Background
The incidence of thyroid cancer, the most common endocrine malignancy, rapidly increased in recent years [
1,
2]. Thyroid cancer is histologically classified of papillary thyroid cancer (PTC, 80–85%), follicular thyroid cancer (FTC, 10–15%), medullary thyroid cancer (MTC, 3–5%) and anaplastic thyroid cancer (ATC, < 2%) [
3,
4]. Mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) signal is particularly highly selected in PTC and a part of ATC [
3,
5]. Though the new mechanisms underlying the formation of thyroid cancer were revealed recently [
6‐
8], BRAFV600E mutation is the most common MAPK/ERK pathway-related genetic alteration in these types of thyroid cancer [
3,
5,
9]. BRAF, as a member of the RAF family of serine/threonine protein kinases, is recruited and phosphorylating activated by RAS family. Active BRAF signals through MEK to activate MAPK/ERK pathway, which, in turn, induces a range of biochemical processes including cell differentiation, proliferation, growth, and apoptosis [
10]. The residues’ glycine-rich segments G595-V600 hydrophobic interacts with segments G463-V470, keep the BRAF protein as an inactive conformation [
9,
10]. The BRAFV600E mutation replaces V600 valine with E600 glutamic acid, a larger, charged side chain, causing the phosphomimic effect [
9]. The phosphomimic effect breaks the hydrophobic interaction and destabilizes the inactive conformation, resulting in the constitutively active state and strongly activated MAPK/ERK signal [
10,
11]. Therefore, targeting BRAFV600E could be the most promising treatment among patients with BRAF mutant (BRAF
MT) thyroid cancer [
12,
13].
Vemurafenib (PLX4032), a selective oral inhibitor of the BRAFV600E kinase, is associated with approximately 50% of response rate and improving survival among patients with BRAFV600E mutation–positive metastatic melanoma, which is another BRAFV600E mutation dominated malignancy [
11,
14]. However, patients diagnosed with BRAFV600E-mutated thyroid cancers seldom benefit from PLX4032 due to the existence of MAPK/ERK as well as phosphatidylinositol 3-kinase/Protein Kinase B (PI3K/AKT) pathways feedback activation [
15]. PLX4032 release the transcription repressor CTBP proteins from HER3 promoter and induce HER3 gene expression. Autocrine-secreted NRG1 binding to HER3, triggers HER3/HER2 heterodimerization and receptor phosphorylation, inducing PI3K and reactivating MAPK signaling, thus promoting resistance to growth inhibition [
15]. Therefore, finding a new combining therapy to relieve this drug resistance and benefit these patients is of great significance.
Vitamin C, as an essential nutrient for humans, has been debated for years because of its controversial role in cancer therapy. More recently, it has been shown that vitamin C at pharmacologic plasma concentrations acquired intravenously can selectively kill the tumor cells but had little cytotoxic effect on normal cells; and these effect was mainly through a reactive oxygen species (ROS) dependent way [
16‐
18]. These findings have inspired other studies to provide new insights into the function of combining therapy with vitamin C in treating different types of cancers [
19‐
21]. Nevertheless, the function and the mechanism of combining therapy with vitamin C and PLX4032 in thyroid cancer has not been investigated yet. Although vitamin C shows effectiveness in treating the PLX4032-resistant melanoma cells; the authors did not explore the detailed mechanism [
22]. Our previous study identified that Vitamin C could inhibit the MAPK/ERK as well as PI3K/AKT pathways [
23]. We therefore hypothesize that the combination of Vitamin C and PLX4032 may synergistically induce the death of BRAF
MT thyroid cancer cell by relieving the feedback activation of MAPK/ERK as well as PI3K/AKT pathways.
In this study, we demonstrate that Vitamin C sensitizes BRAFMT thyroid cancer cells to PLX4032. Vitamin C relieves the feedback activation of MAPK/ERK as well as PI3K/AKT pathways induced by PLX4032. As a result, combining Vitamin C with PLX4032 suppresses the malignant progression of thyroid cancer.
