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Article

Inhibition of TP53 Mutant Oral Cancer by Reactivating p53

by
Yei-Jin Kang
1,
Dae-Won Kim
2,
Xiangguo Che
3,
Je-Yong Choi
3,* and
Seong-Gon Kim
1,*
1
Department of Oral and Maxillofacial Surgery, Gangneung-Wonju National University, Gangneung 25457, Korea
2
Department of Oral Biochemistry, College of Dentistry, Gangneung-Wonju National University, Gangneung 28644, Korea
3
Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, Korea Mouse Phenotyping Center, School of Medicine, Kyungpook National University, Daegu 41944, Korea
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2022, 12(12), 5921; https://doi.org/10.3390/app12125921
Submission received: 19 May 2022 / Revised: 8 June 2022 / Accepted: 8 June 2022 / Published: 10 June 2022
(This article belongs to the Section Applied Biosciences and Bioengineering)
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Abstract

:

Featured Application

4-Hexylresorcinol exhibits a therapeutic effect on TP53 mutant oral cancer cells by increasing the acetylation and phosphorylation of p53.

Abstract

Background: Mutation of p53 is a frequent event, and mutant p53 exhibits low levels of acetylation and phosphorylation. This study aimed to investigate the effect of the histone deacetylase (HDAC) inhibitor, 4-hexylresorcinol (4HR), on the acetylation and phosphorylation of mutant p53 carcinoma cells and its therapeutic effects in a xenograft model. Methods: To determine the effect of 4HR on the acetylation and phosphorylation of p53, western blot analysis was performed using YD-9 and YD-15 cells. p53 siRNA was used to examine whether 4HR acts in a p53-dependent or independent manner. This was evaluated using a xenograft model. Results: In in vitro experiments when the concentration of 4HR was increased, the expression levels of HDAC4, acetylated p53 (Ac-p53), and phosphorylated p53 (p-p53) increased. Transfection with TP53 siRNA successfully suppressed p53 protein and TP53 mRNA expression. When 4HR was administered to a xenograft model, the tumour expansion rate was suppressed compared with the control, and the mice exhibited a higher survival rate. Conclusions: Our findings reveal that 4HR is a potential agent that restores loss of function in mutant p53 cancer cells via acetylation and phosphorylation of p53 as well as inhibition of HDAC4.

1. Introduction

Many types of cancers exhibit p53 mutations [1]. Since p53 is important for cellular apoptosis, cancer cells with p53 mutations may be resistant to chemotherapy [2]. In particular, oral carcinomas have a much higher incidence of p53 mutations [3,4]. Thus, a treatment strategy focused on the functional recovery of mutant p53 could be a promising approach [5]. Chemical chaperones can bind to proteins and induce conformational changes [6]. Mutant p53 may hide the amino acids which are important for its phosphorylation and acetylation [7]. Chemical chaperone-induced conformational changes may expose these amino acids which have not been exposed in the mutant state.
p53 is a transcription factor involved in the regulation of apoptosis-associated gene expression [1]. These genes are involved in p53-dependent apoptosis [1]. To induce the expression of apoptosis-associated genes, p53 needs to be phosphorylated and acetylated in the cytoplasm and translocated into the nucleus [7,8]. Many class I histone deacetylase (HDAC) inhibitors have shown anticancer effects by increasing the acetylation of important proteins, including p53 [9].
The chemical chaperone, 4-hexylresorcinol (4HR), can induce dormancy in microorganisms against environmental stress [10]. Chronic administration of 4HR per os can prevent spontaneous tumour development in experimental animals [11]. 4HR induces cellular apoptosis in cancer cells [12]. 4HR has been recognised as a class I HDAC inhibitor [13]. In addition, 4HR can increase endoplasmic reticulum (ER) stress which may be due to the induction of protein conformational changes [14]. Increased ER stress following 4HR administration may induce p53 independent apoptosis [15]. Therefore, 4HR treatment can induce cancer cell apoptosis in a p53-dependent as well as independent manner. Phosphorylation of p53 is closely associated with p53 function [16]. Acetylation of p53 increases its DNA-binding affinity and activates target gene expression [17]. Cancer cells having mutations in the p53 DNA binding domain may show difficulty in activation of target gene expression. Therefore, increased phosphorylation and acetylation will be important markers for evaluating the functional recovery of p53 in TP53 mutant cancer. However, phosphorylation and acetylation levels of p53 after 4HR treatment in TP53 mutant cancer has not been studied.
The objectives of this study were to (1) evaluate phosphorylation and acetylation levels of p53 after 4HR treatment in p53 mutant oral cancer cells, (2) evaluate HDAC activity after 4HR treatment in p53 mutant oral cancer cells, and (3) evaluate the expression levels of apoptosis-associated proteins after 4HR treatment in p53-silenced oral cancer cells.

