International Immunopharmacology 88 (2020) 106905

Contents lists available at ScienceDirect

International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

The therapeutic potential of resveratrol in a mouse model of melanoma lung T

metastasis
Amirhossein Davoodvandia,b, Maryam Darvishc, Sarina Borrand, Majid Nejatie,
Samaneh Mazaherif, Omid Reza Tamtajib, Micheal R. Hambling, Nahid Masoudianh,
Hamed Mirzaeib,
⁎

a
Student Research Committee, Kashan University of Medical Sciences, Kashan, Iran
b
Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran
c
Department of Medical Biotechnology, Faculty of Medicine, Arak University of Medical Science, Arāk, Iran
d
School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
e
Anatomical Sciences Research Center, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran
f
Department of Analytical Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Iran
g
Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, 40 Blossom Street, Boston, MA 02114, USA
h
Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran

ARTICLE INFO ABSTRACT

Keywords: Resveratrol is an anticancer phytochemical polyphenol isolated from a natural origin, without any significant
Resveratrol side effects. Resveratrol was investigated in immunocompetent mice with regards to its possible effect on lung
Melanoma cancer metastasis. Cytotoxicity was assessed in three melanoma cell lines (B16F10, B6, and A375) by admin-
Lung metastases istration of 20 and 40 μM resveratrol. B16F10 cells were transfected with pT-tdTomato vector to express red
Mouse model
fluorescent protein (RFP). RFP-B16F10 cells were injected IV into 3 groups of 20 C57BL/6 mice (ten for tests and
others for survival). The three groups include PBS, no treatment, and resveratrol 40 mg/kg IP (4X/week for
3 weeks). Lung tissues were analyzed by TUNEL assay, Western blot, and immunohistochemistry. The in vitro
growth of all melanoma cell lines was significantly suppressed by 40 μM resveratrol for 3 days. The mean
survival rate of mice was enhanced and the lung tumor growth was inhibited by in vivo IP injection of 40 mg/kg
resveratrol. Increased CXCL10 and IFN-γ levels and decreased angiogenesis and less tumor infiltration by Tregs
were found in the lung tumors. In conclusion, lung metastasis of melanoma was effectively inhibited by re-
sveratrol treatment.

1. Introduction red wine. Resveratrol has been reported to exert anticancer effects by
suppressing angiogenesis and promoting apoptosis, and also acts as an
Melanoma tends to aggressively invade adjoining tissue and spread antioxidant [3,4]. Moreover, it has been suggested to influence cell
to distant organs (e.g. draining lymph nodes, skin or visceral organs metabolism in various ways. Resveratrol suppressed protein kinase B
including liver (14–20%), lung (18–36%), bone (11–17%) and brain and mitogen-activated protein kinase (MAPK) in ovarian cancer cells,
(12–20%). Melanoma is not usually diagnosed in a timely manner [1,2]. thereby down-regulating hypoxia-inducible factor 1α, vascular en-
Lung metastasis, which is the most frequent location in this cancer, is dothelial growth factor (VEGF), also promoted glioma cell apoptosis
sometimes found in patients who were initially diagnosed with non- [5]. Furthermore, it has been shown to inhibit the generation of ca-
metastatic cancer. There is a 10% and 17% risk of relapse at 5 and pillary-like tubes in human umbilical vein endothelial cells, and also
15 years after surgery, respectively (2). The development of novel reduced angiogenesis and slowed tumor growth in a lung cancer model
therapeutic approaches for metastatic melanoma is therefore highly [6]. In addition, it showed a dose-dependent effect against angiogenesis
desirable. in other tumor models [7]. Finally, in vivo and in vitro investigations
Resveratrol is naturally extracted from grapes and is also present in have shown that resveratrol could suppress melanoma growth by

⁎
Corresponding author at: Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical
Sciences, Kashan, Iran.
E-mail address: [email protected] (H. Mirzaei).

https://doi.org/10.1016/j.intimp.2020.106905
Received 1 July 2020; Received in revised form 28 July 2020; Accepted 13 August 2020
Available online 06 September 2020
1567-5769/ © 2020 Elsevier B.V. All rights reserved.
A. Davoodvandi, et al. International Immunopharmacology 88 (2020) 106905

Fig. 1. A schema of PiggyBac vectors that are

used for melanoma cell transfection. The plasmid
backbone is not shown in the figure. tdTomato:
tandem dimer Tomato cDNA; pA: poly(A) sites;
CAG: cytomegalovirus enhancer 1 chicken b-
actin promoter; PB: acceptor site in the PiggyBac
system; neo: neomycin resistance gene; PGKp:
mouse phosphoglycerate kinase promoter;
Transposase: PB transposase gene.

