Downregulation of tetrahydrobiopterin inhibits tumor angiogenesis in BALB/c-nu mice with hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a highly vascular tumor, and treatment options for patients of advanced-stage are limited. Nitric oxide (NO), which is derived from endothelial nitric oxide synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment. Tetrahydrobiopterin (BH4) is an essential cofactor eNOS and represents a critical determinant of NO production. To examine whether treatment of 2,4-diamino-6-hydroxypyrimidine (DAHP) inhibits angiogenesis of HCC, BALB/c-nu mice were injected with HepG-2 cells with DAHP. Supplemental DAHP treatment decreased K-ras mRNA transcripts, inhibition of phosphorylation of eNOS and Akt, inhibition of guanosine triphosphate cyclohydrolase (GTPCH), and decreased significantly NO synthesis, and then inhibited angiogenesis, compared with the results observed in the saline group. Histopathology demonstrated angiogenesis and tumor formation were significantly inhibited in HCC. DAHP downregulates GTPCH protein expression, corresponding to decreased levels of BH4 and the contents of NO. In addition, DAHP downregulates eNOS and Akt protein expression, corresponding to decreased eNOS phosphorylation at Ser1177 and Akt phosphorylation, compared with the saline control. We suggest that DAHP, recognized as a specific competitive inhibitor of GTPCH, can decrease tumor BH4 and NO by the inhibition of the wild-type Ras-PI3K/Akt pathway, and then inhibiting angiogenesis, and may provide a novel and promising way to target BH4 synthetic pathways to inhibit angiogenesis and to control potential progression of HCC. Whether DAHP has a therapeutic potential will require more direct testing in humans.

Figure 1

DAHP impaired tubule formation of HUVEC in vitro. Treatment with DAHP inhibited tubule formation of HUVEC.

Figure 1

DAHP impaired tubule formation of HUVEC in vitro. Treatment with DAHP inhibited tubule formation of HUVEC.

Figure 2

DAHP modulates BH4 levels and NO content in tumor tissue. (A) Total tumor biopterin and BH4 tissue levels were assessed by HPLC. The administered DAHP decreases BH4 levels of tumor tissue in HCC. (B) Nitrite/nitrate levels in the tumor tissue was determined by the Greiss reaction. These levels indirectly measure NO content. The administration of DAHP significantly decreased nitrite/nitrate levels in tumor tissue.

Figure 2

DAHP modulates BH4 levels and NO content in tumor tissue. (A) Total tumor biopterin and BH4 tissue levels were assessed by HPLC. The administered DAHP decreases BH4 levels of tumor tissue in HCC. (B) Nitrite/nitrate levels in the tumor tissue was determined by the Greiss reaction. These levels indirectly measure NO content. The administration of DAHP significantly decreased nitrite/nitrate levels in tumor tissue.

Figure 3

DAHP inhibits tumor formation in BALB/c-nu mice. (A) Volume of tumor significantly decreased in DAHP group compared with the saline group. (B) Treatment with DAHP inhibited formation of the tumor.

Figure 3

DAHP inhibits tumor formation in BALB/c-nu mice. (A) Volume of tumor significantly decreased in DAHP group compared with the saline group. (B) Treatment with DAHP inhibited formation of the tumor.

Figure 4

DAHP affects intratumoral expression of angiogenesis marker CD31. CD31 staining score for endothelial cells was notably lower in DAHP groups compared to the saline group, indicating less angiogenic activity, which is consistent with the downregulation of K-ras, GTPCH, p-eNOS and p-Akt.

Figure 4

DAHP affects intratumoral expression of angiogenesis marker CD31. CD31 staining score for endothelial cells was notably lower in DAHP groups compared to the saline group, indicating less angiogenic activity, which is consistent with the downregulation of K-ras, GTPCH, p-eNOS and p-Akt.

Figure 5

DAHP inhibits the expression of K-ras mRNA and GTPCH in tumor tissue. (A) K-ras mRNA in tumor tissue was measured by quantitative real-time RT-PCR. Treatment with DAHP decreased K-ras mRNA of the tumor. (B) Lysates of tumor tissue were fractionated by SDS-PAGE and immunoblotted with monoclonal antibody to GTPCH.

Figure 5

DAHP inhibits the expression of K-ras mRNA and GTPCH in tumor tissue. (A) K-ras mRNA in tumor tissue was measured by quantitative real-time RT-PCR. Treatment with DAHP decreased K-ras mRNA of the tumor. (B) Lysates of tumor tissue were fractionated by SDS-PAGE and immunoblotted with monoclonal antibody to GTPCH.

Figure 6

DAHP inhibits the phosphorylation of eNOS and Akt by downregulation of BH4 synthesis depending on PI3K signaling in tumor tissue. Lysates of tumor tissue were fractionated by SDS-PAGE and immunoblotted with monoclonal antibody to p-eNOS (Ser1177) and p-Akt (Ser473). Band intensity of p-eNOS and p-Akt were quantified on Quantity One 4.6 software. Treatment with DAHP inhibited the expression of p-eNOS and p-Akt in the tumor.

Figure 6

DAHP inhibits the phosphorylation of eNOS and Akt by downregulation of BH4 synthesis depending on PI3K signaling in tumor tissue. Lysates of tumor tissue were fractionated by SDS-PAGE and immunoblotted with monoclonal antibody to p-eNOS (Ser1177) and p-Akt (Ser473). Band intensity of p-eNOS and p-Akt were quantified on Quantity One 4.6 software. Treatment with DAHP inhibited the expression of p-eNOS and p-Akt in the tumor.