2. Materials and Methods

2.1. Cell Culture

YD-15 cells, derived from the tongues of patients initially diagnosed with mucoepidermoid carcinoma, were purchased from the Korean Cell Line Bank (Seoul, Korea). The YD-15 cell has mutant p53, and its mutation site is the DNA-binding domain of p53 [18]. Though the YD-15 cell has poor tumourigenicity in xenograft models [18], there are few oral cancer cell lines having p53-mutation-associated information. In addition, YD-9 cells (squamous cell carcinoma derived from the buccal cheek) were used for comparative purposes. The YD-9 cell has wild-type p53. The subsequent culture conditions were in accordance with those recommended by the Korean Cell Line Bank. Cells were grown in six-well culture plates in a humidified CO2 incubator at 37 °C. The freezing medium was 52.5% Roswell Park Memorial Institute 1640 (RPMI 1640) medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 40% foetal bovine serum (FBS) and 7.5% dimethyl sulfoxide (DMSO). The culture medium was RPMI 1640 supplemented with L-glutamine (300 mg/L), 25 mM HEPES, 25 mM NaHCO3, (90%) and 10% heat-inactivated foetal bovine serum (FBS). Two-thirds of the medium was removed and replaced with fresh medium every three days.

2.2. Western Blot Analysis

The inhibitor 4HR was solubilised in 0.1% dimethyl sulfoxide (DMSO). When YD-15 and YD-9 cells were grown to approximately 70% confluency, they were treated with 1, 10, and 100 µM 4HR for 2, 8, or 24 h. Control cells were treated with 0.1% DMSO in the culture medium. Cultured cells were harvested using 0.01% trypsin and 1 mM ethylene-diamine-tetra-acetic acid. Cellular lysis was performed using protein lysis buffer (PRO-PREPTM, iNtRON Biotechnology INC, Sungnam, Korea). The following primary antibodies were purchased: HDAC1 (CAT#: sc-81598, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), HDAC3 (CAT#: sc-376957, Santa Cruz Biotechnology Inc.), HDAC4 (CAT#: sc-46672, Santa Cruz Biotechnology Inc.), HDAC5 (CAT#: sc-133225, Santa Cruz Biotechnology Inc.), p53 (CAT#: sc-126, Santa Cruz Biotechnology Inc.), p-p53 (Ser315) (CAT#: sc-135772, Santa Cruz Biotechnology Inc.), Bax (CAT#: sc-7480, Santa Cruz Biotechnology Inc.), Bad (CAT#: sc-8044, Santa Cruz Biotechnology Inc.), Bcl-2 (CAT#: sc-7382, Santa Cruz Biotechnology Inc.), and Ac-p53 (Lys319) (CAT#: PA5-99334, Invitrogen, Thermo Fisher Scientific Corporation, Waltham, MA, USA). The lysates were subjected to western blotting to detect the expression levels of HDAC1, HDAC3, HDAC4, HDAC5, p53, p-p53, and Ac-p53. Protein quantification was performed as described previously [13].

2.3. HDAC Activity and Caspase-9 Activity Assays

HDAC enzyme activity after treatment with 4HR was assessed using a commercially available kit (CAT#: ab156064, Abcam, Cambridge, UK). YD-15 cells were treated with 1, 10, and 100 μM of 4HR, and cellular lysates were collected after 2, 8, and 24 h. The subsequent procedure was performed according to the manufacturer’s protocol. The activities of HDAC1, 2, 3, and 8 (Class I HDACs) were measured using this kit according to the product datasheet. The HDAC assay buffer and substrate were added to the reaction wells. The inhibitor and developer were added to the wells and thoroughly mixed. Then, the prepared samples were added to each well and incubated for 20 min at room temperature. The stop solution was then added, and the mixture was incubated for 10 min at room temperature. Fluorescence intensity was measured using a plate reader.
Caspase-9 activity was measured using a commercially available colorimetric kit (CAT#: ab65608, Abcam, Cambridge, UK). Lysates from YD-15 or YD-9 cells were centrifuged at 10,000× g for 1 min. The cytosolic extract was transferred to a fresh tube and the protein concentration was measured. The extracted protein (100 μg) was diluted with 50 μL of cellular lysis buffer. The reaction mixture was added to each sample and incubated at 37 °C for 2 h. Fluorescence intensity was measured with a plate reader.