triggering cell apoptosis, and inhibiting angiogenesis and metastasis (Fig. 2). Transgenic B16F10 cells were prepared by seeding 2 × 105
[8,9]. cells per well in triplicate in 6-well plates. 24 h later, transfection with
In the present study, a lung metastasis mouse model was used to pT-neo, pT-tdTomato, and pTrans vectors were performed on these
analyze the anti-melanoma effects of resveratrol. cells. Lipofectamine 2000™ transfection reagent (Invitrogen, Carlsbad,
CA) was used according to the manufacturer’s instructions. Transfection
2. Materials and methods results were analyzed 24, 48 and 72 h later by fluorescence microscopy.
1000 μg/mL of G418 were used for culturing cells over two weeks,
2.1. Reagents 400 μg/mL neomycin (Invitrogen, Carlsbad, CA) was added to RFP
melanoma cells.
Resveratrol and MTT were obtained from Sigma (St. Louis, MO,
USA). The RPMI and fetal bovine serum (FBS) were purchased from 2.4. Cell proliferation assay
GIBCO (Germany) Company.
The groups of the current study were included i. No Treatment
2.2. Plasmid construction group which were not received any therapeutic reagent, ii. PBS group
which were received PBS (Because we solved resveratrol in this agent)
Piggybac vectors, pT-neo, pTrans, and pT-tdTomato, generated by a and iii. Treatment group which were received resveratrol. Cells were
standard cloning process and provided by Dr. Masairo Sato were used to treated with 0.31, 0.62, 1.25, 2.5, 5, 10, 20, 40, and 80 mg/ml re-
transfect the B16F10 cell line, (Fig. 1). In summary, the pBP vectors sveratrol and cultured at 37 °C for 24 h. The MTT (3-(4,5-di-
contained two acceptors of PB with inverted repeats. Neomycin-re- methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was re-
sistant expression units with a CAG promoter-controlled expression unit peatedly (3 times or more) used for evaluation of cell survival after 24 h
of tdTomato (tandem dimer Tomato) cDNA were included in pPB-based of culture. Appropriate dosages were selected to be 20 and 40 μM of
vector of pT-neo (pTpB) and pPB-based vector pT-tdTomato, respec- resveratrol. Cells with a density of 7.5 × 103 cells per well were cul-
tively. Additionally, transposase expression was produced by CAG tured in 96-well plates, 20 and 40 μM of resveratrol was added and
promoter-containing pTrans vectors. incubated at 37 °C for 24 h, the medium was replaced and the cells were
incubated for a further 72 h, when MTT was added to wells for 4 h, and
2.3. Cell culture, cell transfection, and cell selection dimethyl sulfoxide (DMSO) was added. The optical density was assessed
at 570 nm using a multi well plate reader (model 3550; Bio-Rad). The
The B16F10, B6 and A375 cell lines were provided by the Pasteur background absorbance was medium in the absence of cells. Mean
Institute of Iran (https://www.pasteur.fr/en). Cancer cells were cul- values of the experimental triplicate samples were calculated. The
tured at 37 °C and 5% CO2. pT-tdTomato, pT-neo and pTrans were control group without any treatment was assigned as 100% and the
transfected into B16F10, which converted them to RFP-melanoma cells results were reported as percentage viability. Graphpad Prism was
employed to evaluate mean IC50 values +/- standard deviation in all
the 3 aforementioned groups.

2.5. In vivo studies

8-week-old female C57BL/6 mice were obtained from the Pasteur

Institute of Iran. The protocol was approved by the Institutional Animal
Care and Use Committee guidelines (IR.NIMAD.REC.1398.134). The
groups of the current study were included i. No Treatment group which
were not received any therapeutic reagent, ii. PBS group which were
received PBS (because we solved resveratrol in this agent) and iii.
Treatment group which were received resveratrol. All mice were re-
ceived 3 × 105 B16F10 cells and lung metastasis were established in all
of them. Each group (IP injection of PBS, no treatment, and IP injection
Fig. 2. A schema of Piggybac vectors transfection in B16F10 cells. (A) of resveratrol) comprised 20 mice (ten for test group and others for
Untransfected B16F10, (B) Transfected B16F10 with RFP (tdTomato). survival group) and the experiment was repeated twice. B16F10 cells
Bar = 100 μm. (3 × 105) were intravenously injected into each mouse tail vein,

