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. 2018 Nov;17(11):2341-2352.
doi: 10.1158/1535-7163.MCT-17-1296. Epub 2018 Aug 30.

Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin

Yong Zhang  1 , Yinhui Dong  1   2 , Michael W Melkus  1 , Shutao Yin  1   2 , Su-Ni Tang  1 , Peixin Jiang  1 , Kartick Pramanik  1   3 , Wei Wu  1   3 , Sangyub Kim  3 , Min Ye  4 , Hongbo Hu  2 , Junxuan Lu  5   3   6 , Cheng Jiang  5   3
Affiliations

Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin

Yong Zhang et al. Mol Cancer Ther. 2018 Nov.

Abstract

Bufalin is a major cardiotonic compound in the traditional Chinese medicine, Chansu, prepared from toad skin secretions. Cell culture studies have suggested an anticancer potential involving multiple cellular processes, including differentiation, apoptosis, senescence, and angiogenesis. In prostate cancer cell models, P53-dependent and independent caspase-mediated apoptosis and androgen receptor (AR) antagonism have been described for bufalin at micromolar concentrations. Because a human pharmacokinetic study indicated that single nanomolar bufalin was safely achievable in the peripheral circulation, we evaluated its cellular activity within range with the AR-positive and P53 wild-type human LNCaP prostate cancer cells in vitro Our data show that bufalin induced caspase-mediated apoptosis at 20 nmol/L or higher concentration with concomitant suppression of AR protein and its best-known target, PSA and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Bufalin exposure induced protein abundance of P53 (not mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased senescence-like phenotype (SA-galactosidase). Small RNAi knocking down of P53 attenuated bufalin-induced senescence, whereas knocking down of P21CIP1 exacerbated bufalin-induced caspase-mediated apoptosis. In vivo, daily intraperitoneal injection of bufalin (1.5 mg/kg body weight) for 9 weeks delayed LNCaP subcutaneous xenograft tumor growth in NSG SCID mice with a 67% decrease of final weight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apoptosis or suppression of AR and PSA. Our data suggest potential applications of bufalin in therapy of prostate cancer in patients or chemo-interception of prostate precancerous lesions, engaging a selective activation of P53 senescence. Mol Cancer Ther; 17(11); 2341-52. ©2018 AACR.

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Conflict of interest statement

Conflict of interest statement: The authors declare no potential conflicts of interest.

Figures

Figure 1.
Figure 1.. Effects of bufalin on apoptosis in LNCaP, DU145 and PC-3 PCa cells and on AR, P53 and DDR signaling in relationship to apoptosis in LNCaP cells.
A, Chemical structure of bufalin. B. Concentration-dependent reduction of number of adherent LNCaP cells exposed to bufalin for 48 h. The data were shown as Mean ± SEM of crystal violet stain optical density, n=3 wells. C. Western blot detection of caspase-driven apoptotic marker (cleaved PARP) and P53 protein in LNCaP (wild type P53), DU145 (mutant P53) and PC-3 (P53 null) cells after bufalin treatment for 48 h. D, Western blot detection of c-PARP, AR, PSA, SRC-1, SRC-3 in LNCaP cells exposed to bufalin for 48 h. E. Growth suppression by bufalin on PC-3 cells estimated by crystal violet stain optical density, n=3 wells, Mean ± SEM. F, Western blot detection of P53, P21CIP1 and key DDR proteins in LNCaP cells exposed to bufalin for 48 h. G, Western blot detection of LC3 lipid modification as a marker of autophagy in LNCaP cells exposed to bufalin for 24 h and 72 h.
Figure 2.
Figure 2.. Effects of bufalin on cell cycle distribution and senescence-like phenotype in LNCaP cells.
A, Flow cytometry analyses of cell cycle distribution in LNCaP cells exposed to bufalin for 48 h and of mitotic specific protein marker histone H3 Ser 10 phosphorylation. B, Representative light-microscopic image of SA-β-gal activity using x-gal as substrate in LNCaP cells exposed to 20 nM of bufalin treatment for 3 days. C, Representative fluorescent-microscopic image of SA-β-gal activity using C12FDG as substrate in LNCaP cells exposed to 10 nM of bufalin for 9 days. D, Flow cytometry detection of SA-β-gal activity using C12FDG as substrate in LNCaP cells exposed to 20 nM of bufalin treatments for 4 days. Bar graph shows the summary of SA-β-gal activity enhancement by bufalin. The experiments were done in triplicate. The data were shown as Mean ± SEM. *: p < 0.05. E. Durable growth inhibition of LNCaP cells by 48 h bufalin exposure in a re-seeding experiment. After 48 h of pretreatment with ethanol or 20 nM of bufalin, the cells were rinsed with PBS and harvested by trypsinization, then the same number of “live” cells were re-plated into new 6-well plates with fresh complete medium. Adherent cells/colonies were visualized by crystal violet staining over 2 to 14 days.
Figure 3.
Figure 3.. Impact of knocking down P53/P21CIP1 axis on bufalin induction of apoptosis and senescence in LNCaP cells.
A, Western blot detection of P53 and P21CIP1 knocking down by siRNA and caspase-mediated PARP cleavage by bufalin after 24 h exposure. B, Western blot detection of P53 knocking down by siRNA 24 h after transfection and 5 days after initiation of bufalin treatment. C, Flow cytometry detection of SA-β-gal activity using C12FDG as substrate for scramble-RNA transfected vs. si-TP53-knockdown cells exposed to bufalin for 5 days.
Figure 4.
Figure 4.. Effect of once daily i.p. injection of bufalin on the growth of subcutaneously inoculated LNCaP xenograft tumors in NSG SCID mice.
A, Mouse body weight. B. Growth curve of LNCaP xenograft tumors by tumor volumes, non-tumor bearing mice were excluded (effective N=15,16). C, Ranking order of tumor weight of vehicle- and bufalin-treated mice. D. Mean tumor weight at necropsy. The data were shown as Mean ± SEM. vehicle-treated (N=16); bufalin-treated. (N=15). *: p < 0.05.
Figure 5.
Figure 5.. Biomarkers analyses of bufalin-treated LNCaP xenograft tumors.
A, H&E-stained 3rd-8th ranked LNCaP xenograft tumors from ethanol vehicle- and bufalin-treated groups, respectively. Large eosinophilic areas without nuclei in vehicle group represent necrotic regions (marked N in middle panel). Scale bars, 50 micron. B, Representative IHC images of Ki-67 proliferation marker and apoptosis marker (c-caspase 3), p-P53.Ser15 and AR in tumors from vehicle- or bufalin-treated mice. Black scale bars, 200 micron; Yellow scale bars, 100 micron. C, Western blot detection of cleaved-PARP, cleaved-caspase-3, p-P53.Ser15, AR and PSA in tumors from vehicle- or bufalin-treated mice. LNCaP cells exposed to 1 μM doxorubicin (a DNA damage drug) treatment for 24 h were used as a positive control for apoptosis (LN+Rx). PC3 extract and LNCaP extract (LN) were used as negative and positive control for AR and PSA, respectively. D, Representative images of SA-β-gal staining of cryo-sections of LNCaP xenograft tumors and the normal prostate of NSG SCID mice treated with vehicle or bufalin.
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