippo pathway upstream target gene, ras association domain family (RASSF1), and its downstream target genes (MST1, MST2, and LATS1) were significantly upregulated by UA, while the expression of YAP1 gene, together with oncogenes (FOXM1, KRAS, and BATF), were significantly decreased. Similar to the gene expression profiling results, the protein levels of RASSF1, MST1, MST2, LATS1, and p-YAP were increased, whereas those of CTGF were decreased by UA in gastric cancer cells. The p-YAP expression induced in gastric cancer cells by UA was reversed with RASSF1 silencing. In addition, the protein levels in the Hippo pathway were increased in the UA-treated xenograft tumor tissues as compared with that in the control tumor tissues; thus, UA significantly inhibited the tumorigenesis of gastric cancer in vivo in xenograft animals. Collectively, UA diminishes the proliferation and metastasis of gastric cancer via the regulation of Hippo pathway through Rassf1, which suggests that UA can be used as a potential chemopreventive and therapeutic agent for gastric cancer.

Studies in humans have shown that 3,3′-diindolylmethane (DIM), which is found in cruciferous vegetables, such as cabbage and broccoli, is effective in the attenuation of gastrointestinal cancers. This review presents the latest findings on the use, targets, and modes of action of DIM for the treatment of human gastrointestinal cancers. DIM acts upon several cellular and molecular processes in gastrointestinal cancer cells, including apoptosis, autophagy, invasion, cell cycle regulation, metastasis, angiogenesis, and endoplasmic reticulum (ER) stress. In addition, DIM increases the efficacy of other drugs or therapeutic chemicals when used in combinatorial treatment for gastrointestinal cancer. The studies to date offer strong evidence to support the use of DIM as an anticancer and therapeutic agent for gastrointestinal cancer. Therefore, this review provides a comprehensive understanding of the preventive and therapeutic properties of DIM in addition to its different perspective on the safety of DIM in clinical applications for the treatment of gastrointestinal cancers.

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.

Colorectal cancer (CRC) is one of the deadliest malignant tumors worldwide and its prevalence is increasing in South Korea. The efficacy of combined treatment with natural product‑derived and chemotherapy agents including curcumin combined with 5‑fluorouracil, resveratrol combined with cisplatin and epigallocatechin‑3‑gallate (EGCG) combined with cisplatin in preventing cancer progression and killing cancer cells has emerged. The Akt and Hippo signaling pathways serve a key role in colorectal tumor growth; however, the exact role of the crosstalk between Akt and Hippo signaling pathways in CRC remains poorly elucidated. The combined effect of UA and DOX on the cell proliferation, apoptosis, migration and cell cycle of CRC cells were investigated by performing Cell proliferation assay, a soft agar colony formation assay, flow cytometry, wound healing assay and western blotting assay. Subsequently, the expression of AKT and Hippo signaling pathway‑associated proteins were also assessed by western blot assay. Moreover, a xenograft nude mouse model was constructed to verify the effects of UA and DOX on the tumorigenesis of HCT116 cell in vivo. The present study reported that ursolic acid (UA) strongly enhanced the antitumor action of doxorubicin (DOX) via blocking the Akt/glycogen synthase kinase‑3β (Gsk3β) signaling pathway and activating tumor‑suppressive Hippo signaling (mammalian Ste20‑like kinase 1 and 2, salvador family WW domain containing protein 1 and MOB kinase activator 1), thereby downregulating downstream effector yes‑associated protein 1 (Yap) and connective tissue growth factor (CTGF) protein expression levels in CRC cells. Furthermore, The PI3K inhibitor LY294002 further suppressed Akt activity and enhance the function of Hippo pathway‑associated proteins in DOX + UA treated cells; this effect led to subsequent oncogenic Yap and CTGF inhibition following combined treatment, whereas Akt activator SC79 exerted an opposite effect in CTGF expression. In vivo, treatment with UA combined with DOX markedly suppressed the progression of CRC without any toxic effects on a xenograft mouse model by disrupting Akt signaling and activating the Hippo signaling pathway. These results demonstrated that UA and DOX treatment successfully induced Akt/Gsk3β inactivation via Hippo signaling pathway activation to promote Yap degradation, resulting in the inhibition of colorectal tumorigenesis. In conclusion, these findings suggested that combination therapy with UA and DOX may be more effective than DOX alone. UA may be a novel anticancer strategy and could be considered for investigation as a complementary chemotherapy agent in the future.

Ursolic acid (UA) possesses various pharmacological activities, such as antitumorigenic and anti-inflammatory effects. In the present study, we investigated the mechanisms underlying the effects of UA against esophageal squamous cell carcinoma (ESCC) (TE-8 cells and TE-12 cells). The cell viability assay showed that UA decreased the viability of ESCC in a dose-dependent manner. In the soft agar colony formation assay, the colony numbers and size were reduced in a dose-dependent manner after UA treatment. UA caused the accumulation of vacuoles and LC3 puncta, a marker of autophagosome, in a dose-dependent manner. Autophagy induction was confirmed by measuring the expression levels of LC3 and p62 protein in ESCC cells. UA increased LC3-II protein levels and decreased p62 levels in ESCC cells. When autophagy was hampered using 3-methyladenine (3-MA), the effect of UA on cell viability was reversed. UA also significantly inhibited protein kinase B (Akt) activation and increased p-Akt expression in a dose-dependent manner in ESCC cells. Accumulated LC3 puncta by UA was reversed after wortmannin treatment. LC3-II protein levels were also decreased after treatment with Akt inhibitor and wortmannin. Moreover, UA treatment increased cellular reactive oxygen species (ROS) levels in ESCC in a time- and dose-dependent manner. Diphenyleneiodonium (an ROS production inhibitor) blocked the ROS and UA induced accumulation of LC3-II levels in ESCC cells, suggesting that UA-induced cell death and autophagy are mediated by ROS. Therefore, our data indicate that UA inhibits the growth of ESCC cells by inducing ROS-dependent autophagy.