2
A. Davoodvandi, et al. International Immunopharmacology 88 (2020) 106905

thereby establishing lung cancer metastasis. Establishment of lung tu- 1:1000 dilution (catalog no. ab37168; Abcam, Cambridge, MA) was
mors was further confirmed by injection of transfected REF-B16F10. added for another 2 h at the same temperature. The bands were finally
40 mg/kg resveratrol IP injection was begun 10 days later. Resveratrol visualized using modified chemiluminescence.
injection took place on four days each week for three weeks (12 in-
jections). 10 mice in each group were followed up in order to analyze 2.9. TUNEL) assay
their survival rate.
The apoptosis rate was also evaluated using the TUNEL assay. 10%
2.6. Immunohistochemsitry neutral buffered formalin solution was applied for fixation of the lung
tissue which was further embedded in paraffin. According to the
Three weeks later, paraffin sections were prepared from each manufacture’s instruction, the presence of apoptotic cells within the
sample removed from the euthanized mice. Dewaxing and rehydration samples were examined by an in situ cell death detection kit
were performed using xylene and graded alcohol, respectively. 0.3% (Fluorescein; Roche) to detect apoptotic cells. Nicked DNA was treated
(vol/vol) hydrogen peroxide in methanol was applied to inhibit en- with deoxynucleotidyl transferase, digoxigenin nucleotide. Ultimately,
dogenous peroxidase activity, and then the slides were washed in water. 10 fields with the same size were randomly selected from each tissue
Antigen retrieval was conducted by incubating the slides in citrate section for analysis.
buffer with pH 6.0 for 20 min using a steamer. For blocking the non-
specific bindings, 5% (vol/vol) normal serum was employed to incubate 2.10. Statistical analysis
the slides, based on the species in which the secondary antibody was
produced. The following primary antibodies and their dilutions were The data was analyzed using SPSS version 19 (SPSS Inc, Chicago,
applied: anti-CD31, 1:50 dilution (catalog no. ab28364, Abcam; IL). The groups were differentiated by conducting ANOVA, Student t
Cambridge, MA); anti-CD137 polyclonal antibody, 1:600 dilution test, and Log-rank tests. Survival of mice was determined as the time
(catalog no. ab203391, Abcam; Cambridge, MA); polyclonal antibody elapsed from injection of melanoma cells into their blood. P values
anti-FOXp3, 1:100 dilution (catalog no. ab54501; Abcam, Cambridge, below 0.05 were considered significant.
MA); anti-CXCL10 polyclonal (catalog no. ab9938; Abcam, Cambridge,
MA) and polyclonal antibody anti-IFN-γ polyclonal antibody (catalog
3. Results
no. ab216642, Cambridge, MA). The related secondary antibodies,
which were conjugated to horseradish peroxidase, 1:1000 dilution
3.1. Transfection of B16F10 with RFP
(catalog no. ab97200; Abcam, Cambridge, MA), were applied to the
slides, following the incubation of primary antibodies. Bound anti-
B16F10 cells were transfected using the 3 aforementioned vectors
bodies were visualized by peroxidase-based Vectastain Elite ABC Kit
and the efficiency of the process was ascertained by fluorescent mi-
(Vector Laboratories, Peterborough, UK). Tissue sections were stained
croscopy after 3 days, to confirm the transfectant production by the
by DAB (3, 3-diaminobezidine tetrahydrochloride) (Roche Applied
Piggybac system. Ten cell culture fields were chosen at random and the
Science, Mannheim, Germany) substrate solution in a dark chamber at
tdTomato-containing B16F10 cells were counted. The B16F10 cells
25 °C. The process was stopped after 10 min by washing the slides with
showed a 95 to 100% transfection rate (Fig. 2). Additionally, neomycin
water, and the slides were counterstained by hematoxylin. Ethanol and
was used for 2 weeks or more to achieve stable clones of transfected
xylene were later applied to the sections for tissue dehydration and
B16F10 cells.
clearance, respectively. DPX (Shandon, Thermo scientific, USA) was
then used to mount the slides, which were subsequently examined
3.2. Resveratrol inhibits melanoma cell growth
under microscopic magnification using X100 objective. Finally, the
total cell number and the positively stained number per field was es-
B16F10, A375 and B6 cells were assessed using MTT to determine
timated by counting ten randomly chosen fields (12).
the effects of resveratrol at 20 and 40 µM on cell survival. It can be seen
Fig. 3 that the growth of melanoma cells was significantly suppressed
2.7. Enzyme-linked immunosorbent assay
by 40 µM resveratrol (p < 0.001), especially in the B16F10 cell line
which showed higher susceptibility to 40 µM resveratrol in comparison
The mouse blood samples were tested before and after the experi-
ment. The blood samples were transferred to an empty tube in a sterile
environment without anticoagulant. The sample tubes were first kept in
an upright position for 30 min and then centrifuged at 1500 g at 4 °C for
10 min. The ELISA microplates were pre-coated with CXCL10/IFN-γ
specific polyclonal antibody, and sample incubation conducted for 2 h.
Then, samples were treated with anti-CXCL10 and IFN-γ antibody
conjugates. After another 2-hour incubation (at 25 °C) and washing, the
substrate solution was added. Color development was performed at
25 °C for 30 min and the color intensity was read at 450 nm. Based on a
four-parameter logistic curve fit, a standard curve was produced to
provide the assay results.

