Potentiation of etoposide-induced apoptosis in HeLa cells by co-treatment with KG-135, a quality-controlled standardized ginsenoside formulation.

Our previous studies demonstrated that KG-135, a quality-controlled red ginseng-specific formulation containing approximately equal amounts of three major ginsenosides (Rk1, Rg3 and Rg5), down-regulated G1 cyclin-dependent kinase in HeLa cells. In the present work, we have found that KG-135 potentates cytotoxicity of etoposide by modulating apoptotic signaling. Co-treatment of etoposide and KG-135 markedly elevated the expression and phosphorylation at the serine 15 residue of p53 as well as the cellular levels of Bax and p21(Waf1/Cip1). The increased accumulation and phosphorylation of p53 (Ser15) were attenuated by treatment of cells with wortmannin, a pan-phosphatidylinositol-3 kinase inhibitor. Moreover, co-treatment of etoposide and KG-135 enhanced mitochondrial localization of Bax. Our results indicate that etoposide-induced apoptosis in HeLa cells can be potentiated in the presence of KG-135 through a mechanism that involves the stabilization of p53 and the stimulation of Bax- and p21-mediated apoptotic signaling pathways. These findings suggest that KG-135 represents a useful candidate adjuvant for the treatment of cancers that could potentially minimize the adverse effects of current clinical chemotherapeutics.

Xylocydine, a novel Cdk inhibitor, is an effective inducer of apoptosis in hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) frequently includes abnormalities in cell cycle regulators, including up-regulated cyclin-dependent kinase (Cdks) activities due to loss or low expression of Cdk inhibitors. In this study, we show that xylocydine, a cyclin-dependent kinase (Cdk) specific inhibitor, is a good anti-cancer drug candidate for HCC treatment. Xylocydine (50muM) selectively down-regulates the activity of Cdk1 and Cdk2, accompanied by significant cell growth inhibition in HCC cells. Xylocydine also strongly inhibits the activity of Cdk7 and Cdk9, in vitro as well as in cell cultures, that is temporally associated with apoptotic cell death in xylocydine-induced HCC cells. This is associated with inhibition of phosphorylation of RNA polymerase II at serine residues 5 and 2, which are targets of Cdk7 and Cdk9, respectively. The effects on apoptosis are concomitant with changes in the levels of anti-apoptotic proteins, Bcl-2, XIAP, and survivin, which are markedly down-regulated, and pro-apoptotic molecules, p53 and Bax, which are elevated in HCC cells after treatment with xylocydine. The up-regulated level of p53 was associated with increased stability of the protein, as levels of Ser15 and Ser392 phsophorylated p53 are similarly elevated in the inhibitor treated cells. We demonstrated that xylocydine can effectively suppress the growth of HCC xenografts in Balb/C-nude mice by preferentially inducing apoptosis in the xenografts, whereas the drug did not cause any apparent toxic effect on other tissues. Taken together, these data suggest that the novel Cdk inhibitor xylocydine is a good candidate for an anti-cancer drug for HCC therapy.

ATR (AT mutated Rad3 related) activity stabilizes Cdc6 and delays G2/M-phase entry during hydroxyurea-induced S-phase arrest of HeLa cells.

The Cdc6 protein, a key DNA replication initiation factor, contributes to the long-term maintenance of the S-phase checkpoint by anchoring the Rad3-Rad26 complex to chromatin. Here, we demonstrate that ATR (AT mutated and Rad3 related) activity is essential for maintaining high chromatin levels of the Cdc6 protein, thereby delaying entry into mitosis during hydroxyurea (HU)-induced S-phase arrest of HeLa cells. Downregulation of ATR (AT mutated and Rad3 related) (i.e., using ATR-siRNA) reduced the protein levels of chromatin Cdc6 and significantly increased the cellular levels of phospho-histone H3 (Ser 10), an index of mitosis. Downregulation of Cdc6 was completely restored by pretreatment with MG132, a proteasome inhibitor. Moreover, mitotic entry of MG132-pretreated cells was significantly downregulated. Our results also show that ATR (AT mutated and Rad3 related) kinase phosphorylates Cdc6 at serine residue 6. Thus, this ATR (AT mutated and Rad3 related)-mediated phosphorylation of Cdc6 is likely associated with stabilization of Cdc6 protein, thereby maintaining high levels of chromatin Cdc6 and delaying premature mitotic entry. This novel mechanism likely contributes to the functional regulation of chromatin Cdc6, which delays the cell cycle of hydroxyurea-induced cells to enter mitosis at the S-phase checkpoint.

Phosphorylation of Smac by JNK3 attenuates its interaction with XIAP.