2.4. The Effect of p53 siRNA on 4HR-Induced Apoptosis

Cancer cells were washed twice with phosphate-buffered saline (PBS), and fresh α-MEM supplemented with 10% FBS was added. To determine the optimal concentration of p53 siRNA, 1–10 pg/mL of p53 siRNA was transfected, and the expression levels of p53 were examined after 8 h of treatment. The levels of TP53 inhibition were evaluated by qRT-PCR. The primers used for qRT-PCR are listed in Supplementary Table S1. Since 4HR-mediated cellular apoptosis can be either p53-dependent or independent, treatment with p53 siRNA can clarify the mechanism of tumour cell apoptosis after 4HR treatment. Cancer cells were washed twice with phosphate-buffered saline (PBS), and fresh α-MEM supplemented with 10% FBS was added. For the experimental group, p53 siRNA was transfected and the concentration was determined by the previous experiment. The control group was transfected with the same concentration of random siRNA. After 8 h of siRNA transfection, HR (1 and 10 μM) was added to the cells. Total protein or RNA were collected 24 h after 4HR administration. Western blotting for Bax, Bad, and Bcl-2 was performed.

2.5. Confocal Microscopy

After fixation of cells, the slides were washed with phosphate-buffered saline (PBS) containing Tween20. Then, the slides were incubated with a blocking reagent (DAKO, Glosturp, Denmark) for 30 min. The Cytochrome c Apoptosis ICC Antibody Kit (CAT#: ab110417, Abcam) contains cytochrome c monoclonal antibody and ATP synthase V subunit alpha monoclonal antibody. Cytochrome c and ATP synthase V are localised in the mitochondria, and only cytochrome c is released from the mitochondria during apoptosis. Cytochrome c is conjugated to FITC and ATP synthase V to TXRD, and cells with cytochrome c leakage show isolated green fluorescence. The antibodies were added to the slides and incubated in a humidified dark chamber for 1 h. After washing, the slides were mounted. The mounted slides were examined using the Stellaris 5 confocal microscope (Leica Microsystems, Wetzlar, Germany) at the Center for Scientific Instruments, Gangneung-Wonju National University.

2.6. Xenograft Study

Seven-week-old male athymic nude mice were purchased from Orient Bio (Seoul, Korea). The mice were used in accordance with the Animal Care and Use Guidelines of the College of Dentistry at Gangneung-Wonju National University (IACUC No. GWNU- 2021-3, approval date 15 January 2021). To produce tumours, YD-15 cells were harvested from sub-confluent cultures. A viable cell suspension was used for administration. The mice were anaesthetised using O2 and isoflurane. Overall, 20 mice were intradermally injected with YD-15 cells (3.0 × 105 cells/per animal) using a 26-gauge hypodermic needle with 1 mL syringe according to a method described previously [13]. Subsequently, the nude mice were randomly assigned to two groups: 4HR and control groups. After the tumour mass was identified, the 4HR groups (each group, n = 10) received daily subcutaneous injections of 4HR (10 mg/kg body weight) for 28 days, while the control group received daily injections of the vehicle (β-cyclodextrin). Moreover, changes in tumour mass and body weight were examined every three days. The day of euthanasia was recorded, and the data were used for survival analysis. Euthanasia was performed when body weight decreased by 20%, tumour necrosis was observed, or a back hump was thought to be due to pain.

2.7. Immunohistochemical Staining

Primary antibodies for immunohistochemical analysis were purchased and the details regarding the antibodies are provided in the supplementary information. The dilution rate was 1:100 for all primary antibodies. The main tumour mass from each mouse was used for immunohistochemical staining. Sections of 5 μm thickness were prepared for immunohistochemistry. Immunohistochemical staining was performed using the Universal LSAB+ Kit (Dako, Glostrup, Denmark), and the subsequent procedure was performed according to the manufacturer’s instructions. Immunostaining without primary antibodies was used as a negative control. The slides were evaluated for intensity at the boundary of the maximum area of staining. For adjustment, the intensity scales were subtracted from the grey scale (255-x) and the adjusted value of the negative control was subtracted from the adjusted value of each sample. The difference between the average values of each group was analysed using the independent sample t-test and the significance was set at p < 0.05.