2.8. Western blotting on mouse tissues

Western blotting for caspase 8 was performed in all the groups for
evaluating the apoptosis rate in fresh lung tissue samples. The proteins
were separated by SDS-PAGE and transferred to membranes. 5% non-
fat dry milk in TBST buffer was used to block the membranes at 25 °C
for 2 h. Anti-caspase 8 antibody (catalog no. 9746; Cell Signaling, USA), Fig. 3. Effect of resveratrol (20 μM and 40 μM) on cell survival in B16F10, B6,
1:1000 dilution was then added to the membranes at 4 °C overnight. and A375 cell lines. (**, p < 0.01, and ***, p < 0.001). Error bars are standard
Subsequently, horseradish peroxidase-conjugated secondary antibody deviation.

3
A. Davoodvandi, et al. International Immunopharmacology 88 (2020) 106905

Fig. 4. Establishment of tumors in mouse lungs and resveratrol administration. Pre- and post-treatment analysis of RFP-B16F10 cells by fluorescence microscopy and
effects of resveratrol on CXCL10 and IFN-γ expression. Lung tumors were found to be established 10 days after injection. (A) Fluorescence micrographs of lung
sections 5 and 10 days after RFP-B16F10 injection (Before treatment). (B) Fluorescence micrographs of lung sections after 14 and 21 days application of resveratrol
(After treatment). ELISA assays of pre- and post-treatment serum levels of (C) CXCL10 and (D) IFN-γ. Error bars are standard deviation. (Magnification, 10×).

with the B6 and A375 cell lines. quantitative analysis and Western blotting. The resveratrol-treated
group and the control group showed significantly different apoptosis
3.3. Homing of tumor in the lungs and treatment with resveratrol rates (assessed by TUNEL staining, p < 0.01, Fig. 6A and 6B) and
amount of tumor vasculature (calculated as mean vessel density using
The establishment of lung tumors at ten days after injection, was anti-CD31 staining, p < 0.01, Fig. 6D and 6F). In addition, the re-
evaluated using RFP-transfected B16F10 cells. Lung tissues were re- sveratrol-treated group showed higher apoptosis compared to the no
moved from the euthanized mice after 49 days of resveratrol treatment. treatment group according to WB for caspase 8, although the PBS group
Tumors were microscopically confirmed to be established in the lungs also had increased caspase 8 (Fig. 6C).
according to Fig. 4A and 4B. CXCL10 and IFN-γ were shown to be
systemically expressed in resveratrol-treated mice, using ELISA assays 3.6. Effects of resveratrol on immune cells
on mice serum samples. Study findings revealed that the levels of IFN-γ
and CXCl10 in the resveratrol-treated group were not significantly A comparison was made between the resveratrol groups, no treat-
different compared to those of control (Fig. 4C and 4D). ment group and PBS group in respect of the number of Tregs and ac-
tivated T cells as shown in Fig. 7. Resveratrol-treated group showed a
3.4. Antitumor effects of resveratrol significantly lower content of Tregs and a significantly higher number
of activated T cells compared to the other groups (**, p < 0.01) (see
Sixty C57BL/6 mice were equally distributed into three groups that Fig. 8).
received B16F10 cells (3 × 105) through tail vein injection. 10 days
later, resveratrol treatment was begun via intraperitoneal injection for 3.7. Effect of resveratrol treatment on CXCLl10 and IFN-γ expression
3 weeks, 4 times per week. Then, half the mice were euthanized for
tissue analysis, and the other half were followed for survival. The data Resveratrol-treated, PBS and no treatment groups were evaluated
in Fig. 5 show that the number of metastatic colonies in the lungs were for their CXCL10 and interferon-γ expression levels as shown in Fig. 7.
significantly lower (p < 0.05, Fig. 5B)) and the survival rate was While there was no significant difference between the no treatment
significantly longer (p < 0.01, Fig. 5C)) in mice injected with B16F10 group and the PBS control, the tumor microenvironment of the re-
cells and treated with resveratrol. Over 70% of the resveratrol-treated sveratrol group showed significantly more CXCL10 and IFN-γ
mice survived for longer than 1 month, whereas all the mice in the (p < 0.05) compared to the no treatment group.
control group had died within this time. The survival rate of the re-
sveratrol-treated mice at the 50th day was 30%. 4. Discussion

3.5. Effects of resveratrol on apoptosis, and angiogenesis Although many studies have attempted elevate the survival rate of
subjects afflicted with metastatic melanoma, not many have been suc-
Fig. 6 shows the results of the TUNEL assay, immunohistochemistry, cessful, and advanced melanoma still has a relatively poor prognosis.

4
A. Davoodvandi, et al. International Immunopharmacology 88 (2020) 106905

Fig. 5. In vivo studies. Mice received RFP-

B16F10 cells (3 × 105) through tail vein injec-
tion. They were then randomized into three dif-
ferent groups (no treatment control, PBS control,
and resveratrol treatment), each comprising 20
mice. Treatment groups received 40 mg/kg re-
sveratrol administered 4 times per week for
3 weeks. After 3 weeks, ten mice in each group
were euthanized for laboratory tests and the re-
mainder (n = 10) were followed up for survival.
The in vivo study was conducted twice for con-
firmation. (A) Lung tissue sample removed from
euthanized mice after 21 days of treatment; and
(B) number of lung metastatic colonies per 1 cm3
(*, p < 0.05). Image C represents the long-term
survival (** p < 0.01). Error bars are standard
deviation.

