A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

<div><p>BF211 is a synthetic molecule derived from bufalin (BF). The apoptosis-inducing effect of BF211 was stronger than that of BF while the acute toxicity of BF211 was much lower than that of BF. BF211 exhibited promising concentration-dependent anti-cancer effects in nude mice inoculated with A549 cells <i>in vivo</i>. The growth of A549 tumor xenografts was almost totally blocked by treatment with BF211 at 6 mg/kg. Notably, BF and BF211 exhibited differences in their binding affinity and kinetics to recombinant proteins of the α subunits of Na+/K+-ATPase. Furthermore, there was a difference in the effects of BF or BF211 on inhibiting the activity of porcine cortex Na+/K+-ATPase and in their time-dependent effects on intracellular Ca2+ levels in A549 cells. The time-dependent effects of BF or BF211 on the activation of Src, which was mediated by the Na+/K+-ATPase signalosome, in A549 cells were also different. Both BF and BF211 could induce apoptosis-related cascades, such as activation of caspase-3 and the cleavage of PARP (poly ADP-ribose polymerase) in A549 cells, in a concentration-dependent manner; however, the effects of BF211 on apoptosis-related cascades was stronger than that of BF. The results of the present study supported the importance of binding to the Na+/K+-ATPase α subunits in the mechanism of cardiac steroids and also suggested the possibility of developing new cardiac steroids with a stronger anti-cancer activity and lower toxicity as new anti-cancer agents.</p></div

<i>In vivo</i> anti-cancer effects of BF and BF211 in nude mice inoculated with A549 cells.

<p>(A) The tumor growth curve of nude mice treated with vehicle control, rapamycin (positive control), BF or BF211 at similar concentrations as indicated. (B) The tumor growth curve of nude mice treated with vehicle control, rapamycin (positive control), and BF211 at concentrations of 2, 4, and 6 mg/kg.</p

Acute toxicity of BF211 in mice.

<p>Acute toxicity of BF211 in mice.</p

Effects of BF or BF211 on caspase-3 activation and PARP cleavage in A549 cells.

<p>(A) <b>The r</b>epresentative western blot assay results probing the levels of caspase-3 and PARP in cells treated with BF or BF211 at different concentrations for 24 h. (B) The quantification of the western blot assay results of the activation of caspase-3 (percentage of cleaved caspase-3 from the total caspase-3) and cleavage of PARP (percentage of cleaved PARP from the total PARP) in cells treated with BF or BF211 at different concentrations for 24 h. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments. *<i>p</i><0.05 vs. BF-treated group. (C) The activity of caspase-3 in cells treated with BF or BF211 at different concentrations for 24 h. The data comprise the statistical results (n = 3, mean ± SEM) of three independent experiments. *<i>p</i><0.05 vs. BF-treated group.</p

Inhibitive effects of BF or BF211 on the activity of porcine cortex Na+/K+-ATPase and the intracellular Ca2+ level of A4549 cells.

<p>(A) The activity of Na⁺/K⁺-ATPase extracted from porcine cerebral cortex in the presence of BF or BF211 at different concentrations was determined. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments. (B) The level of intracellular Ca2+ level in A549 cells treated with 50 nM BF or BF211 for different time periods. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments.</p

Expression of Na+/K+-ATPase α subunits in A549 cells and binding affinity of BF or BF211 to Na+/K+-ATPase α subunits.

<p>(A) The results of the RT-PCR analysis of the expression levels of the α subunits of Na+/K+-ATPase in A549 cells. The data comprise the statistical results (n = 3, mean ± SEM) of three independent experiments. (B) The representative results of the western blot assay probing the protein levels of the α1 and α3 subunits of Na+/K+-ATPase in A549 cells. (C) The real time binding affinity measurements of BF or BF211 to the recombinant Na+/K+-ATPase α subunits. The binding ability between the compound and the recombinant protein is reflected in recorded response unit (RU) values.</p

Parameters of binding between BF or BF211 with α1, α2 or α3 recombinant subunit protein of Na+/K+-ATPase.

<p>Parameters of binding between BF or BF211 with α1, α2 or α3 recombinant subunit protein of Na+/K+-ATPase.</p

Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells

<div><p>Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix <i>Stephaniae tetrandrae</i> S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity <i>in vitro</i>. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit.</p></div

Biacore assay results of binding affinity between recombinant human proteasome β1 subunit and fangchinoline or tetrandrine.

<p>Biacore assay results of binding affinity between recombinant human proteasome β1 subunit and fangchinoline or tetrandrine.</p

Fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis in PC-3 cells.

<p>*P<0.05 compared with control (dose at 0 μM).</p><p>Fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis in PC-3 cells.</p