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Jiang et al. Hepatoma Res 2019;5:5 I http://dx.doi.org/10.20517/2394-5079.2018.97 Page 9 of 11

cancer cells apoptosis in a p53-dependent manner. To functionally validate SPx, we subjected them and
their unstapled control to a cell viability assay in a FBS-free medium using a pair of cell lines with the same
-/-
+/+
genetic background carried wild-type p53 (HCT116 p53 ) or deleted p53 (HCT116 p53 ). While the control
peptide exhibited no anti-proliferative activity against both cell lines at concentrations of up to 100 μmol/L,
+/+
SP3 displayed dose- and p53-dependent growth inhibitory activity against HCT116 p53 , but not HCT116
p53 , with an IC50 value of ~18.6 μmol/L at 72 h [Figure 4A and B]. Besides, SP3 also showed an obvious
-/-
suppression for a hepatocellular carcinoma cell line carried wild-type p53, named Sk-Hep-1, with an IC50
value of ~24.8 μmol/L in the absence of serum [Figure 4C and Supplementary Figure 2]. Consistent with this
+/+
result, the induction of apoptosis of HCT116 p53 and SK-Hep-1 cells by SP3 was verified by fluorescence-
activated cell sorting [Figure 4D-I]. Taken together, these findings support that SP3 actively traversed the
cell membrane and killed tumor cells by reactivating the p53 pathway. It is worth pointing out that as is
often the case with other stapled peptide activators, although its efficient blocking the interaction between
+/+
p53-MDM2/MDMX, are rather weak in killing HCT116 p53 and SK-Hep-1 cells. The weak in vitro activity
implies that stapling alone is insufficient to achieve optimal therapeutic efficacy of helical peptides, dictated
by cell internalization, endosomal escape, proteolytic stability, spatio-temporal distribution, etc.

DISCUSSION
The tumor suppressor protein p53 induces powerful cancer cell antiproliferation and apoptotic responses
[4]
to cellular stress, plays a pivotal role in preventing damaged cells from cancerous . Not surprisingly, the
impairment of p53 signaling pathway is a hallmark of almost all human cancers, where either the TP53 gene
is mutated or wild-type p53 is functionally inactivated by the E3 ubiquitin ligase MDM2 and its homolog
MDMX [37,38] . In many tumor cells harboring wild-type p53, the up-regulated MDM2 and/or MDMX often
cooperate to inhibit p53 transactivation activity and urge p53 for degradation, conferring tumor development
[2]
and progression . A great number of studies have validated that MDM2 and/or MDMX antagonism as a
viable therapeutic regimen for cancer therapy, and several small-molecule antagonists specific for MDM2
are in various phases of clinical trials [39,40] . As for HCC, our results in Figure 1 further illustrates that p53-
MDM2/MDM2 is an important target for therapy, thus, the development of potent antagonists specific for
MDM2 and MDMX is meaningful for HCC therapy.

Growing evidence suggests that the interplay between MDM2 and MDMX confers robust p53 inactivation
in tumorigenesis and that antagonizing both MDM2 and MDMX affords a powerful, synergistic and
sustained inhibition of tumor growth [41,42] . However, traditional small-molecule drugs are always limited
by the comparatively small interaction area, resulting in the failure as dual specific inhibitors to target both
MDM2 and MDMX simultaneously. To this end, a peptide therapeutics termed PMI was developed to
competes with p53 for MDM2 and MDMX binding at high affinity [14,43] . However, major pharmacological
hurdles still impede the development of anticancer peptide therapeutics with optimal therapeutic efficacy,
including: short circulation half-life due to proteolytic degradation and poor cellular uptake. To overcome
these technical obstacles, we developed a novel peptide stapling method to link the side chains of Cys and
Cys at (i, i+4) positions by two bromine methyl group in benzene para-, ortho- or meta- positions to form
the dimethylbromobenzene-cysteine structure. Of note, this stapling method is appropriate for all α-helix
after mutating two nonfunctional residues into Cys at (i, i+4) positions. After a series of characterization
and functional verification, SP3, a stapled PMI crosslinked the side chains by 1,4-dimethylbromobenzene,
can potently inhibit the growth of cancer cell in a p53 dependent manner. Of note, the remaining position
of the benzene in the dimethylbromobenzene can be further modification for more hydrophilic and more
appropriate charge characteristics.

In this work, we found that the expression of p53, MDM2 and MDMX were closely related to the survival
of ~70% HCC patients carrying wild-type p53, and provided strong evidence that reactivating p53 from
MDM2 and MDMX was a potentially feasible program for HCC therapy. After that, we have developed a