A combination of indol-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy.

BACKGROUND: The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies. RESULTS: Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not. CONCLUSION: Although in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.

Contribution of adenosine to the increase in skeletal muscle blood flow caused by manual acupuncture in rats.

BACKGROUND AND AIM: Adenosine is believed to play an important role in local acupuncture analgesia. The aim of this study was to investigate the contribution of adenosine to the increase in skeletal muscle blood flow (MBF) caused by manual acupuncture (MA). METHODS: Thirty-two male Sprague-Dawley rats (310-360 g) were anaesthetised and divided into four equal groups (n=8 each): Saline, Saline+MA, Theophylline, and Theophylline+MA. In the two MA groups, the sparrow-pecking MA technique was applied at 30 repetitions per min for 1 min to a depth of 15-18 mm using a stainless steel acupuncture needle (0.20×40 mm). The stimulus point was located on the right tibialis anterior (TA) muscle 7-8 mm below the knee. Animals in the two theophylline groups were intra-arterially injected with 8-(p-sulphophenyl) theophylline, a non-selective adenosine receptor antagonist, at a dose of 30 mg/kg before MA. Animals in the two saline groups received control saline. Fluorescent microspheres (15 µm in diameter, yellow-green fluorescent) were used for MBF measurement in all four groups. RESULTS: MA of the TA muscle significantly increased MBF (Saline+MA vs Saline: p=0.001; Saline+MA vs Theophylline: p=0.008). Pre-treatment with theophylline appeared to inhibit this increase (Theophylline vs Theophylline+MA; p=1.000). MBF in the Theophylline+MA group was 43% lower than in the Saline+MA group, although this was not significantly different (p=0.104). CONCLUSIONS: The results suggest that adenosine leads to an increase in MBF caused by MA. Adenosine may play a role in acupuncture analgesia by washing out algesic substances. Further studies are needed in order to elucidate the precise mechanism.

[Corrigendum] Brassinin induces G1 phase arrest through increase of p21 and p27 by inhibition of the phosphatidylinositol 3-kinase signaling pathway in human colon cancer cells.

After the publication of the article, the authors noted that they had made the following errors. In Fig. 5B, total PDK1 is incorrect in the published paper. The corrected version of Fig. 5B is shown below. Also in Fig. 6B, the DMSO-treated group and the brassinin (400 µM)-treated group are incorrect, and the corrected Fig. 6B is shown below. The corrected figures demonstrate the same findings as the original figures. These corrections do not alter the interpretation of the results and conclusions. [the original article was published in the International Journal of Oncology 40: 816-824, 2012 DOI: 10.3892/ijo.2011.1246].

Brassinin induces G1 phase arrest through increase of p21 and p27 by inhibition of the phosphatidylinositol 3-kinase signaling pathway in human colon cancer cells.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is activated in a broad spectrum of human cancers, including colon cancer. The natural product brassinin is a type of indole compound derived from cruciferous vegetables, and has been shown to have anti-proliferative effects against cancer for both in vivo and in vitro models. Here, we show for the first time that brassinin inhibits cell growth in human colon cancer cells by arresting the cell cycle at the G1 phase via inhibition of the PI3K signaling pathway. Brassinin increased the expression of p21 and p27, resulting in hypophosphorylation of the retinoblastoma gene (RB). Knockdown of p21 or p27 by each siRNA significantly repressed G1 phase arrest induced by brassinin. The increase of p21 and p27 was associated with inhibition of the PI3K signaling pathway. In addition, exogenous expression of constitutively active Akt represses the cell cycle arrest at G1 phase induced by brassinin. These results suggest the possibility that brassinin inhibits the PI3K signaling pathway and upregulates the expression of p21 and p27, thereby inducing G1 phase arrest.

Cucurbitacin B induces G2 arrest and apoptosis via a reactive oxygen species-dependent mechanism in human colon adenocarcinoma SW480 cells.

Cucurbitacin B (cucB) is a triterpenoid constituent of Cucurbitaceae vegetables and a promising phytochemical for cancer prevention. However, the mechanism of anti-tumor activity of cucB remains unknown, especially in colon cancers. Here, we demonstrate for the first time that cucB inhibited growth of human colon cancer SW480 cells through a reactive oxygen species (ROS)-dependent mechanism. CucB induced G(2) phase arrest and apoptosis in a dose-dependent manner. At the molecular level, cucB reduced the expression of cyclin B1 and cdc25C proteins and activated caspases in SW480 cells. On the other hand, the state of phosphorylation of signaling transducer and activator of transcription 3 (STAT3) was unchanged. We found that cucB increased intracellular ROS levels, and N-acetylcysteine, a well-known antioxidant, reduced the changes in expression of the molecules, and suppressed both G(2) arrest and apoptosis. These results suggested that cucB induced G(2) arrest and apoptosis through a STAT3-independent but ROS-dependent mechanism in SW480 cells.

Histone deacetylase inhibitors and 15-deoxy-Delta12,14-prostaglandin J2 synergistically induce apoptosis.

PURPOSE: The clinically relevant histone deacetylase inhibitors (HDI) valproic acid (VPA) and suberoylanilide hydroxamic acid exert variable antitumor activities but increase therapeutic efficacy when combined with other agents. The natural endogenous ligand of peroxisome proliferator-activated receptor gamma 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a potent antineoplastic agent. Therefore, we investigated whether these HDIs in combination with 15d-PGJ(2) could show synergistic antitumor activity in colon cancer DLD-1 cells. EXPERIMENTAL DESIGN: Cell viability was determined using a Cell Counting Kit-8 assay. Apoptosis and reactive oxygen species (ROS) generation were determined using flow cytometry analysis. Western blotting and real-time reverse transcription-PCR analysis were carried out to investigate the expression of apoptosis-related molecules. Mice bearing DLD-1 xenograft were divided into four groups (n = 5) and injected everyday (i.p.) with diluent, VPA (100 mg/kg), 15d-PGJ(2) (5 mg/kg), or a combination for 25 days. RESULTS: HDI/15d-PGJ(2) cotreatments synergistically induced cell death through caspase-dependent apoptosis in DLD-1 cells. Moreover, HDIs/15d-PGJ(2) caused histone deacetylase inhibition, leading to subsequent ROS generation and endoplasmic reticulum stress to decrease the expression of antiapoptotic molecules Bcl-X(L) and XIAP and to increase that of proapoptotic molecules CAAT/enhancer binding protein homologous protein and death receptor 5. Additionally, VPA/15d-PGJ(2) cotreatment induced ROS-dependent apoptosis in other malignant tumor cells and was more effective than a VPA or 15d-PGJ(2) monotherapy in vivo. CONCLUSIONS: Cotreatments with the clinically relevant HDIs and the endogenous peroxisome proliferator-activated receptor gamma ligand 15d-PGJ(2) are promising for the treatment of a broad spectrum of malignant tumors.