Materials and methods
Cell culture
Thyroid cancer cell lines 8305C and BCPAP were obtained from Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology. 8505C were obtained from Otwo Biotechnology Co., Ltd. All the cells were routinely cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific), 1% Non-Essential Amino Acids (Gibco, Thermo Fisher Scientific) and 1% Sodium Pyruvate (Gibco, Thermo Fisher Scientific) at 37 °C.
Reagents
For in vitro study, BRAF inhibitor PLX4032 (Selleck Chemicals, Cat # S1267) and PLX4720 (Selleck Chemicals, Cat # S1152) were dissolved in dimethyl sulphoxide (DMSO), N-acetylcysteine (Sigma, Cat #A7250) and Vitamin C (Sigma, Cat # A4034) was dissolved in ddH2O, both in a high concentration. They were adding into the culture medium to dilute at the indicated concentration. For in vivo study, PLX4032 was dissolved in 0.5% hydroxypropyl methylcellulose (Sigma), and administered by oral gavage. Vitamin C was dissolved in ddH2O and administered by intraperitoneal injection at a total volume of 0.2 ml.
Cell viability assay
Cells (3000 to 4000/well) were seeded in 96-well plates. After a 24-h’s culture, cells were pre-treated with indicated doses of Vitamin C or ddH2O for 6 h. Then the different doses of PLX4032 or PLX4720 were added to the medium for the indicated period. Next, the medium was refreshed and 20 μL of 5 mg/mL 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was added into the medium and the plates were further incubated for 4 h, followed by adding 150 μl of DMSO. The plates were then read on a microplate reader using a test wavelength of 570 nm and a reference wavelength of 670 nm. All MTT assays were done in triplicate. IC50 values were calculated with the Reed-Muench method [
24].
Cell proliferation assay
5-Ethynyl-2′-deoxyuridine (EdU) assay kits were obtained from Solarbio, Beijing (Cat # CA1170). Briefly, cells (2–4 × 105) were seeded in 12-well plates. After a 24-h’s treatment with 6 μM PLX4032 or 0.25 Mm vitamin C, individually or in combination, the medium was changed into 50uM EdU containing medium to further culture for 2 h. Then the cells were fixed with 4% paraformaldehyde and washed with PBS, followed by dyeing with Hoechst33342. The images were obtained with a fluorescent inverted microscope (Leica, Wetzlar, Germany, Cat # DMI8), and color mergence was performed using ImageJ image software (ImageJ version 1.44p, NIH, MD). The percent of positive staining cells were calculated from five random views.
Monolayer culture was performed to measure colony formation. Thyroid cancer cells were seeded into 12-well plate at a concentration of 3000 cells per well, and cultured with various doses of Vitamin C and PLX4032 or PLX4720, individually or in combination. The medium was refreshed every 72 h. After 10–14 days of cultivation, colonies were fixed with 4% paraformaldehyde, and then washed with PBS and stained with a crystal violet solution. Each assay was carried out in triplicate.
Cell apoptosis assay
Cells were seeded at a concentration of 60–70% in 6 well plate, and cultured with indicated doses of Vitamin C (0.25 mM) and PLX4032 (6 μM), individually or in combination (Vitamin C 0.25 mM and PLX4032 4 μM) for 48 h. All the cells were collected by centrifuged followed by washed twice with PBS and stained with Annexin V-FITC/PI Apoptosis Detection Kit (Roche Applied Science) according to the manufacturer’s protocol. Apoptotic cells were measured by flow cytometer (BD Biosciences, NJ). Each experiment was carried out in triplicate.
Cell cycle analysis
Cells were seeded at a concentration of 40% in 6 well plate and serum starved for 24 h. After individually (Vitamin C 0.25 mM or PLX4032 6 μM) or in combination co-culture with 0.25 mM Vitamin C and 4 μM PLX4032 for 48 h, the cells were washed twice by PBS and fixed in 70% ethanol on ice for 30 min. Cells were then stained with PI solution (50 μg/mL PI, 50 μg/mL RNase A, 0.1% Triton-X, 0.1 mM EDTA). Cell cycles were analyzed based on DNA content using a flow cytometer (BD Biosciences, NJ).
