Resveratrol suppresses malignant progression of oral squamous cell carcinoma cells by inducing the ZNF750 pathway

Deletion or mutation of zinc nger protein 750 (ZNF750) has been linked to oral squamous cell carcinoma (OSCC), but it is not clear whether ZNF750 is a therapeutic target for OSCC. This study examined whether activation of zinc nger protein 750 (ZNF750) pathway may be involved in the ability of resveratrol to inhibit malignant progression of CAL-27 oral squamous cell carcinoma cells. CAL-27 cells were treated with resveratrol and transfected with plasmids expressing a ZNF750 mimic or ZNF750 inhibitor. Cell proliferation was assessed using the CCK-8 assay and a BrdU ELISA, and cell cycle distribution and apoptosis were examined using ow cytometry. Colony formation was also assessed. Western blotting was used to examine the effects of resveratrol on levels of angiogenin, vascular endothelial growth factor (VEGF), prolyl hydroxylase 2 (PHD2), G protein signal-regulated protein 5 (RGS5), integrin A5 (ITGA5), integrin B1 (ITGB1), CD44 and ZNF750. Quantitative PCR was used to examine effects on mRNA levels of platelet derived growth factor (PDGFB) and tumor vascular marker CD105. Resveratrol down-regulated angiogenin, VEGF, RGS5, CD105, and the cell adhesion molecules ITGA5, ITGB1 and CD44 in CAL-27 cells. Conversely, it up-regulated ZNF750, PHD2 and PDGFB. These changes were associated with reduced proliferation, reduced colony formation and increased apoptosis. ZNF750 silencing partly reversed these effects of resveratrol. The ability of resveratrol to suppress progression of oral squamous cell carcinoma may involve activation of the ZNF750 pathway and modication of the tumor vascular microenvironment.


Introduction
Targeted molecular therapies inhibit oncogenic signaling pathways necessary for progression of malignant cancers (Chiorean and Coveler. 2015); for example, monoclonal antibodies may bind key protein actors, and small-molecule kinase inhibitors may inhibit phosphorylation-mediated activation of key proteins. Various genetic variants have been associated with development of human oral squamous cell carcinoma (OSCC) (Zhong et al. 2018;Choi et al. 2018;Junttila et al. 2013), raising the possibility that the corresponding proteins can be targeted to inhibit their involvement in oncogenic pathways.
Deletion or mutation of zinc nger protein 750 (ZNF750) has been linked to OSCC as well as to malignant phenotype in squamous epithelial cell cultures (Lin et al. 2014). Indeed, ZNF750 appears to be a lineagespeci c tumor suppressor gene in OSCC (Hazawa et al. 2017) as well as a promising biomarker of poor outcomes in patients with the disease (Nambara et al. 2017;Otsuka et al. 2017). ZNF750 may suppress malignant progression of OSCC by regulating the tumor vascular microenvironment (Pan et al. 2018).
The tumor vascular microenvironment contributes to tumorigenesis by providing oxygen, nutrients and soluble growth factors. Hypoxia and limited nutrients in this microenvironment can trigger production of angiogenic cytokines such as VEGF, triggering vascular abnormality and cancer progression (Pan et al. 2018). The tissue oxygen sensing PHD2 is an important mediator of vascular normalization and regulator of RGS5, which is responsible for vessel maturation. PDGFB is responsible for vessel maturation (Goel et al. 2011). CD44, a transmembrane adhesion receptor for hyaluronan and growth factors, contributes to the migration and invasion by tumor cells (Cho et al. 2012). Moreover, ITGB1 and ITGA5 mainly participant in cancer cell adhesion, cell migration and invasion (Zhao et al. 2014;Ohyagi-Hara et al. 2013).
Given the ability of the natural phytoalexin resveratrol to reduce the risk of OSCC as well as other cancers (Valenzano et al. 2006;Uzawa et al., 2005), we wondered whether this compound may interact with the endogenous tumor-suppressing activity of ZNF750 in OSCC cells. Therefore, the current study examined whether the inhibitory effects of resveratrol on OSCC malignant phenotype involve activation of ZNF750.
Cell culture and treatments CAL-27 OSCC cells were provided by the Human Science Research Resources Bank and cultured in DMEM supplemented with 10% fetal bovine serum at 37 ℃ in an environment of saturated humidity and 5% CO 2 . Cells in logarithmic growth phase were used in all experiments. Resveratrol was dissolved in DMSO and added to cells at nal concentrations of 10, 20 or 40 µM. Control cells were given the same volume of DMSO vehicle. Cells were incubated with resveratrol for 48 h. Each experiment was performed in triplicate.

