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. 2021 May 28:14:3487-3501.
doi: 10.2147/OTT.S308371. eCollection 2021.

Acetone Extract of Cornus officinalis Leaves Exerts Anti-Melanoma Effects via Inhibiting STAT3 Signaling

Ruiqi Xu  1   2 , Mengnan Zeng  1   2 , Yuanyuan Wu  1   2 , Shengchao Wang  1   2 , Beibei Zhang  1   2 , Jingke Zhang  1   2 , Yuxuan Kan  1   2 , Benke Li  1   2 , Bing Cao  1   2 , Xiaoke Zheng  1   2 , Weisheng Feng  1   2
Affiliations

Acetone Extract of Cornus officinalis Leaves Exerts Anti-Melanoma Effects via Inhibiting STAT3 Signaling

Ruiqi Xu et al. Onco Targets Ther. .

Abstract

Purpose: This research aims to investigate the intervention and mechanism of 50% acetone extract of C. officinalis leaves (SZYY) on melanoma xenografts.

Patients and methods: Tumor size and cardiac function were measured via ultrasound. The accumulation of 2-deoxy-D-glucose (2-DG) in tumor tissue was examined with near-infrared in vivo imaging. Flow cytometry was performed to assess apoptosis and reactive oxygen species (ROS) levels in tumor and immune cells in spleen. The levels of inflammatory cytokines in serum were detected by cytometric bead array. The expression of proliferation-, apoptosis-, and angiogenesis-related proteins in tumor cells was measured to evaluate the underlying mechanisms. Subsequently, the effects of four compounds separated from SZYY on the proliferation and migration of A375 cells and STAT3 signaling were examined. The peak identification and contents of the four components were performed via high-performance liquid chromatography (HPLC). Finally, we evaluated the inhibitory effects of STAT3 overexpression on the cytotoxic activity of four constituents in A375 cells.

Results: SZYY inhibited the growth and glycolysis of melanoma xenograft in mice, improved cardiac function, increased the percentages of macrophages, neutrophils, and lymphocytes in spleen, reduced the levels of IL-6, IL-17A, TNF-α, and IFN-γ in serum, promoted apoptosis and oxidative stress in tumor tissues, and inhibited STAT3 phosphorylation and expression of angiogenic factors. Chemical analysis showed that SZYY is rich in loganin, rutin, triohimas C, and triohimas D, which all could restrain the proliferation and migration of A375 cells and inhibit the phosphorylation and nuclear translocation of STAT3. Moreover, STAT3 overexpression could diminish the cytotoxic activity of four compounds on A375 cells.

Conclusion: SZYY could exert anti-melanoma effects via inhibiting STAT3 signaling to induce apoptosis and inhibit tumor angiogenesis. Its active ingredients might be loganin, rutin, triohimas C, and triohimas D.