2.8. Cancer and p53 Signalling Phospho Antibody Array

Whole blood samples were collected from the mice and centrifuged. Serum was collected and used for analysis. The collected sera were sent to Ebiogen Inc. (Seoul, Korea), which performed the subsequent procedure. Cancer Signaling Phospho Antibody Array (Cat#: PCS248, Full Moon Biosystems) and p53 Signaling Phospho Antibody Array (Cat#: PFT196, Full Moon Biosystems) were used to detect phosphorylation/activation of key signalling proteins. The array results were delivered as a Microsoft Excel file via Ebiogen Inc. (Seoul, Korea) [19,20].

2.9. Statistical Analysis

The data from the cell and animal experiments were analysed using Sigma Scan Pro 5.0 and SPSS software version 25 (IBMⓇ, Armonk, New York, NY, USA). Statistical significance of the differences between two average values was tested using the independent sample t-test, whereas multiple values were compared using one-way analysis of variance. Post hoc analyses were performed using the Bonferroni test. Survival was compared using the Kaplan–Meier method, and differences between the groups were evaluated using the log-rank test. The significance level was set at p < 0.05.

3. Results

3.1. Increased Phosphorylation of p53 upon Treatment with 4HR

Treatment of YD-15 cells with 4HR resulted in increased expression of p-p53 (Ser 315) (Figure 1). The relative expression level of p-p53 in the untreated control group was 0.011 ± 0.004 (Figure 1). Upon treatment with 1 μM 4HR, the relative expression levels were 0.027 ± 0.010, 0.143 ± 0.030, and 0.580 ± 0.041 at 2, 8, and 24 h after treatment, respectively; for 10 μM 4HR, this was 0.419 ± 0.035, 0.617 ± 0.040, and 1.074 ± 0.037 at 2, 8, and 24 h, respectively; for 100 μM 4HR, this was 0.786 ± 0.045, 0.964 ± 0.029, and 1.285 ± 0.030 at 2, 8, and 24 h, respectively. The difference among groups was statistically significant (p < 0.001). In the post hoc comparison test, the difference between the untreated control and 4HR-treated groups was statistically significant (p = 0.003 for 1 μM 4HR treatment at 8 h and p < 0.001 for the others except for 1 μM 4HR treatment at 2 h). A similar expression pattern was observed in YD-9 cells (Supplementary Figure S1).

3.2. Increased Acetylation of p53 upon Treatment with 4HR

Treatment of YD-15 cells with 4HR increased the expression levels of Ac-p53 (Lys319) (Figure 2). The relative expression level of Ac-p53 in the untreated control was 0.022 ± 0.012 (Figure 2). Upon 1 μM 4HR treatment, the relative expression levels were 0.024 ± 0.011, 0.038 ± 0.009, and 0.542 ± 0.090 at 2, 8, and 24 h after treatment, respectively; for 10 μM 4HR, this was 0.027 ± 0.011, 0.746 ± 0.064, and 1.051 ± 0.073 at 2, 8, and 24 h, respectively; and for 100 μM 4HR, this was 0.032 ± 0.009, 1.103 ± 0.131, and 1.278 ± 0.122 at 2, 8, and 24 h, respectively. The difference between groups was statistically significant (p < 0.001). In the post hoc comparison test, the difference between the untreated control and 4HR-treated groups was statistically significant (p < 0.001 for 10 and 100 μM 4HR treatment at 8 h and p < 0.001 for all groups at 24 h).
The expression levels of HDAC4 decreased after 4HR treatment (Figure 3A). The differences were statistically significant (p < 0.05). The relative expression level of HDAC4 in the untreated control was 1.270 ± 0.219. Upon 1 μM 4HR treatment, the relative expression levels were 0.893 ± 0.095, 0.487 ± 0.100, and 0.394 ± 0.024 at 2, 8, and 24 h after treatment, respectively; for 10 μM 4HR, this was 0.667 ± 0.096, 0.444 ± 0.038, and 0.175 ± 0.042 at 2, 8, and 24 h, respectively; for 100 μM 4HR, this was 0.568 ± 0.150, 0.583 ± 0.063, and 0.043 ± 0.007 at 2, 8, and 24 h, respectively. The difference between groups was statistically significant (p < 0.001). In the post hoc comparison test, the difference between the untreated control and 4HR-treated groups was statistically significant (p = 0.010 for 1 μM 4HR treatment at 2 h and p < 0.001 for the others). A similar expression pattern was observed in YD-9 cells (Supplementary Figure S2). HDAC activity was inhibited upon treatment with 4 HR (Figure 3B). The differences were statistically significant (p < 0.05). In the post hoc comparison test, the difference between the untreated control and 4HR-treated groups was statistically significant (p = 0.003 for 10 μM 4HR treatment at 24 h and p < 0.001 for 100 μM 4HR treatment).