Most pharmaceutical herbal compounds that have been historically and the EMT (epithelial-to-mesenchymal transition) at cellular and
used in traditional medicine are commonly regarded as non-toxic, but molecular levels. In addition, some of the targets of resveratrol are in-
some reports have contradicted this notion for particular preparations. volved in the development and progression of melanoma, i.e., p21,
Resveratrol is a natural phytochemical polyphenol isolated from grapes, MAPK, Bcl2 and some microRNAs. Herein, we explored the effect of
which has been used for some therapeutic purposes, particularly as an resveratrol on metastatic melanoma in an immunocompetent mouse
anti-cancer therapy (e.g. in melanoma). Resveratrol promotes apoptosis model. The experimental results indicated that the apoptosis rate of
and stimulates immune responses, while also inhibiting angiogenesis melanoma cells was appreciably elevated by resveratrol treatment,

Fig. 6. TUNEL assay, immunochemistry, and WB were conducted to assess the apoptosis and angiogenesis in the lungs of B16F10 injected mice. Injections of 40 mg/
kg resveratrol IP commenced 10 days after tumor injection. Three weeks of four times per week resveratrol therapy was administered to the mice. The lung tissues
were embedded in paraffin and then sectioned. (A) The lung slides were stained with TUNEL, and visualized via Axio vision 4.0 fluorescence microscopy. (B) Ten
areas were randomly chosen and the positive cells were counted to produce apoptotic indices (**, p < 0.01). (C) 21 days after treatment the lungs were removed and
WB for caspase 8 was performed. (D) The endothelial cells were stained by anti-CD31 polyclonal antibody. (E) The number of blood vessels in 10 random fields of the
stained tissue were calculated to estimate the total number of blood vessels (**, p < 0.01). Error bars are standard deviation. (Magnification, 100×).

5
A. Davoodvandi, et al. International Immunopharmacology 88 (2020) 106905

Fig. 7. Resveratrol treatment on activated T cells and regulatory T cells. 40 mg/kg resveratrol was injected IP for 3 weeks of 4X per week, starting 10 days after tumor
injection. (A) CD137 as marker of activated T cells and (B) the number of Activated T cells (C) FOXP3 as markers of Tregs, in sectioned lung tissues, were stained by
polyclonal antibodies and (D) the number of Tregs were quantitatively analyzed via counting the cells in ten random fields of CD137/FOXP3-stained tissue sections
(**, p < 0.01). Error bars are standard deviation. (Magnification, 100×).