Transfection with plasmids encoding ZNF750 mimic or ZNF750 inhibitor
To test the effects of ZNF750 overexpression and knockdown, CAL-27 cells were transiently transfected with empty vector (#20190112, Santa Cruz Biotechnology, AB, USA) or the same vector encoding the ZNF750 mimic (#20190123) or the ZNF750 inhibitor (#20190126). Cells were transfected using Lipofectamine 3000 (Invitrogen) based on the manufacturer's instructions. ZNF750 overexpression and knockdown were con rmed by Western blot.

Cell viability and proliferation
After CAL-27 cells had been treated with resveratrol, the culture medium was removed completely, and 100 µL of medium containing 10 µL of CCK-8 reagent was added to each well. Plates were incubated for 2 h, then optical density (OD) at 540 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Relative cell viability (%) was calculated as OD experiment / OD control × 100%.
Treated cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and analyzed by BrdU ELISA based on the manufacturer's instructions. OD at 507 nm was measured using a microplate reader (Bio-Rad).

Colony formation
After CAL-27 cells had been treated with resveratrol, numbers of cell colonies were counted under a reversed microscope. For each condition, the numbers of colonies in 10 elds of view were averaged.

Apoptosis
After CAL-27 cells had been treated with resveratrol, cells were harvested and adjusted to a concentration of 1×10 6 cells/mL. The cell suspension (0.5 mL) was stained with 1.25 µL Annexin V-FITC at room temperature for 15 min in the dark, then 10 µL PI was added. Cells were sorted by apoptosis stage using a BD ow cytometer.

Levels of target proteins
After CAL-27 cells had been treated with resveratrol, cells were harvested and total protein was extracted using RIPA lysis buffer. Total protein concentration was estimated using the Bradford method (Thermo Fisher Scienti c). Proteins (50 µg) were fractionated using 10% sodium sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Nonspeci c binding sites were blocked with 5% skim milk for 1.5 h at room temperature on a shaking table. Then blots were incubated overnight at 4 ℃ with rabbit anti-mouse monoclonal antibodies (all diluted 1:1000) against the following proteins: angiogenin, VEGF, PHD2, RGS5, ITGA5, ITGB1, CD44, and ZNF750. Subsequently blots were washed three times with PBS-Tween 20, and blots were incubated for 2 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:4000). Proteins were detected using luminol reagent and peroxide solution (Millipore, Billerica, MA, USA). Densitometry of images was performed using Image J software.
Statistical analysis Data were reported as mean ± SD. Inter-group differences were assessed for signi cance using Welch's t test (comparisons of two groups) or ANOVA (comparisons of three or more groups). P < 0.05 was considered signi cant.

Impact of resveratrol on expression of genes related to vascular normalization and metastasis
As shown in the Fig. 1, after treatment with different concentrations of resveratrol, it could signi cantly inhibited expression of angiogenin and VEGF in a concentration-dependent manner (Fig. 1A). It also down-regulated RGS5, CD105 and MMP3, while up-regulating PHD2, PDGFB and E-cadherin in CAL-27 cells (Fig. 1B-D).

Impact of resveratrol on expression of cell adhesion molecules ITGA5, ITGB1 and CD44
We further assess the effects of resveratrol on the expression levels of cell adhesion molecules in CAL-27 cells. The results from Fig. 2 showed that resveratrol signi cantly down-regulated ITGA5, ITGB1and CD44, which are involved in cell adhesion and cell migration, in a concentration-dependent manner.

Impact of resveratrol on proliferation and apoptosis
Resveratrol signi cantly decreased the viability of CAL-27 cells in a concentration-dependent manner based on the CCK-8 assay (Fig. 3A), which coincided with signi cant up-regulation of apoptosis based on ow cytometry (Fig. 3B). Resveratrol also signi cantly decreased numbers of colonies and BrdU-positive cells in a concentration-dependent manner (Fig. 3C-D).