Keywords: Cornus officinalis; STAT3; angiogenesis; apoptosis; melanoma.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
SZYY inhibited A375 melanoma xenograft growth and improved cardiac function. (A) Relative tumor volume = tumor volume/tumor volume measured the first time. Data obtained by ultrasound. (B and C) Photos of tumor-bearing mice and tumors dissected from mice. The subcutaneous neoplasms are shown as the arrows and circles. (D) Effects on cardiac function (LVEF and LVFS) were detected via ultrasound. (E) Effects on Ki-67 in tumor tissue were detected via Western blot. (F) Representative images showed the fluorescent signal of 2-DG probe in mice at 14 h and quantitative result. X ± SD. n = 3 mice per group. **p < 0.01 vs model group.
Figure 2
Figure 2
SZYY promoted apoptosis and ROS production in the tumor tissues. (A) The percentage of apoptosis in tumor tissues. (B) The production of ROS in tumor tissues. (C) Effects on Mcl-1, Bcl-xL, Bcl-2 and cleaved caspase-3 in tumor tissues were detected via Western blot. X ± SD. n = 3 mice per group. **p < 0.01 vs model group. Con: the normal control group; M the model group; Y the positive control group (docetaxel, 20 mg/kg); Low: the low dose group (SZYY, 200 mg/kg); High: the high dose group (SZYY, 400 mg/kg).
Figure 3
Figure 3
SZYY enhanced immune function of the spleen and improve the immune microenvironment of tumor-bearing mice. (A) Flow cytometry strategy. Relative counts of leukocyte subgroups were quantified by gating on Ly-6G+ for neutrophils, F4/80+ for macrophages and CD4+/CD8+ for lymphocytes. (B) The representative peak graphs of IL-6, TNF-α, IFN-γ and IL-17A were obtained by FACS software. (C) Quantitative analysis of neutrophils (Ly-6G+), macrophages (F4/80+) and lymphocytes (CD4+/CD8+) in spleen. (D) Quantitative analysis of IL-6, TNF-α, IFN-γ and IL-17A in serum. X ± SD. n = 3 mice per group. **p < 0.01 vs model group.
Figure 4
Figure 4
SZYY restrained phosphorylation/activation of STAT3 and the expression of angiogenic factors in melanoma xenografts. (A) Expression of STAT3 (green), p-STAT3 (red) in melanoma slices was evaluated by immunofluorescent double staining. Scale bar =50μm. (B) Expression of CD31 (green), Survivin (red) in melanoma slices was evaluated by Immunofluorescent double staining. Nuclei, blue. Scale bar =100μm. (C) The activation of STAT3 was measured by Western blot. (D) Expression of VEGF-A, BFGF and HGF in melanoma xenografts was detected by Immunohistochemistry (×40). Scale bar =50μm. X ± SD; n = 3 mice per group; **p < 0.01 vs model group.
Figure 5
Figure 5
Four compounds reduced the viability of A375 cells and inhibited their migration ability. (A) Effects of four compounds on A375 cells proliferation. (B and C) A375 cells migration ability detected via High Content Screening System. X ± SD. n = 6 per group. *p < 0.05, **p < 0.01 vs control group.
Figure 6
Figure 6
High Performance Liquid Chromatography (HPLC) chromatograms of standard and SZYY. (A–D) The chromatograms of loganin, triohimas C, triohimas D, rutin, respectively. (E) Chromatogram of mixed solution of 4 standard substances. (F) Chromatogram of the typical sample of SZYY. Peak identification: 1. Loganin, 2. Triohimas C, 3. Triohimas D, 4. Rutin.
Figure 7
Figure 7
Four compounds restrained phosphorylation/activation and nuclear translocation of STAT3. (A) Phosphorylation of STAT3 in A375 cells was detected by In-cell Western. Histogram illustrated the relative levels of p-STAT3. (B) Representative fluorescent microscopy images of STAT3 nuclear translocation in A375 cells. Scale bar=50 μm. Histogram illustrated nuclear translocation ratio of STAT3 protein=Mean fluorescence intensity in the nucleus/Cells mean fluorescence intensity. (C) JAK2, p-JAK2, Src and p-Src proteins expression levels in A375 cells were examined by In-cell Western. Histogram illustrated the relative levels of p-JAK2. The fluorescence intensity was normalized to GAPDH. X ± SD. n = 3 per group. **p < 0.01 vs control group.
Figure 8
Figure 8
The overexpression of STAT3 weakened the inhibitory effect of SZYY on cells. (A) Protein expression in A375 cells transiently transfected with either Empty vector or h-STAT3 were examined by In-cell Western. Histogram illustrated the relative levels of STAT3 and p-STAT3. The fluorescence intensity was normalized to GAPDH. (B) Effects of STAT3 overexpression on A375 cells proliferation inhibited by SZYY. X ± SD. n = 3 per group. ##p < 0.01 vs Empty vector control group. **p < 0.01 vs STAT3C control group. &p < 0.05, &&p < 0.01 vs cells transfected with the empty vector in each group.
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