which was consistent with previous studies in this area. mutations or else are viral proteins [16]. Moreover, cancer cell pro-
Mechanistically, this phenomenon is due to triggering caspase 8 ex- liferation can be directly inhibited by IFN-γ, either through induction of
pression, as assessed by Western blotting. apoptosis or by p21-and-p27-mediated cell cycle arrest [17,18]. The
Wu et al., reported that the resveratrol-induced apoptotic activity of anti-angiogenesis activity of IFN-γ is considered an indirect anticancer
melanoma cells was mediated by modulation of p53 protein and cas- feature, by which formation of new blood vessels are both prevented
pases [10]. Resveratrol-induced apoptosis in A375 and SK-MEL-31 and reversed, via reducing the availability of non-transformed en-
human melanoma cells, was revealed by Western blot analysis to be dothelial cells in the tumor micro-environment [19–21].
associated with over-expression of B-cell lymphoma 2 (Bcl-2), and Bcl- In other hand, several reports have showed that increased serum
2-associated X protein (BAX) supposedly through the p53 pathway and levels of various cytokines are associated with systemic adverse effects
activation of caspase 3 and 9 [10]. in patients [22,23]. Therefore, it seems that employing resveratrol to
In addition, tumor development can be inhibited by the anti-an- the tumor sites without significant increase in plasma cytokine level
giogenesis activity of resveratrol as shown by several studies [11]. In would prevent undesirable systemic adverse effect. According to our
the case of our study on metastatic lung melanoma, the results de- results, resveratrol is able to restrict the anti-cancer activity of itself to
monstrated that angiogenesis and blood vessel density in the tumor tumor sites without evidence of systemic adverse effects. It seems that
tissue was reduced after resveratrol treatment. more studies for confirming these results are needed.
Melanoma tumors (in common with other solid tumor types) sti- Jeong et al, reported that HS-1793 (a resveratrol analogue) led to a
mulate angiogenesis leading to tumor progression and metastasis [12]. significant increase in number of IFN-γ generating splenocytes, and a
Lee et al, explored the effects of monotherapy with resveratrol and co- corresponding switch in the tumor infiltrating cells from CD206+
administration of resveratrol plus 5-FU on melanoma tumor progression macrophages to CD68+ cells with higher IFN-γ levels [24]. They in-
in a B16 murine model [8]. The combined application of 5-FU plus vestigated the physiological and morphological features of the macro-
resveratrol proved to be more effective than either monotherapy in phages in the tumor, since TAM (tumor associated macrophages) are
terms of suppressing cell proliferation, tumor growth and angiogenesis. known to possess a M2 phenotype. M2 macrophages were developed
Mechanistic investigations revealed that the tumor inhibition, was as- from human THP-1 monocytes through stimulation by PMA (phorbol-
sociated with dysregulation of five factors, namely, VASP (vasodilator- 12-myristate-13-acetate) and then polarized by Th2 cytokines (IL-4/IL-
stimulated phosphoprotein), VEGF, AMPK, cyclooxygenase-2, and va- 13). These cells were CD204(high), CD206(high), IL-6(high), TGF-
sodilator-stimulated phosphoprotein. The findings of the present study β(high), IL-12(low), IL-10(low), MMP-9(high) and VEGF(high). How-
revealed that IFN- γ and CXCL10 were up-regulated by administration ever, once these TAMs were exposed to IFN-γ, the morphological fea-
of resveratrol in melanoma tumors. CTLs and NK cells are regarded as tures and cellular markers shifted to those of a M1 phenotype, with
key antitumor effector cells of the immune system, and are activated by higher levels of immunostimulatory and pro-inflammatory cytokines.
IFN-γ [13]. IFN-γ can also lead to over-expression of class I-A MHC These findings suggested the mechanism by which HS-1793 resveratrol
(major histocompatibility complex) and tumor antigen-presenting analogue could suppress tumor progression. In conclusion, HS-1793
MHC, which, in turn, boosts tumor cell immunogenicity [14,15]. The therapy switched the M2, immunosuppressive, tumor progression