Animal studies
8305C (5 × 106) cell lines were injected subcutaneously into the right armpit region of 5-week-old female nude mice purchased from SLAC laboratory Animal Co., Ltd. (Shanghai, China) to establish xenograft mouse model. Mice were then randomly divided into four groups (5 mice each group) when tumor volume grew to 20–30 mm3. PLX4032(50 mg per kg of body weight, once a day) and Vitamin C (3 g per kg of body weight, once a day) was administered individually or in combination. The treatment was administered for 2 weeks, body weight and tumor volume were measured with calipers every other day (Volume = width×length×width/2). Mice were sacrificed via cervical dislocation. Tumors were collected and weighted 5 h after the final dose of drugs. Animal experiments were approved by The Animal Experimental Center of Peking University Shenzhen Hospital. Ethical approval document of this research was provided at supplementary information, the approval number was 2020–328.
Immunohistochemical (IHC) staining and H&E staining
Tumor tissues were embedded in paraffin, sectioned at 4 μm, then cell proliferation ability was assessed by quantification of Ki-67 staining (percentage of positive cells). In brief, antihuman Ki-67 antibody (BD Pharmingen, CAT 550609) and p-ERK (Cell Signaling Technology, CAT#4370) were 1:200–300 diluted, and immunostaining was done according to a standard protocol using DAB Substrate Kit (ZSGB-BIO). To ensure the comparability of immunohistochemical staining, a common reference standard was included as an internal or intra-assay control in each batch. Ki-67 protein expression was scored with 0, 1, 2, 3, which represent negative, weak positive, positive, and strong positive, respectively. Ki-67 protein expression scored with 1, 2, 3 were calculated as positive, the percent of positive staining cells were then calculated. ERK phosphorylation was quantitated by integral optical density (IOD) using Image-pro plus 6.0 (Media Cybernetics). Each stained section was evaluated under the same magnification, light brightness, and exposure intensity. To evaluate the effect of different treatments on different organs, we performed hematoxylin and eosin (H&E) staining of liver, kidney, and heart sections.
Western blot analysis
The indicated cells were treated with PLX4032 4 μM and Vitamin C 0.5 mM, individually or in combination for 0,6,12,24 h. NAC 0.5 mM was added for 24 h in rescue experiment. Then cells were lysed in prechilled RIPA buffer containing protease inhibitors. Equal amounts of protein lysates were separated by SDS–PAGE and transferred onto PVDF membranes (Roche Diagnostics). The membranes were then incubated with primary antibodies. This was followed by incubation with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and immunoblotting signals were visualized using the Western Bright ECL detection system (Tanon). The information of primary antibodies was listing as below: anti-tERK (Cell Signaling Technology, CAT #4695); anti-Pan-AKT (Bioworld Technology, CAT BS1810); anti-Actin (Santa Cruz CAT sc-1616).
Statistical analysis
Synergy of the action of both compounds were statistical analyzed with Chou-Talalay method using ComboSyn Software (ComboSyn Inc. Paramus, NJ) [
25,
26]. All the rest statistical analyses were performed with the SPSS statistical package (16.0, SPSS Inc. Chicago, IL). Unpaired student’s t test was used to compare the means of two groups of data. One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test and Two-way ANOVA with Bonferroni post-test were used to compare differences between three or more groups. All values were expressed as the mean ± standard deviation (SD).
P < 0.05 was considering statistically significant differences. Each experiment was carried out and calculated in triplicate.
Discussion
BRAFV600E mutation is recognized as the driving pathogenesis of most PTC and some ATCs, and thyroid-specific knock-in of BRAFV600E will induce an aggressive PTC [
3,
30,
31]. This make BRAFV600E a prognostic molecular marker and promising therapeutic target for thyroid cancer, although in clinical trials the selective BRAFV600E inhibitor PLX4032 were not as promising as expected [
32,
33]. The MAPK/ERK signal axis rebounding after the application of BRAFV600E specific inhibitors plays the key role in drug resistance [
15].