3.3. HR-Induced Apoptosis-Associated Protein Expression via Both p53-Dependent and Independent Pathways

Treatment with 4HR increased the release of cytochrome c (Figure 4a,b). In addition, the activity of caspase-9 was increased upon 4HR treatment (Figure 4c,d).
Transfection with TP53 siRNA successfully suppressed p53 protein expression (Figure 5). The expression levels of Bcl2 were not dependent on the presence of p53. Interestingly, the expression levels of Bax were increased upon transfection with TP53 siRNA (Figure 5). Interestingly, treatment with 4HR increased the expression levels of Bad protein without p53. Thus, 4HR-mediated apoptosis in YD-15 cells seemed to be induced via both p53-dependent and independent pathways.

3.4. HR Administration Exhibited Tumour-Suppressing Effects in the Xenograft Model

YD-15 cells were injected into the sublingual space (Figure 6A). As the tumour grew, skin ulceration was observed in both the groups. The average tumour size and survival rate were compared between the groups (Figure 6A). The survival rate of the 4HR group was higher than that of the control group. However, these differences were not statistically significant (Figure 6B). Xenografted carcinoma cells were observed by haematoxylin and eosin staining (Figure 6C). Immunohistochemistry showed that the expression levels of Ac-p53 and p-p53 were higher in the 4HR group than in the control group. However, the expression level of HDAC4 was lower in the 4HR group than in the control group.
In the protein array, the systemic effect of 4HR administration was evaluated by collecting blood serum from each group. The 4HR group showed increased expression of ERK8 (2.836), STAT4 (1.434, caspase-9 (1.539), MDM2 (1.890), histone H2A (1.286), p-mitogen-activated protein kinase-activated protein kinase-2 (MK2) (1.440), and p-p53 (Ser315) (1.203). In contrast, the 4HR group showed decreased expression of p-BRCA1 (0.777), p-HDAC4 (0.756), and HDAC5 (0.799) (Table 1).