Impact of resveratrol on ZNF750 expression
In order to assess the mechanism in the inhibitory effect of resveratrol on the growth of CAL-27 cells, we further observe the expression level of ZNF750 in cells after treatment with resveratrol. As shown in Fig. 4, we found that resveratrol signi cantly up-regulated ZNF750 in a concentration-dependent manner.

Regulation of OSCC cell growth by resveratrol via ZNF750 induction
Our experiments above suggested that resveratrol up-regulated the tumor supressor ZNF750 in OSCC cells, leading us to ask whether this activation might help explain the compound's ability to inhibit the malignant phenotype in these cells. We transfected CAL-27 cells with plasmids expressing a ZNF750 mimic or inhibitor and then treated the cells with resveratrol (Fig. 5A-B). Expression of the mimic led to signi cantly lower viability and greater apoptosis than expression of the empty vector, while expression of the inhibitor led to signi cantly higher viability and less apoptosis (Fig. 5C-D). These results suggest that resveratrol inhibits the growth of OSCC cells by activating the ZNF750 pathway.

Discussion
Drugs derived from medicinal herbs show various potent biological functions against multiple cancers (Treasure 2005;Cao et al. 2017). Resveratrol is derived from certain plants and shows anti-in ammatory, anti-oxidative stress, and anti-tumor properties (Rauf et al. 2017;Thaung et al. 2017;Xia et al. 2017;Wu et al. 2010;Jiang et al. 2017;Yousef et al. 2017). It can modify the tumor microenviroment and sensitize cancer cells to chemo-and radiotherapy . It can also inhibit proliferation and induce apoptosis in several tumor cell types (Kim et al. 2017;D'Asti et al. 2016). The present study extends these activities of resveratrol to OSCC cells, and it suggests that the anti-tumor e cacy against this disease involves activation of ZNF750-dependent tumor-suppressive pathways.
Previous work has demonstrated that ZNF750 can suppress the malignant progression of OSCCs by regulating the tumor vascular microenvironment (Goel et al. 2011). We con rmed and extended those ndings by showing that, in OSCC cells, resveratrol down-regulates angiogenin, VEGF, RGS5, CD105, and the cell adhesion molecules ITGA5, ITGB1 and CD44 (D'Asti et al. 2016). At the same time, it up-regulates PHD2 and PDGFB. These results support the idea that resveratrol inhibits malignant progression in OSCC by regulating the tumor vascular microenvironment. Our observation that resveratrol also up-regulates ZNF750 suggests that this endogenous tumor suppressor mediates at least some of the changes in the tumor microenvironment. It is well known that suppressed genes have been enriched for terms related to cell proliferation. Earlier researches reported that ZNF750 is typically mutated or deleted in squamous cell carcinoma (Hazawa et al. 2017;Nambara et al. 2017;Otsuka et al. 2017). The loss of ZNF750 is related to impaired differentiation and failure to fully inhibit the proliferative genetic program, both of which are important markers of tumor (Okuno et al. 2014). Previous study have demonstrated that ZNF750 could suppress the malignant progression of OSCCs by regulating tumor vascular microenvironment (Goel et al. 2011). Thus, this study further observe the role of ZNF750 in the inhibitory effect of resveratrol on the proliferation of OSCC CAL-27 cells. Western blot analysis displayed that resveratrol induced the upregulation of ZNF750 expression. Then OSCC CAL-27 cells were transfected with ZNF750 knockdown partially reversed the effects of resveratrol on OSCC cell growth. This idea is strengthened by our observation that ZNF750 knockdown partially reversed the effects of resveratrol on OSCC cell growth.
Our results justify further work into how resveratrol may induce ZNF750 expression and how this affects downstream signaling and gene expression pathways. This future research may help develop novel therapeutic targets against OSCC and potentially other cancers in which resveratrol shows anti-tumor e cacy.
In summary, resveratrol could regulate the tumor vascular microenvironment to suppress the oral squamous cells carcinoma malignant process through the activation of ZNF750 pathway. These results of the present study provide a reference for clinical treatment of oral squamous cell carcinoma.