# Inhibition of _Tp53_ Mutant Oral Cancer by Reactivating p53
## 4 Discussion
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 4 HR 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 xenografts 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 4 HR 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 4 HR increased serum ERK8 levels compared to the untreated control (Table 1). In a previous study, 4 HR 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 4 HR 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 4 HR 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 4 HR 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 4 HR administration might be associated with functional recovery of p53. The expression level of HDAC4 was reduced significantly by the administration of 4 HR (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 4 HR administration (Figure 2). Ac-p53(Lys319) is important for the stress mediated p53 stabilization [34]. 4 HR administration may have therapeutic potential in a different cancer type. In a previous study, 4 HR showed anti-tumour effects via stimulating the differentiation of SCC-9 cells (tongue cancer cells) [35; 36]. In addition, 4 HR inhibits NF-kappaB phosphorylation and has a synergistic anti-tumour effect with cisplatin in KB cells (nasopharyngeal carcinoma) [12]. 4 HR administration suppresses the cellular growth of oral mucosal melanoma cells [37].