Studies On The Mechanisms Of Pseudolaric Acid B-Induced Growth Inhibition In Tumor Cells

Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae), known as "TU-Jin-Pi" in Chinese, which has been used to treat dermatological fungi infections. PAB exerts potent antitubulin, antitumor, antifertility. In this study the mechanism of PAB in murine fibrosarcoma L929 cells and human breast cancer MCF-7 cells was investigated.In human breast cancer MCF-7 cells, PAB induced apoptosis, autophagy, senescence and mitotic arrest. The half maximal inhibitory concentration IC50 was 3.4 and 1.35μM at 36 and 48 h after PAB treatment, respectively. The MCF-7 cells exposed to PAB showed typical characteristics of apoptosis, including the morphologic changes and DNA fragmentation. And MCF-7 cells treated with 4μM PAB for 36 h underwent apoptosis, but not necrosis. The apoptosis induced by PAB was independent of the Fas death receptor pathway. Together with PAB, mitogen-activated protein kinases (MAPKs) including p38, JNK and ERK did not participate in PAB-induced necrosis. P38 had no obvious function on apoptosis after 4μM PAB treatment for 36 h, but PAB induced JNK phosphorylation and inhibited ERK phosphorylation in the apoptotic process. In this study the inhibitor of protein tyrosine kinases (PTKs), genistein, inverted the inhibitory effect of PAB, instead promoting the survival of MCF-7 cells. Like genistein, another PKTs inhibitor AG1024 had similar effect on PAB-treated MCF-7 cells, indicating that PAB activated PTKs to induce apoptosis. Together with PAB, genistein increased the expression of p-ERK, and decreased the expression of JNK and p-JNK in PAB-treated MCF-7 cells at 36 h. In apoptotic process induced by PAB, parts of live cells escaped from apoptosis through autophagy, at last became senescence. The survival cells at 36 h had positive MDC staining, and the expression of Beclin 1 was up-regulated, the conversion of LC3 I to LC3 II happened after PAB treatment, which marked autophagy. And when MCF-7 cells was treated with PAB for 3 d, then culture for 5 d in fresh medium, the survived cells became larger and flatter, and the SA-β-galactosidase staining was positive. Meanwhile at 36 h PAB up-regulated nuclear cyclinB1 and cdc2 to induce mitotic arrest, and the expression of p21 and p53 was up-regulated after PAB treatment, therefore p21 and p53 might be involved in mitotic arrest. And this mitotic arrest might be related to autophagy, apoptosis and senescence.In murine fibrosarcoma L929 cells, PAB induced autophagy, senescence and mitotic arrest, but no apoptosis. At 36 h, the inhibitory ratio of 80μM PAB was 65.37±4.12%, and 80μM PAB had positive MDC staining, up-regulated the expression of Beclin 1, and promoted the conversion of LC3 I to LC3 II, which marked autophagy, But PAB did not induce the appearance of apoptotic bodies. PAB had no effect on mitochondrial membrane potential (MMP), but up-regulated the expression of Bax, Bcl-xl and Bcl-2. Immunoprecipitation analysis showed that PAB inhibited the binding of Bcl-2 with Beclin 1. Additionally PAB inhibited the localization of Bax in mitochondria, but Bcl-2 and Bcl-xl were still in mitochondria to sustain MMP. Meanwhile PAB promoted the phosphorylation of cytoplasmic Bcl-2. Therefore the phosphorylation of Bcl-2 in cytoplasm and the mitochondrial location of Bcl-2 might be the reasons why PAB inhibited the binding of Bcl-2 with Beclin 1. PAB did not activate caspase 9 and caspase 8, which were the downstream of mitochondrial pathway of apoptosis and the downstream of Fas death receptor pathway, respectively, for induction of autophagy. Meanwhile caspase 3, the downstream of caspase 9 and caspase 8, was not activated or involved in autophagy after PAB treatment, but when the inhibitor of caspase family proteins was applied, it was found that together with PAB, the inhibtor of caspase family proteins inhibited the conversion from LC3 I into LC3 II, decreased autophagic ratio and increased apoptotic and necrotic ratio contrasting with PAB. While together with PAB, staurosporine, the inhibitor of PKC, enhanced apoptosis. Another PKC inhibitor Ro-31-8220 has the similar effect with staurosporine. After PAB treatment, L929 cells displayed the shape of giant cells, but addition of staurosporine abolished the effect of PAB. PAB promoted the generation of multinuclear cells and the accumulation of 4 n cells, but together with staurosporine, PAB did not induce those phenomenons. It was speculated that the appearance of giant cell might be due to the appearance of multinuclear cells and the accumulation of 4 n cells after PAB treatment, and PKC was involved in cytostatic effect, multinuclear cell formation and the accumulation of 4 n cells induced by PAB. PAB up-regulated the expression of nuclear cyclinB1 and cdc2, and inhibited their degradation in nuclei, proving PAB effect on L929 cell arrest at mitosis. Meanwhile, PAB increased the expression of p53 and p21, indicating that p53 and p21 promoted mitotic arrest of PAB-treated L929 cells. But in presence of staurosporine, these PAB effects were abolished, indicating that PKC was involved in mitotic arrest through regulating the expression of p53, p21, nuclear cyclinB1 and cdc2. Meanwhile when L929 cells were treated with PAB for 3 d, the survived cells became larger and flatter, and the SA-β-galactosidase staining was positive, meaning senescence.