br P plays an important role in
P53 plays an important role in a variety of life activities, including apoptosis (Lopes et al., 2018; Mantovani et al., 2017). To investigate the eﬀect of buforin IIb on p53 function, we firstly examined the mRNA and protein level of p53. As shown in Fig. 3A, the transcriptional ac-tivity of p53 and its protein level are increased by buforin IIb in a dose-dependent manner. Meanwhile, we further examined the mRNA ex-pression of p53 target genes by qPCR, the mRNA level of p53 target genes (Dr5, fas, p21 and puma) were all up-regulated under the treat-ment of buforin IIb (Fig. 3B). Moreover, after knockdown p53 expres-sion, we found the induction of p53 target genes, bax and bcl-2 by buforin IIb was clearly inhibited (Fig. 3C). Collectively, these findings demonstrated that buforin IIb could enhance p53 expression and pro-mote p53 functions to induce apoptosis.
3.4. Role of p53 in cytotoxicity of buforin IIb
To confirm the relationship between cytotoxicity and p53 under the treatment of buforin IIb on PC-3 and Du-145 cells, we detected the cell viability and the apoptosis proteins by knockdown p53, Protease Inhibitor Cocktail were transfected with p53 siRNA for 24 h and then treated with buforin IIb for another 24 h. As shown in Fig. 4A, compared to the control group, the cell viability of p53 knockdown groups was increased after buforin IIb treatment, especially the #2 p53 siRNA (Fig. 4A). Furthermore, we also found that Bax and PARP cleavage were decreased while Bcl-2 was increased after p53 knockdown (Fig. 4B). Together These results sug-gested that buforin IIb may act in a p53-dependent manner.
Buforin IIb (21-aa peptide) derived from histone H2A, has been reported to exhibit a strong antimicrobial and anticancer activity (Park et al., 2000; Lee et al., 2008). The protein structure prediction software ProtParam was used to analyze the physicochemical parameters of buforin II and buforin IIb. The GRAVY index refers to the hydrophilicity of a polypeptide and Aliphatic index can be used to speculate on the
Fig. 4. Role of p53 on PCa cell toxicity of buforin IIb. (A) Eﬀect of p53 knockdown on cytotoxicity of buforin IIb. PCa cells were transfected with p53 siRNA (si p53 #1, si p53 #2 and si p53 #3) or negative siRNA for 24 h, and then treated with 8 μM buforin IIb for another 24 h. Cell viability were normalized with the cells without buforin IIb. (B) Eﬀect of p53 knockdown on the induction of apoptosis proteins and PARP cleavage by buforin IIb. β-Actin was used as internal control. Error bars represent the mean ± SD. **p < 0.01; ***p < 0.001.
thermal stability of a peptide. As shown in supplementary Table1, bu-forin II and buforin IIb are cationic peptides with good thermal stabi-lity. Buforin IIb has more positive charges and the thermal stability of buforin IIb is also stronger than that of buforin II. From the data of GRAVY and hydrophobicity ratio, both peptides showed high hydro-phobicity, and the amphiphilicity of buforin IIb is stronger than that of Buforin II. In addition, an increase in the antimicrobial activity of a linear α-helical antimicrobial peptide has been shown to correlate closely with an increase in α-helical secondary structure (Park et al., 2000). Park et al. and Jang et al. reported that the hybrid peptide bu-forin IIb, which showed stronger antimicrobial activity than Buforin II (Table S2), had the higher α-helical content (55% and 72%, respec-tively) (Table S3) (Park et al., 2000; Jang et al., 2012). Furthermore, the hemolytic properties of buforin IIb was less than 25% even at high concentrations of 80 μM (Table S4). In previous works, buforin IIb ex-hibited relatively higher cytotoxicity against breast cancer cells than against normal cells. The selectivity of buforin IIb could be related to the presence of O-, N-glycoproteins and gangliosides on the surface of breast cancer cells, which are binding target proteins of ABPs (Han et al., 2013). However, the molecular mechanism for buforin IIb un-derlying anticancer activity in PCa is still unknown. In the present study, we investigated the anticancer eﬀects and mechanism of action of buforin IIb in PC-3 and Du-145 cells.
Our research indicated that buforin IIb markedly inhibited PC-3 and Du-145 cell proliferation in a dose-dependent manner, and its IC50 was less than 8 μM (Fig. 1B). In addition, we also found that buforin IIb significantly reduced PC-3 and Du-145 cells clonogenicity (Fig. 1C). Furthermore, buforin IIb showed little eﬀect on the human normal cell line (RWPE and 293T) up to 60 μM (Fig. 1D), and our previous data have showed buforin IIb exhibited 20% cytotoxicity to mice hepato-cytes at a concentration of 60 μM (Han et al., 2013), which is consistent with the Jang's data, Jang et al. found that buforin IIb killed 35.8% of fibroblasts at 200 μg/ml (Jang et al., 2012). These results showed that buforin IIb could selectively inhibit the proliferation ofAIPC cell lines. In tumor cells, the induction of apoptosis is critical events of che-motherapeutic agents, Therefore, we tested cell apoptosis in buforin IIb -treated PC-3 and Du-145 cells. The results showed that buforin IIb induced PCa cell apoptosis (Fig. 2A). Apoptotic cell death was verified by cleavage of apoptotic proteins pro-caspase 9/8/3 and PARP (Fig. 2B). The apoptotic process always activates the expression of p53 and its target genes, thereby inhibiting cell proliferation. Our data showed that the mRNA and protein levels of p53 were increased by buforin IIb in treated cells (Fig. 3A), the same trend was found in P53 dependent target genes (Fig. 3B and C). Fas initiate the extrinsic pathway, resulting in complete apoptosis in a cell through DISC as-sembly (Wang et al., 2010; Verlekar et al., 2018). Puma could make