4. Discussion

Loss of function mutation in TP53 is frequently found in oral cancers [3,4]. As p53 is a transcription factor, its function is highly dependent on acetylation and phosphorylation levels [16,17]. Rapid apoptosis of cell can be induced by phosphorylation of p53 [21]. Accordingly, oral cancer having aberrant p53 usually shows low levels of p53 acetylation and phosphorylation [3,4]. In this study, 4HR treatment increased the acetylation and phosphorylation of p53 in YD-15 cells (Figure 1 and Figure 2). The expression levels of HDAC4 and HDAC activity were decreased upon 4HR treatment (Figure 3). YD-15 cells have a p53 mutation in their DNA-binding domain [18]. The expression of Bax was increased upon 4HR treatment in YD-15 cells (Figure 5). Apoptosis induced by 4HR was confirmed by cytochrome c leakage in YD-15 cells after 4HR treatment (Figure 4). In the xenograft model, 4HR administration increased the survival rate, but the difference was not significant (Figure 6). A protein array was performed using the sera from xenografted animals. The phosphorylation of p53, as well as the expressions of MDM2 and MAPKAPK2 (MK2) were increased, but that of BRCA1, HDAC4, and HDAC5 were decreased (Table 1). Collectively, 4HR treatment in oral cancer cells with a TP53 mutation reactivated p53 by acetylation and phosphorylation.
Transcription factors are activated by acetylation and phosphorylation [22]. Accordingly, p53 activation and its translocation into the nucleus are achieved through p53 acetylation and phosphorylation [23]. Since histone deacetylase (HDAC) removes the acetyl group from the target protein, its inhibitor increases the acetylation of target proteins [9], and 4HR is a class I and II HDAC inhibitor [13]. In this study, treatment with 4HR increased the acetylation of p53 in YD-15 cells (Figure 2). Phosphorylation is also important for signal transduction [24]. Treatment with 4HR also increased the phosphorylation of p53 in both YD-9 and YD-15 cells (Figure 1 and Supplementary Figure S1). When TP53 expression was silenced by siRNA, the expression levels of Bax were still elevated compared to that in the siRNA control group (Figure 5). Some cancers show gain-of-function mutation in TP53 [25]. The TP53 mutation in YD-15 cells might be gain-of-function mutation which might contribute to tumour survival. Accordingly, inhibiting TP53 expression by siRNA increased the expression levels of Bax, which is important for cellular apoptosis (Figure 5). Treatment with 4HR increased the expression levels of Bad in the siRNA-TP53 group (Figure 5). Thus, 4HR could increase the expression levels of Bad in both p53-dependent and -independent manners. Since 4HR treatment increased ER and mitochondrial stress [14], the increased expression of Bad and Bax might be mediated by ER and mitochondrial stress.
Administration of 4HR in the xenograft model resulted in an improved survival rate (Figure 6B). However, the difference between the groups was not statistically significant (p > 0.05). The main reason for the insignificant results might be due to small sample size. Although YD-15 cells have the mutant p53 protein, their ectopic tumour formation rate is notoriously low [18]. Despite 10 animals being included in each group, the success rate of tumour growth was very low (approximately 50%). In a previous study, the tumourigenicity of YD-15 cells was 0/5 in athymic nude mice [18]. The experiment began when tumour growth was observed. Therefore, the tumour size was not exactly the same at the initial point, with the tumours in the 4HR group being slightly larger (data not shown). In addition, angiogenesis in YD-15 cells might be poor in xenografts [26]. This was observed as spontaneous ulcer formation and central necrosis as the tumour growth progressed (Figure 6A). The high mortality rate was also an obstacle for long-term observation. We repeated tumour xenografting twice, but we did not observe sufficient number of tumours for the result to be considered significant, as reported in previous studies [18]. Despite this limitation, the expression levels of p-p53 and Ac-p53 were elevated in the 4HR administrated group (Figure 6C).
Activation of p53 was also observed upon protein array analysis of blood serum samples. The expression levels of serum ERK8 have been reported to be decreased in aggressive breast and lung cancers [27]. ERK8 inhibits the motility of tumour cells [27]. The administration of 4HR increased serum ERK8 levels compared to the untreated control (Table 1). In a previous study, 4HR administration significantly decreased tumour cell metastasis [28]. Elevated levels of serum caspase-9 and STAT4 might be associated with p53 activation (Table 1). Moreover, the phosphorylation of p53 at Ser315 was increased (Table 1). Phosphorylation at Ser315 is associated with p53 tetramer formation [29] and its DNA binding [7]. Rapid apoptosis is induced upon phosphorylation of p53 [21]. MK2 can induce cell cycle arrest and DNA repair in TP53-mutant cancer cells [30]. Since YD-15 cells are TP53-mutant cancer cells [18], increased levels of MK2 induced upon 4HR treatment would be therapeutically beneficial. Furthermore, protein array results showed HDAC inhibition (Table 1), similar to the results obtained from the in vitro experiment (Figure 3). The expression levels of HDAC4, p-HDAC4, and p-HDAC5 were decreased upon 4HR administration (Table 1).
In accordance with the result of the Western blot, an increase of p-p53(Ser315) was observed in protein array (Table 1). Ser315 in p53 is important to oligomerization and DNA-binding capacity [7,29]. Aurora kinase A phosphorylate p53 at Ser315, and this induces localization to nucleus protecting from MDM2 and p53 degradation [31]. Low levels of p-p53(Ser315) is the phenotypes of loss-of-function mutation of p53 [31]. Additionally, phosphorylation of p53 on Ser9 was reduced by 4HR administration in the protein array (Table 1). Ser9 in p53 is MDM2 binding site. Phosphorylation at Ser9 in p53 suppresses p53 function [32]. Therefore, a decrease of p-p53(Ser9) by 4HR administration might be associated with functional recovery of p53.
The expression level of HDAC4 was reduced significantly by the administration of 4HR (Figure 3A). This was also identified in the results of the protein array (Table 1). Ac-p53 is needed to recruit HDAC4 as a co-suppressor of the G2/M promoter [33]. Cells blocked for long periods in G2/M may be prone to enter the apoptotic pathway. The acetylation site for p53 can be found in its C-terminal domain, DNA binding domain, and some lysines between those two domains [17]. Ac-p53(Lys319) is localized between those two domains [17]. In this study, the expression level of Ac-p53(Lys319) was increased by 4HR administration (Figure 2). Ac-p53(Lys319) is important for the stress mediated p53 stabilization [34].
4HR administration may have therapeutic potential in a different cancer type. In a previous study, 4HR showed anti-tumour effects via stimulating the differentiation of SCC-9 cells (tongue cancer cells) [35,36]. In addition, 4HR inhibits NF-kappaB phosphorylation and has a synergistic anti-tumour effect with cisplatin in KB cells (nasopharyngeal carcinoma) [12]. 4HR administration suppresses the cellular growth of oral mucosal melanoma cells [37]. The administration of 4HR can decrease the incidence of leukemia, hepatocellular carcinoma, and circulatory system tumours in the animal model [11].
The limitations of this study are as follows. First, YD-15 cells are known to be poorly tumourigenic when grafted into nude mice [18]. Although there were some successful implantations, surface ulceration and central necrosis were frequent. This might be due to tumour lysis syndrome [38]. Moreover, it has been shown that some tumours invade the vertebra, and cause malignant spinal cord compression [39]. These events are obstacles to evaluate long-term therapeutic effects. Since YD-15 cells have a known p53 mutation, they were used in this study [18]. According to a previous study, high-grade mucoepidermoid carcinoma may exhibit sensitivity similar to that of squamous cell carcinoma [40]. The genes associated with cellular differentiation is increased by 4HR administration on SCC-9 [36], and this may be associated with p53 activation. The merit of this study was the identification of the therapeutic effect of 4HR in oral cancer cells with p53 mutation. Second, the number of acetylated lysine residues holds the key of mutated p53 function [17]. There are many sites for acetylation in p53, but only Ac-p53(Lys319) was examined in this study. In addition, p53 acetylation level in YD-9 cells which has wild-type p53 had not been studied. Investigating the acetylation level of p53 C-terminal domain and DNA binding domain after 4HR administration will be an interesting topic. It should be clarified in future studies.