antigens expressed in tumor cells are relatively different compared to phenotype to the M1 anti-tumor phenotype, by stimulating IFN-γ se-
those found in normal cells, and some neo-antigens arise from gene cretion [24].

6
A. Davoodvandi, et al. International Immunopharmacology 88 (2020) 106905

Fig. 8. Resveratrol treatment on CXCL10 and IFN-γ expression. 40 mg/kg of resveratrol IP injection began 10 days after tumor cell injection and lasted for 3 weeks, 4
times per week. The staining of mouse lung sections was performed using rat specific polyclonal antibody for (A) CXCL10 and (D) IFN-γ. (B) CXCL10 and (E) IFN-γ
were quantitatively analyzed via counting the cell numbers in ten random fields in stained tissue sections (*, p < 0.05). Representative Western blotting is shown for
(C) CXCL10 and (F) IFN-γ. Error bars are standard deviation. (Magnification, 100×).

CXCL10 is a member of the CXCR3 receptor-binding CXC chemokine vivo. In summary, resveratrol inhibited the CD4+ CD25+ cell popu-
family, and has various biological activities including regulation of lation in the tumor tissue and could be useful in vaccination-based
apoptosis, chemotaxis, cell growth, along with angiogenesis. Moreover, cancer therapy [26].
CXCL10 is involved in various human disorders involving the immune In conclusion the present study indicated that activated T cells were
system, infections, chronic inflammation and tumor growth and mi- increased and Tregs were decreased by resveratrol treatment in the
gration. This CXC is an major biomarker for screening the prognosis of mouse metastatic lung melanoma model. Moreover, resveratrol pro-
these diseases [25]. The current study has revealed for the first time duced a higher survival rate in the mice, and could be added to clinical
that resveratrol induces a significant increase in CXCL10 level in a therapeutic regimens without any additional side effects or toxicity.
cancer tumor. Furthermore, it is likely that the existence of multiple
CXCL10 receptors is the cause of the disparate and even opposing ef- Declaration of Competing Interest
fects of CXCL10 on tumor progression (e.g. the growth-inhibitory effect
of CXCR3-B versus the tumor proliferative effect of CXCR3-A [25]). MRH declares the following potential conflicts of interest. Scientific
The present study demonstrated that activated T cells were appre- Advisory Boards: Transdermal Cap Inc, Cleveland, OH; BeWell Global
ciably increased after resveratrol treatment, while tregswere decreased. Inc, Wan Chai, Hong Kong; Hologenix Inc. Santa Monica, CA;
The ability of the immune system to modulate tumor growth is highly LumiThera Inc, Poulsbo, WA; Vielight, Toronto, Canada; Bright
dependent on the Treg-T cell balance. Photomedicine, Sao Paulo, Brazil; Quantum Dynamics LLC, Cambridge,
Yang et al., injected EG7 tumor cells into a C57BL/6 mouse model to MA; Global Photon Inc, Bee Cave, TX; Medical Coherence, Boston MA;
investigate the fluctuations in the levels of Treg cells and im- NeuroThera, Newark DE; JOOVV Inc, Minneapolis-St. Paul MN; AIRx
munomodulatory cytokines [26]. Administration of resveratrol dose- Medical, Pleasanton CA; FIR Industries, Inc. Ramsey, NJ; UVLRx
dependently suppressed the CD4+ CD25+ cell (Treg) population Therapeutics, Oldsmar, FL; Ultralux UV Inc, Lansing MI; Illumiheal &
among CD4+ cells ex vivo. Also intracellular FACS analysis revealed Petthera, Shoreline, WA; MB Lasertherapy, Houston, TX; ARRC LED,
that resveratrol induced a significant decrease in FoxP3+ expressing San Clemente, CA; Varuna Biomedical Corp. Incline Village, NV; Niraxx
cells among the CD4+ CD25+ population ex vivo. IP injection of Light Therapeutics, Inc, Boston, MA. Consulting; Lexington Int, Boca
40 mg/kg resveratrol led to down-modulation of the im- Raton, FL; USHIO Corp, Japan; Merck KGaA, Darmstadt, Germany;
munosuppressive cytokine TGF-beta, and inhibition of the CD4+ Philips Electronics Nederland B.V. Eindhoven, Netherlands; Johnson &
CD25+ cell population in the spleens of tumor-bearing mice. More- Johnson Inc, Philadelphia, PA; Sanofi-Aventis Deutschland GmbH,
over, the immunostimulatory effect of resveratrol was associated with Frankfurt am Main, Germany. Stockholdings: Global Photon Inc, Bee
upregulation of IFN-gamma expression in CD8+ T cells ex vivo and in Cave, TX; Mitonix, Newark, DE. Other authors declare no conflict of

