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Page 4 of 11 Jiang et al. Hepatoma Res 2019;5:5 I http://dx.doi.org/10.20517/2394-5079.2018.97

Fluorescence polarization-based competitive binding assays were performed in Microfluor® 2, 96-well
black plates (Thermo Fisher Scientific) and readings were taken using a Tecan Infinite M2000 fluorescence
plate reader. Serially diluted Lupbin or corresponding peptide were prepared in Tris-HCl buffered saline
(10 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, pH 7.0) and incubated with 200 nmol/L 15-29 p53-FITC/

MDM2 or 50 nmol/L 15-29 p53-FITC/MDMX in a total volume of 150 μL per well. After 2 h incubation at
room temperature, fluorescence polarization was measured at λex = 470 nm and λem = 530 nm. Nonlinear
regression analyses were performed to give rise to IC50 values.

Cell culture and cell viability analysis
+/+
Human colon cancer cell line HCT116 (wild-type p53) was purchased from ATCC, and maintained in
-/-
McCoy’s 5A medium with 10% FBS. The isogeneic HCT116 (p53 deletion) cells were presented by Prof.
Bert Vogelstein of Johns Hopkins University (Baltimore, MD), and maintained in McCoy’s 5A medium with
10% FBS. human hepatoma cell line SK-Hep-1 was also purchased by ATCC, and maintained in DMEM
with 10% FBS. All cells were maintained at 37 °C in an atmosphere of 5% CO . For cell viability test, three
2
cell lines were plated in 96-well plates at a density of 2,500 cells/well (100 μL). After 24 h, cells were treated
with drug sample at the indicated concentrations and times in FBS-free mediums, respectively. The in vitro
cytotoxicity was then measured by using a standard MTT (Thermo Fisher scientific) assay after 72 h drug
treatment.

Apoptosis analysis
Necrosis/apoptosis was evaluated by flow cytometric analysis using the FITC Annexin V Apoptosis
Detection Kit (BD Biosciences). Briefly, cells were treated with samples for 48 h. Cells were then harvested,
6
washed twice with cold PBS, and re-suspended in 1× binding buffer at a concentration of 1 × 10 cells/mL.
One hundred microliters of the solution (1 × 10 cells) was transferred to a 5 mL culture tube, followed by
5
addition of 5 µL of FITC Annexin V and 5 µL of PI. After gentle vortexing and a 15-min incubation in the
dark at room temperature, 400 µL of 1× binding buffer was added to the tube, and cells were analyzed by
fluorescence-activated cell sorting (FACS).

RESULTS
Wild-type p53 is a feasible target for HCC therapy
Research has shown that the tumor suppressor p53 has an important role in tumor progression, and that it
[2]
is mutated or functionally inactivated in most human cancers . As for HCC, p53 was nonsynonymously
mutated in 259 (29.9%) of 867 hepatoma cases in TCGA [Figure 1A], suggesting that in a substantial
proportion of HCC, TP53 (which encodes p53) is wild type but the protein is inactivated. To explore
the importance of p53 and its two agonists- MDM2 and MDMX- in the HCC process, we analyzed the
relationship between these protein expression and survival of HCC patients carried wild-type p53. As
shown in Figure 1B and C, decreased expression of p53 was significantly associated with poor patient
overall survival (P = 0.043) and disease-free survival (P = 0.037). It is well-known that the tumor suppressor
activity and in vivo stability of p53 are abrogated by regulatory molecules such as the E3 ubiquitin ligase
MDM2 and its homologue MDMX (also known as HDMX and MDM4) [Figure 1D] [28,29] . This offers an
attractive strategy for cancer therapy based on p53 reactivation by blocking the interaction between p53 and
MDM2 (MDMX). In this case, the two major negative regulators of p53 now seem to be “druggable”, and
[2]
recent studies in cancer patients have provided proof-of-concept for this approach . To further verify the
feasibility of p53 restoration via MDM2 and MDMX blocking for HCC therapy, we attempted to evaluate
the association of MDM2 and MDMX expression with survival in 209 HCC patients carried wild-type p53.
As expected, the 5-year overall survival [Figure 1E] and disease-free survival [Figure 1F] rates of MDM2
high-expressed cases are significantly higher than that of MDM2 low-expressed cases. Meanwhile, MDMX
showed the same tendency as MDM2 [Figure 1G and H]. Collectively, all these results demonstrated that
high-level p53 is beneficial to the survival of HCC patient, thus p53 restoration was a potentially feasible
program for HCC therapy in p53-wild-type patients.