Alphastatin downregulates vascular endothelial cells sphingosine kinase activity and suppresses tumor growth in nude mice bearing human gastric cancer xenografts

Abstract

Aim: To investigate whether alphastatin could inhibit human gastric cancer growth and furthermore whether sphingosine kinase (SPK) activity is involved in this process.

Methods: Using migration assay, MTT assay and Matrigel assay, the effect of alphastatin on vascular endothelial cells (ECs) was evaluated in vitro. SPK and endothelial differentiation gene (EDG)-1, -3, -5 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). SPK activity assay was used to evaluate the effect of alphastatin on ECs. Matrigel plug assay in nude mice was used to investigate the effect of alphastatin on angiogenesis in vivo. Female nude mice were subcutaneously implanted with human gastric cancer cells (BGC823) for the tumor xenografts studies. Micro vessel density was analyzed in Factor VIII-stained tumor sections by the immunohistochemical SP method.

Results: In vitro, alphastatin inhibited the migration and tube formation of ECs, but had no effect on proliferation of ECs. RT-PCR analysis demonstrated that ECs expressed SPK and EDG-1, -3, -5 mRNAs. In vivo, alphastatin sufficiently suppressed neovascularization of the tumor in the nude mice. Daily administration of alphastatin produced significant tumor growth suppression. Immunohistochemical studies of tumor tissues revealed decreased micro vessel density in alphastatin-treated animals as compared with controls.

Conclusion: Downregulating ECs SPK activity may be one of the mechanisms that alphastatin inhibits gastric cancer angiogenesis. Alphastatin might be a useful and relatively nontoxic adjuvant therapy in the treatment of gastric cancer.

Figure 1

Alphastatin inhibit HUVECs migration induced by HGF( crystatl violet stain, × 100). All data shown as mean ± SD. ^aP < 0.05; ^bP < 0.01 vs control group.

Figure 2

Alphastatin has no effect on HUVECs proliferation.

Figure 3

Alphastatin inhibit HUVECs tubule formation in response to PMA. All data shown as mean ± SD. ^aP < 0.05 vs control group.

Figure 4

Expression of SPK , EDG-1, EDG-3, EDG-5 in HUVECs.

Figure 5

Alphastatin downregulate HUVECs SPK activity in a dose-dependent manner. HUVECs SPK activity reached a minimum value at 6 h. All data shown as mean ± SD. A: ^aP < 0.05; ^bP < 0.01 vs 0 nmol/L alphastatin; B: ^aP < 0.05 vs 0 h group.

Figure 6

Alphastatin inhibit new capillaries formation on Matrigel plug (original magnification × 100). A: Many newly formed capillaries full of red blood cells (arrow); B: Granuloma formation without significant neovasculature.

Figure 7

Factor-VIII staining revealed decreased blood vessel density in alphastatin-treated group vs PBS group (× 400).

Figure 8

Animals injected with alphastatin exhibited a significantly reduced tumor volume. All data shown as mean ± SD. ^aP < 0.05; ^bP < 0.01 vs PBS group.