BJ-1108, a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog, Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway

Abstract

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT1 receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.

Fig 1. 5-HT-induced angiogenesis is mediated through 5-HT1, but not through 5-HT2 and 5-HT7 receptors.

(A) Structural formula of 5-HT and BJ-1108. (B) After incubation with 5-HT for 3 days, the newly formed blood vessel branches on the 13-day-old chick CAM were counted. Data represent the mean ± S.E.M. of at least seven chick embryos. *P <0.05, compared with the untreated control group, (C) HUVECs in Matrigel-coated plates were treated with 5-HT for 14 h, and digital images were then taken under the microscope. (D) Migration pattern of HUVECs after treatment with VEGF or 5-HT for 5 h. The bar graph represents the mean ± S.E.M. of at least three independent experiments. *P<0.05, compared with the untreated control group. (E) Tube formation of HUVECs treated with 5-HT for 14 h in the absence or presence of 0.1 μM cyanopindolol (CYPD), Cinanserin (CISN), or SB269970 was quantitated by measuring tube length on the digital images. The bar graph represents the mean ± S.E.M. of at least three independent experiments. *P<0.05, compared with the untreated control group. #P<0.05 compared with the 5-HT-stimulated group.

Fig 2. 5-HT-induced ROS was suppressible by inhibitors of Gβγ, Src, PI3K, and NOX in HUVECs.

(A) Intracellular ROS level detected using DCF-DA in HUVECs treated with 5-HT (10 μM) for the indicated time was determined by fluorescence microscopy. (B-D) Superoxide production was measured by lucigenin chemiluminescence assay. HUVECs treated with 5-HT for 3 h in the absence or presence of 5-HT receptor antagonists (B), various ROS-producing enzyme inhibitors (C), and inhibitors of PI3K, Src and Giβγ (D). (E) Tube formation of HUVECs treated with 5-HT with or without inhibitors against NOX (Apo, DPI), Src (AZM-475271), PI3K (wortmannin), and MAPK (U0126) was quantitated by measuring tube length on the digital images. The bar graph represents the mean ± S.E.M. of at least three independent experiments. *P<0.05, compared with the untreated control group. #P<0.05 compared with the 5-HT-stimulated group.

Fig 3. BJ-1108, a novel pyridinol analog, inhibits 5-HT-induced angiogenesis, in a PI3K-dependent manner.

(A) The CAM of a 10-day-old chick embryo was treated with 5-HT, and BJ-1108 was applied 30 min later. The bar graph represents the mean ± S.E.M. of at least seven chick embryos. *P <0.05, compared with the PBS-treated control group. #P<0.05 compared with the 5-HT-stimulated group. (B) The tube length of HUVECs pretreated with BJ-1108 prior to 5-HT treatment for 14 h was quantitated. (C) KDR (VEGF receptor 2 tyrosine kinase) activity in vitro was measured by KDR kinase enzyme system and ADP-Glow^™ kinase assay kit. Sunitinib malate was used as a positive control. The bar graph represents the means ± S.E.M. of at least three independent experiments. *P<0.05, compared to control and #P<0.05, compared to sunitinib. (D) Following pretreatment of HUVECs with BJ-1108 prior to treatment with 5-HT for 5 min, the expression level of phosphorylated form of signaling molecules was analyzed by immunoblotting. Antibodies against phospho-src (at Tyr416), phospho-p85-PI3K (at Tyr488), phospho-AKT (at T308), phospho-mTOR (at Ser2448), phospho-ERK (at T202), and phospho-p38 (at T180), were used for detection of the phospho form of respective proteins. (E) c-Src kinase activity was measured using the Cyclex c-Src Kinase Assay/Inhibitor screening kit. AZM-475271 was used as a positive control. (F) PI3K enzyme activity of the catalytic subunit, p110 was measured using a competitive PI3K activity assay kit, and wortmannin was used as a positive control. The bar graph represents the means ± S.E.M. of at least three independent experiments. *P<0.05, compared to control and #P<0.05, compared to wortmannin.

Fig 4. Inhibitory effect of BJ-1108 on 5-HT-induced superoxide was not due to antioxidant activity or direct inhibitory action on NOX activation.

(A) Superoxide production was measured by lucigenin chemiluminescence assay in HUVECs pretreated with BJ-1108 for 1 h prior to 5-HT for 3 h. (B-D) HUVECs were treated with mevalonate (MEV, 500 μM), or geranylgeranyl pyrophosphate (GGPP, 20 μM) for 3 h. Before treatment with MEV or GGPP, HUVECs were pretreated with NOX inhibitors (apocynin, VAS2870, DPI) (B), and BJ-1108 or Vitamin C (C, D) for 1 h. The bar graph represents the mean ± S.E.M. of at least three independent experiments. *P<0.05, compared to control and #P<0.05, compared to MEV or GGPP. (E) Comparison of DPPH radical scavenging activity of BJ-1108, Vitamin C, and apocynin. (F) Comparison of XO-derived superoxide scavenging activity of BJ-1108, Vitamin C, and apocynin. Data shown are expressed as the mean ± SEM of three independent experiments. *P<0.05, compared to Vitamin C.

Fig 5. Inhibitory effects of BJ-1108 on human breast cancer cells (MDA-MB-231 and MCF-7)-induced angiogenesis and tumor growth.

MDA-MB-231 and MCF-7 human breast cancer cells (2×10⁶ cells/CAM) were inoculated on top of CAM, and BJ-1108 was mixed with cell suspension before inoculation. (A) Digital images of CAM sections were captured, and (B) imported into the image analysis program for quantitation of the number of new vessel branches formed. (C) Tumor mass was isolated and weighed. Data represent the mean ± S.E.M. of at least seven chick embryos. *P<0.05, compared with vehicle-treated control.

Fig 6. Proposed action mechanism by which BJ-1108 suppresses 5-HT-induced angiogenesis.

In HUVECs, 5-HT activates Src, PI3K, NOX, AKT, mTOR, ERK and p38. However, ERK and p38 pathways are not involved in 5-HT-induced angiogenesis. BJ-1108 inhibits 5-HT-induced angiogenesis by suppressing 5-HT₁ receptor-activated PI3K/Akt/NOX signaling pathway.