Here we demonstrate that JNK3 can phosphorylate Smac. Smac phosphorylation attenuates its ability to activate apoptosome activity in HeLa S-100 cell lysates. Addition of the X-linked inhibitor of apoptosis protein (XIAP) to the S-100 markedly suppresses apoptosome activity, and this suppressive effect of XIAP is neutralized by adding unphosphorylated Smac, but not phosphorylated Smac. Furtherover, JNK3-mediated phosphorylation of Smac markedly attenuates the interaction between Smac and XIAP, as measured by BIACORE assays and non-denaturing gel shift assays. When JNK3 activity is down-regulated in etoposide-induced HeLa cells by transiently overexpressing a dominant negative version of JNK3 (DN-JNK3), the caspase-3 activity as well as PARP cleavages are markedly enhanced. And the interaction of Smac with XIAP also increases by down-regulating JNK3 activity under the same conditions. These results suggest that JNK3 activity can attenuate the progression of apoptosis through a novel mechanism of action, the down-regulation of interaction between Smac and XIAP.

Regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 by protein kinase Cdelta-mediated phosphorylation.

Cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1(-/-))-null mice have an increased incidence of tumor formation. Here, we demonstrate that p21(WAF1/CIP1) is unstable in HeLa cells treated with siRNA duplexes that target PKCdelta. PKCdelta phosphorylates p21(WAF1/CIP1 )at a serine residue ((146)Ser) located in its C-terminal domain. In cells treated with 12-O-tetradecanoylphorbol 13-acetate, the levels of both p21(WAF1/CIP1) and its (146)Ser-phosphorylated form increased significantly. We also show that a substitution, resulting from a single nucleotide polymorphism (SNP) at (149)Asp found in certain cancer patients, strongly compromises PKCdelta-mediated phosphorylation at (146)Ser and results in cells that are relatively resistant to TNFalpha-induced apoptosis. Thus, post-translational phosphorylation of p21(WAF1/CIP1) is important from an apoptotic cell death, and may also have patho-physiological relevance for the development of human cancer.

Ginsenoside-Rh2-induced mitochondrial depolarization and apoptosis are associated with reactive oxygen species- and Ca2+-mediated c-Jun NH2-terminal kinase 1 activation in HeLa cells.

We show here that Ca(2+) and reactive oxygen species (ROS) are involved in the up-regulation of c-Jun NH(2)-terminal kinase 1 (JNK1) activity during apoptosis induced by ginsenoside Rh2 (G-Rh2) in HeLa, MCF10A-ras, and MCF7 cells. Addition of antioxidants such as N-acetyl-l-cysteine or catalase attenuates G-Rh2-induced ROS generation, JNK1 activation, and apoptosis. The overexpression of catalase down-regulates caspase-3 and JNK1 activities. G-Rh2 treatment of cells results in mitochondrial depolarization, second mitochondrial activator of caspase release, and translocation of Bax into the mitochondria, and these events are inhibited by antioxidants. Ca(2+) is also involved in mitochondrial depolarization during G-Rh2-induced apoptosis. These results suggest that ROS and Ca(2+) are important signaling intermediates leading to stress-activated protein kinase/extracellular signal-regulated kinase kinase 1/JNK1 activation and depolarization of the mitochondrial membrane potential in G-Rh2-induced apoptosis.

Caspase-3-mediated cleavage of Cdc6 induces nuclear localization of p49-truncated Cdc6 and apoptosis.

We show that Cdc6, an essential initiation factor for DNA replication, undergoes caspase-3-mediated cleavage in the early stages of apoptosis in HeLa cells and SK-HEP-1 cells induced by etoposide, paclitaxel, ginsenoside Rh2, or tumor necrosis factor-related apoptosis-inducing ligand. The cleavage occurs at the SEVD442/G motif and generates an N-terminal truncated Cdc6 fragment (p49-tCdc6) that lacks the carboxy-terminal nuclear export sequence. Cdc6 is known to be phosphorylated by cyclin A-cyclin dependent kinase 2 (Cdk2), an event that promotes its exit from the nucleus and probably blocks it from initiating inappropriate DNA replication. In contrast, p49-tCdc6 translocation to the cytoplasm is markedly reduced under the up-regulated conditions of Cdk2 activity, which is possibly due to the loss of nuclear export sequence. Thus, truncation of Cdc6 results in an increased nuclear retention of p49-tCdc6 that could act as a dominant negative inhibitor of DNA replication and its accumulation in the nucleus could promote apoptosis. Supporting this is that the ectopic expression of p49-tCdc6 not only promotes apoptosis of etoposide-induced HeLa cells but also induces apoptosis in untreated cells. Thus, the caspase-mediated cleavage of Cdc6 creates a truncated Cdc6 fragment that is retained in the nucleus and induces apoptosis.