5. Conclusions

The HDAC inhibitor HR is a potential agent for recovering loss of function in mutant p53 cells and acts as a pharmacological chaperone which induces phosphorylation of p53 and as a HDAC4 inhibitor which induces acetylation of p53.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app12125921/s1, Supplementary Table S1: The primer design for qRT-PCR, Supplementary Table S2: Initial body weight and tumour size at the time of treatment, Supplementary Figure S1: Western blot for p-p53, Supplementary Figure S2. The administration of 4HR decreased the expression level of HDAC4 in YD-9 cell.

Author Contributions

Conceptualization, Y.-J.K. and S.-G.K.; methodology, D.-W.K.; validation, X.C., D.-W.K., and Y.-J.K.; formal analysis, Y.-J.K.; investigation, J.-Y.C.; data curation, Y.-J.K. and X.C.; writing—original draft preparation, Y.-J.K.; writing—review and editing, J.-Y.C. and S.-G.K.; supervision, J.-Y.C. and S.-G.K.; project administration, S.-G.K.; funding acquisition, J.-Y.C. and S.-G.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development (Project no. PJ01562601 and PJ01562602)” Rural Development Administration, Korea.

Institutional Review Board Statement

Mice were used in accordance with the Animal Care and Use Guidelines of the College of Dentistry at Gangneung-Wonju National University (IACUC No. GWNU- 2021-3, Approval date 15 January 2021).