In this study, we demonstrated Vitamin C sensitizing BRAFMT thyroid cancer cells to PLX4032. Combining PLX4032 with Vitamin C synergistically inhibited cell proliferation, colony formation and tumorigenic potential in nude mice, and induced cell apoptosis and cell cycle arrest in BRAFMT thyroid cancer cells. Our data indicated this synergistic effect was triggered by Vitamin C relieving the feedback activation loops caused by PLX4032. These results pointed out that combining PLX4032 with Vitamin C is a promising therapy in treating BRAFMT thyroid cancer.
PLX4032 was a selective BRAFV600E inhibitor which was proven of high response rate in patients with metastatic melanomas [
34]. In contrast to the high efficacy in patients with metastatic melanomas, PLX4032 as a single agent demonstrated limited response in patients with BRAF
MT colorectal cancers, which was ascribed to the activation of EGF receptor signal [
35,
36]. Single agent of PLX4032 shows minimal activity towards patients with progressive, BRAF mutant PTC [
37]. It was further confirmed by our data that PLX4032 exhibits a slight inhibitory action on the malignant activity of BRAF
MT thyroid cancer cells. Recent studies have proved that using PLX4032 will result in the reactivation of MAPK/ERK signal as well as PI3K/AKT pathways, leading to drug resistance in thyroid cancer [
15]. Supporting by our data, an increased phosphorylation of ERK as well as pan-AKT in BRAF
MT thyroid cancer cells were observed after treating them with PLX4032 for 12–24 h. This was further validated by the IHC of phosphorylated ERK in xenograft. These results revealed that reactivation of MAPK/ERK signal and PI3K/AKT pathways will relieve the function of PLX4032 in BRAF
MT thyroid cancer.
In recent years, Vitamin C was proved to be a promising and potential therapy for certain cancers including thyroid cancer [
16,
17]. In this study, we also proved the antitumor activity of Vitamin C in different BRAF
MT thyroid cancer cells. A series of in vivo studies also supported the antitumor efficacy of vitamin C through parenteral administration [
38,
39]. Our results show that Vitamin C could enhance antitumor activities of the selective BRAFV600E inhibitor PLX4032 in both in vitro and in vivo study, and the Vitamin C dosage could be easily achieved via intravenous administration [
38,
40]. More importantly, Vitamin C has been proved to be able to enhance the effect of some traditional therapies in different cancers [
41,
42]. Therefore, we attempted to reveal the mechanism underlying our combining therapy. Our previous study has reflected that Vitamin C killed thyroid cancer cells, especially with mutant BRAF, through a ROS dependent inhibition of MAPK/ERK and PI3K/AKT pathways [
23]. We thus hypothesized the reason behind was the inhibition of feedback activation of MAPK/ERK and PI3K/AKT pathways. In line with our hypothesis, combination of Vitamin C and PLX4032 greatly relieves the feedback activation of MAPK/ERK signal. It should be noted that, time-dependent inhibition of tumor growth in Fig.
5a revealed there is no difference between PLX4032 group and the combining group during the first 9 days after drug administration; while the tumor volume increased more quickly afterwards. We then assumed that the start of the feedback activation of MAPK/ERK signal has led to the result, while the combining therapy could further inhibit MAPK/ERK signal but not the PLX4032 monotherapy. However, extensive clinical trials may be necessary to determine the safety and efficacy of the combining therapy towards thyroid cancer.
In summary, we found that Vitamin C sensitizes BRAFMT thyroid cancer cells to PLX4032 via relieving the feedback activation loops of MAPK/ERK and PI3K/AKT pathways. These preliminary results indicated that the Vitamin C/PLX4032 combination therapy may be an encouraging treatment in the cure of BRAFMT thyroid cancer.
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