Abstract: 【Abstract】 Objective To explore intrinsic mechanisms underlying the inhibitory effect of resveratrol on neovascularization in cutaneous squamous cell carcinoma from the perspective of ubiquitin/ubiquitin-like protein modification balance. Methods The human cutaneous squamous cell carcinoma cell line A431 was used as the research object. Cultured A431 cells at exponential growth phase were divided into 3 groups （control group, 50 μmol/L resveratrol group, and 100 μmol/L resveratrol group） to be cultured with mediums containing 0, 50, and 100 μmol/L resveratrol, respectively. Cell proliferation activity was assessed by the 3-（4,5）-dimethylthiazol （-z-y1）-2,5-di-phenytetrazoliumromide （MTT） assay after 48-hour culture; the vasculogenic mimicry formation assay was performed to evaluate the vasculogenic mimicry formation ability of A431 cells after 12-hour treatment with resveratrol; Western blot analysis was conducted to detect the relative protein expression levels of ubiquitin, small ubiquitin-related modifier-1 （SUMO1）, hypoxia-inducible factor 1α （HIF-1α）, and vascular endothelial growth factor receptor （VEGFR） in different groups after 48-hour treatment with resveratrol. Then, 24 8-week-old BALB/c male thymectomized mice were randomly and equally divided into 3 groups to be subcutaneously inoculated with A431 cells in the inguinal region, followed by intraperitoneal injections of 1 mg/kg or 2 mg/kg resveratrol （1 mg/kg or 2 mg/kg resveratrol group）, or the same volume of physiological sodium chloride solutions （control group）; the intraperitoneal injections were done once every 3 days in all groups; all the above mice were sacrificed on the 21st day, and the tumors were resected and weighed. Immunohistochemistry assay was performed to determine the CD31 expression in tumor tissues. One-way analysis of variance was used for comparisons among multiple groups, and least significant difference （LSD）-t test was used for multiple comparisons. Results The proliferation rate of A431 cells significantly differed among the control group, 50 μmol/L resveratrol group, and 100 μmol/L resveratrol group （F = 17.75, P = 0.017）, and was significantly lower in the 50 μmol/L resveratrol group （66.53% ± 5.09%） and the 100 μmol/L resveratrol group （35.88% ± 4.28%） than in the control group （100%, LSD-t = 21.17, 29.04, P = 0.011, 0.004, respectively）; the total length of vessel wall-like structures formed by A431 cells significantly differed among the 3 groups （F = 21.37, P = 0.004）, and was significantly lower in the 50 μmol/L resveratrol group （102.73 ± 11.36 μm） and the 100 μmol/L resveratrol group （37.83 ± 4.19 μm） than in the control group （185.26 ± 8.02 μm, both P < 0.05）; the relative protein expression levels of ubiquitin, SUMO1, HIF-1α, and VEGFR also significantly differed among the 3 groups, the ubiquitin protein expression was significantly higher in the 50 μmol/L resveratrol group （2.09 ± 0.13） and the 100 μmol/L resveratrol group （3.53 ± 0.16） than in the control group （0.68 ± 0.11, both P < 0.05）, while the protein expression of SUMO1, HIF-1α, and VEGFR was significantly lower in the 50 μmol/L resveratrol group （1.87 ± 0.13, 0.81 ± 0.06, 0.73 ± 0.09, respectively） and the 100 μmol/L resveratrol group （1.02 ± 0.11, 0.45 ± 0.06, 0.39 ± 0.05, respectively） than in the control group （3.10 ± 0.11, 0.97 ± 0.08, 0.98 ± 0.07, respectively, all P < 0.05）. In the mice experiment, the weight of subcutaneous tumors and the proportion of CD31-positive cells in tumor tissues significantly differed among the control group, 1 mg/kg resveratrol group, and 2 mg/kg resveratrol group （weight: 3.29 ± 0.57 g, 2.91 ± 0.49 g, 2.55 ± 0.52 g; proportion: 76.24% ± 5.51%, 39.45% ± 5.48%, 12.07% ± 3.54%; F = 14.33, 15.34, P = 0.019, 0.021, respectively）, and were significantly lower in the 1 mg/kg resveratrol group and 2 mg/kg resveratrol group than in the control group （all P < 0.05）. Conclusion Resveratrol could inhibit tumor growth and neovascularization in tumor tissues, which were possibly associated with the inhibitory effect of resveratrol on neovascularization in cutaneous squamous cell carcinoma by suppressing the SUMOylation of HIF-1α protein via ubiquitin/ubiquitin-like protein modification pathways.

Key words: Neoplasms, squamous cell, Cell proliferation, Ubiquitin, SUMO-1 protein, Hypoxia-inducible factor 1, alpha subunit, Mice, inbred BALB C, Resveratrol, A431 cell line, Angiogenesis inhibition