Data Availability Statement

Data sharing is not applicable to this article since no dataset was generated or analyzed during the current study.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 12 05921 g001 550
Figure 1. The change in p-p53 expression levels upon 4HR treatment in YD-15 cells. When compared to the untreated control, 4HR treatment increased p-p53 expression levels significantly (* p < 0.05 by Bonferroni test).
Figure 1. The change in p-p53 expression levels upon 4HR treatment in YD-15 cells. When compared to the untreated control, 4HR treatment increased p-p53 expression levels significantly (* p < 0.05 by Bonferroni test).
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Figure 2. The change in Ac-p53 expression levels upon 4HR treatment in YD-15 cells. When compared to the untreated control, 4HR treatment increased Ac-p53 expression levels significantly (* p < 0.05 by Bonferroni test).
Figure 2. The change in Ac-p53 expression levels upon 4HR treatment in YD-15 cells. When compared to the untreated control, 4HR treatment increased Ac-p53 expression levels significantly (* p < 0.05 by Bonferroni test).
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Figure 3. The change in p53 acetylation levels and HDAC activity. (A) The expression levels of HDAC4 decreased after 4HR treatment (A). The differences were statistically significant (p < 0.05). (B) When compared to the untreated control, HDAC activity was significantly inhibited upon 4HR treatment (* p < 0.05 by Bonferroni test).
Figure 3. The change in p53 acetylation levels and HDAC activity. (A) The expression levels of HDAC4 decreased after 4HR treatment (A). The differences were statistically significant (p < 0.05). (B) When compared to the untreated control, HDAC activity was significantly inhibited upon 4HR treatment (* p < 0.05 by Bonferroni test).
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Figure 4. Treatment with 4HR increased apoptosis. The untreated control showed that cytochrome c (green) was co-localised with ATP synthase V (red). Accordingly, the cytoplasm appeared as a bright yellow colour (a). When YD-15 cells were treated with 100 μM of 4HR, the cytoplasm appeared as a green colour due to cytochrome c release from the mitochondria (b). Caspase-9 activity was significantly increased upon 4HR treatment in both YD-15 (c) and YD-9 cells (d) (* p < 0.05).
Figure 4. Treatment with 4HR increased apoptosis. The untreated control showed that cytochrome c (green) was co-localised with ATP synthase V (red). Accordingly, the cytoplasm appeared as a bright yellow colour (a). When YD-15 cells were treated with 100 μM of 4HR, the cytoplasm appeared as a green colour due to cytochrome c release from the mitochondria (b). Caspase-9 activity was significantly increased upon 4HR treatment in both YD-15 (c) and YD-9 cells (d) (* p < 0.05).
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Figure 5. Transfection with TP53 siRNA. TP53 siRNA inhibited p53 expression successfully. The expression levels of Bcl-2 were not dependent on the presence of p53. When 4HR was added to TP53 mutant oral cancer cells (YD-15), the expression levels of Bax were increased. Interestingly, the expression levels of Bax were increased upon transfection with TP53 siRNA. Interestingly, treatment with 4HR increased the expression levels of Bad protein in a p53 independent manner.
Figure 5. Transfection with TP53 siRNA. TP53 siRNA inhibited p53 expression successfully. The expression levels of Bcl-2 were not dependent on the presence of p53. When 4HR was added to TP53 mutant oral cancer cells (YD-15), the expression levels of Bax were increased. Interestingly, the expression levels of Bax were increased upon transfection with TP53 siRNA. Interestingly, treatment with 4HR increased the expression levels of Bad protein in a p53 independent manner.
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Figure 6. The result of xenograft study. (A) YD-15 cells were injected into the sublingual space. As the tumour grew, skin ulceration was observed in both groups. (B) Survival curve. The survival rate of the 4HR group was higher than that of the control group. However, the differences were not statistically significant. (C) Histological finding. The average intensity of each protein was in accordance with the results of the in vitro experiment. However, there was no significant difference due to small sample number.
Figure 6. The result of xenograft study. (A) YD-15 cells were injected into the sublingual space. As the tumour grew, skin ulceration was observed in both groups. (B) Survival curve. The survival rate of the 4HR group was higher than that of the control group. However, the differences were not statistically significant. (C) Histological finding. The average intensity of each protein was in accordance with the results of the in vitro experiment. However, there was no significant difference due to small sample number.
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Table
Table 1. The results of protein array performed using serum samples.
Table 1. The results of protein array performed using serum samples.
Cancer SignalRatio *p53 SignalRatio †
Caspase-91.539p-MK2 (Thr334)1.440
Histone H2A1.286p-MDM2 (Ser166)1.218
STAT41.434p-BRCA1 (Ser1524)0.777
ERK82.836p-HDAC4 (Ser632)0.756
HDAC40.766p-HDAC5 (Ser259)0.799
MDM21.890p-p53 (Ser315)1.203
* 4HR/Control. † phosphorylated (4HR)/unphosphorylated (4HR) ÷ phosphorylated (control)/unphosphorylated (control).
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Kang, Y.-J.; Kim, D.-W.; Che, X.; Choi, J.-Y.; Kim, S.-G. Inhibition of TP53 Mutant Oral Cancer by Reactivating p53. Appl. Sci. 2022, 12, 5921. https://doi.org/10.3390/app12125921

AMA Style

Kang Y-J, Kim D-W, Che X, Choi J-Y, Kim S-G. Inhibition of TP53 Mutant Oral Cancer by Reactivating p53. Applied Sciences. 2022; 12(12):5921. https://doi.org/10.3390/app12125921

Chicago/Turabian Style

Kang, Yei-Jin, Dae-Won Kim, Xiangguo Che, Je-Yong Choi, and Seong-Gon Kim. 2022. "Inhibition of TP53 Mutant Oral Cancer by Reactivating p53" Applied Sciences 12, no. 12: 5921. https://doi.org/10.3390/app12125921

APA Style

Kang, Y.-J., Kim, D.-W., Che, X., Choi, J.-Y., & Kim, S.-G. (2022). Inhibition of TP53 Mutant Oral Cancer by Reactivating p53. Applied Sciences, 12(12), 5921. https://doi.org/10.3390/app12125921

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