7
A. Davoodvandi, et al. International Immunopharmacology 88 (2020) 106905

interest. [6] Y. Kimura, H. Okuda, Resveratrol isolated from Polygonum cuspidatum root pre-
vents tumor growth and metastasis to lung and tumor-induced neovascularization
in Lewis lung carcinoma-bearing mice, J. Nutr. 131 (6) (2001) 1844–1849.
Acknowledgements [7] E. Brakenhielm, R. Cao, Y. Cao, Suppression of angiogenesis, tumor growth, and
wound healing by resveratrol, a natural compound in red wine and grapes, FASEB
This study was supported by National Insititute for Medical J.: Official Publ. Federation Am. Soc. Exp. Biol. 15 (10) (2001) 1798–1800.
[8] S.H. Lee, B.S. Koo, S.Y. Park, Y.M. Kim, Anti-angiogenic effects of resveratrol in
Research Development, NIMAD. combination with 5-fluorouracil on B16 murine melanoma cells, Mol. Med. Rep. 12
(2) (2015) 2777–2783.
Funding [9] S. Bhattacharya, S.R. Darjatmoko, A.S. Polans, Resveratrol modulates the malignant
properties of cutaneous melanoma through changes in the activation and attenua-
tion of the antiapoptotic protooncogenic protein Akt/PKB, Melanoma Res. 21 (3)
HM was supported by National Institute for Medical Research (2011) 180–187.
Development, NIMAD (Grant No.982491) [10] Z. Wu, B. Liu, E C, J. Liu, Q. Zhang, J. Liu, N. Chen, R. Chen, R. Zhu, Resveratrol
inhibits the proliferation of human melanoma cells by inducing G1/S cell cycle
arrest and apoptosis, Mol. Med. Rep. 11 (1) (2015) 400–404.
Availability of data and material [11] M.H. Pourhanifeh, K. Abbaszadeh-Goudarzi, M. Goodarzi, S.G.M. Piccirillo,
A. Shafiee, S. Hajighadimi, S. Moradizarmehri, Z. Asemi, H. Mirzaei, Resveratrol: A
The primary data for this study is available from the authors on new potential therapeutic agent for melanoma? Curr. Med. Chem. (2019).
[12] G.H. Mahabeleshwar, T.V. Byzova, Angiogenesis in melanoma, Semin. Oncol. 34 (6)
direct request. (2007) 555–565.
[13] H.A. Young, K.J. Hardy, Role of interferon-gamma in immune cell regulation, J.
Author contributions Leukoc. Biol. 58 (4) (1995) 373–381.
[14] D. Street, A.M. Kaufmann, A. Vaughan, S.G. Fisher, M. Hunter, C. Schreckenberger,
R.K. Potkul, L. Gissmann, L. Qiao, Interferon-gamma enhances susceptibility of
HM, involved in conception, design, statistical analysis and drafting cervical cancer cells to lysis by tumor-specific cytotoxic T cells, Gynecol. Oncol. 65
of the manuscript. AD, MD, SB, MN, SM, ORT, and MRH contributed in (2) (1997) 265–272.
[15] K. Cornetta, D. Berebitsky, M. Behnia, C. Traycoff, E.F. Srour, G.W. Sledge, A ret-
conception, data collection and manuscript drafting. roviral vector expressing human interferon gamma upregulates MHC antigen ex-
pression in human breast cancer and leukemia cell lines, Cancer Gene Ther. 1 (2)
Ethics approval and consent to participate (1994) 91–98.
[16] N. Vigneron, Human tumor antigens and cancer immunotherapy, Biomed. Res. Int.
2015 (2015) 948501.
This study was considered exempt by the NIMAD Institutional [17] B.L. Harvat, P. Seth, A.M. Jetten, The role of p27Kip1 in gamma interferon-medi-
Review Board. ated growth arrest of mammary epithelial cells and related defects in mammary
carcinoma cells, Oncogene 14 (17) (1997) 2111–2122.
[18] Y.E. Chin, M. Kitagawa, W.C. Su, Z.H. You, Y. Iwamoto, X.Y. Fu, Cell growth arrest
Consent for publication and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by
STAT1, Science (New York, NY) 272 (5262) (1996) 719–722.
Not applicable. [19] Z. Qin, J. Schwartzkopff, F. Pradera, T. Kammertoens, B. Seliger, H. Pircher,
T. Blankenstein, A critical requirement of interferon gamma-mediated angiostasis
for tumor rejection by CD8+ T cells, Cancer Res. 63 (14) (2003) 4095–4100.
References [20] S. Ibe, Z. Qin, T. Schuler, S. Preiss, T. Blankenstein, Tumor rejection by disturbing
tumor stroma cell interactions, J. Exp. Med. 194 (11) (2001) 1549–1559.
[21] C.M. Coughlin, K.E. Salhany, M.S. Gee, D.C. LaTemple, S. Kotenko, X. Ma, G. Gri,
[1] H. Mirzaei, A. Sahebkar, A. Avan, M.R. Jaafari, R. Salehi, H. Salehi, H. Baharvand, M. Wysocka, J.E. Kim, L. Liu, et al., Tumor cell responses to IFNgamma affect tu-
A. Rezaei, J. Hadjati, J.M. Pawelek, et al., Application of mesenchymal stem cells in morigenicity and response to IL-12 therapy and antiangiogenesis, Immunity 9 (1)
melanoma: a potential therapeutic strategy for delivery of targeted agents, Curr. (1998) 25–34.
Med. Chem. 23 (5) (2016) 455–463. [22] B.E. Lippitz, R.A. Harris, Cytokine patterns in cancer patients: A review of the
[2] R. Mardani, M.R. Hamblin, M. Taghizadeh, H.R. Banafshe, M. Nejati, M. Mokhtari, correlation between interleukin 6 and prognosis, Oncoimmunology 5 (5) (2016)
S. Borran, A. Davoodvandi, H. Khan, M.R. Jaafari, et al., Nanomicellar-curcumin e1093722.
exerts its therapeutic effects via affecting angiogenesis, apoptosis, and T cells in a [23] R.J. Dunlop, C.W. Campbell, Cytokines and advanced cancer, J. Pain Symptom
mouse model of melanoma lung metastasis, Pathol. Res. Pract. 216 (9) (2020) Manage. 20 (3) (2000) 214–232.
153082. [24] S.K. Jeong, K. Yang, Y.S. Park, Y.J. Choi, S.J. Oh, C.W. Lee, K.Y. Lee, M.H. Jeong,
[3] L. Belguendouz, L. Fremont, A. Linard, Resveratrol inhibits metal ion-dependent W.S. Jo, Interferon gamma induced by resveratrol analog, HS-1793, reverses the
and independent peroxidation of porcine low-density lipoproteins, Biochem. properties of tumor associated macrophages, Int. Immunopharmacol. 22 (2) (2014)
Pharmacol. 53 (9) (1997) 1347–1355. 303–310.
[4] S. Garvin, K. Ollinger, C. Dabrosin, Resveratrol induces apoptosis and inhibits an- [25] M. Liu, S. Guo, J.K. Stiles, The emerging role of CXCL10 in cancer (Review), Oncol.
giogenesis in human breast cancer xenografts in vivo, Cancer Lett. 231 (1) (2006) Lett. 2 (4) (2011) 583–589.
113–122. [26] Y. Yang, J.H. Paik, D. Cho, J.A. Cho, C.W. Kim, Resveratrol induces the suppression
[5] Z. Cao, J. Fang, C. Xia, X. Shi, B.H. Jiang, trans-3,4,5'-Trihydroxystibene inhibits of tumor-derived CD4+CD25+ regulatory T cells, Int. Immunopharmacol. 8 (4)
hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression (2008) 542–547.
in human ovarian cancer cells, Clin. Cancer Res.: An Official J. Am. Assoc. Cancer
Res. 10 (15) (2004) 5253–5263.