Transient treatment with CDK inhibitors eliminates proliferative potential even when their abilities to evoke apoptosis and DNA damage are blocked

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell-cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.     

Calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of protein kinase A and protein kinase A-mediated stabilization of Mcl-1 and X-linked inhibitor of apoptosis (XIAP)

Human neutrophils underwent spontaneous apoptosis, which was accompanied with proteasome-mediated degradation of Mcl-1 and X-linked inhibitor of apoptosis (XIAP). Calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) prevented spontaneous neutrophil apoptosis and degradation of Mcl-1 and XIAP, and the effects of calpain inhibitors on neutrophils were resistant to cycloheximide. Calpain inhibitors induced protein kinase A (PKA) activation, which was unaccompanied with an increase in intracellular cyclic AMP. Calpain inhibition-mediated delayed neutrophil apoptosis, stabilization of Mcl-1 and XIAP, and phosphorylation of PKA substrates were suppressed by H-89 (specific PKA inhibitor). These findings suggest that calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of PKA and PKA-mediated stabilization of Mcl-1 and XIAP.     

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser². This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.     

Inhibition of Cyclin-dependent Kinase-2 Induces Apoptosis in Human Diffuse Large B-cell Lymphomas

Cyclin-dependent kinase (cdk) inhibitors have the potential to induce growth arrest and apoptosis in cancer cells. The genes encoding cdks involved in G1-S progression are often amplified in B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Here, we evaluated the in vitro cytotoxic activity of the cdk2 inhibitor CVT-313 against several human DLBCL cells. Treatment of DLBCL cells with CVT-313 resulted in apoptosis. CVT-313 treatment reduced cdk2-mediated phosphorylation of the retinoblastoma gene product (Rb) on T821, but did not affect cyclin D-cdk4/6-mediated Rb phosphorylation on S807/811. Depletion of endogenous cdk2 by short interfering (si)RNA also resulted in apoptosis in human LY3, LY8 and LY18 DLBCL cells. Importantly, inhibition of cdk2 with CVT-313 or knockdown of endogenous cdk2 with siRNA resulted in down-regulation of the anti-apoptotic factor Myeloid cell leukemia-1 (Mcl-1), suggesting that decreased levels of cellular Mcl-1 contribute to apoptosis. In support of this, siRNA-mediated knockdown of Mcl-1 was sufficient to induce apoptosis in LY3 and LY18 DLBCL. Further, cdk2 inhibition led to decreased Mcl-1 mRNA levels, which was proceeded by reduced phosphorylation of serine 2 on the carboxyl terminal domain (CTD) of RNA polymerase II. Taken together, these data suggest that cdk2 activity is necessary for the survival of human DLBCL.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.     

Cyclin-dependent Kinase-1 (Cdk1)/Cyclin B1 Dictates Cell Fate after Mitotic Arrest via Phosphoregulation of Antiapoptotic Bcl-2 Proteins

The prevailing model suggests that cell fate after mitotic arrest depends on two independent and competing networks that control cyclin B1 degradation and the generation of death signals. However, recent evidence for Cdk1/cyclin B1-mediated phosphorylation and inactivation of antiapoptotic Bcl-2 proteins suggests the existence of significant cross-talk and interdependence between these pathways. Further, the nature of the mitotic death signals has remained elusive. In this study, we sought to test the hypothesis that fate after mitotic arrest is dictated by the robustness of Cdk1/cyclin B1 signaling to Bcl-2 proteins and to identify signals that may represent a mitotic death signature. We show that when treated with Taxol, slippage-resistant HT29 colon carcinoma cells display robust Cdk1 activity and extensive Mcl-1/Bcl-x_L phosphorylation and die in mitosis, whereas slippage-prone DLD-1 colon carcinoma cells display weak Cdk1 activity and partial and transient Mcl-1/Bcl-x_L phosphorylation and die in subsequent interphase or survive. Furthermore, modulation of this signaling axis, either by inhibition of Cdk1 in slippage-resistant HT29 or by enforcing mitotic arrest in slippage-prone DLD-1 cells, evokes a switch in fate, indicating that the strength of Cdk1 signaling to Bcl-2 proteins is a key determinant of outcome. These findings provide novel insight into the pathways that regulate mitotic death, suggest that the robustness of these signaling events may be useful as a marker to define susceptibility to antimitotic drugs, and encourage a revision in the current model describing fate after mitotic arrest.     

Relationship of Mcl-1 isoforms,ratio p21WAF1/cyclin A,and Jun kinase phosphorylation to apoptosis in human breast carcinomas

Full length Mcl-1 is an anti-apoptotic protein consisting of two closely migrating 42/40kDa species. We now investigated the relationship of these isoforms to the expression of cell cycle stimulatory (cyclin A) and inhibitory (p21WAF1) proteins and to the induction of apoptosis in wt p53 MCF-7 and mutant p53 SKBR3 human breast carcinomas. The latter cells exhibited lower 42kDa Mcl-1, higher expression of cyclin A relative to that of p21WAF1, and apoptosis in response to okadaic acid, a phosphatase 1/2A inhibitor. The proteasome inhibitor MG-115 selectively increased expression of the 40kDa Mcl-1 isoform and induced p21WAF1, but also promoted preferential apoptosis in SKBR3 cells. Neither okadaic acid nor MG-115 caused comparable effects in MCF-7 cells. However, vanadate or acetyl furanonaphthoquinone induced the 40kDa Mcl-1 and greater Jun kinase (JNK) phosphorylation without apoptosis-associated PARP fragmentation in MCF-7 cells. Our data suggest that the higher susceptibility of SKBR3 cells to undergo apoptosis may be partly due to their greater proliferative potential (cyclin A), low expression of the anti-apoptotic 42kDa Mcl-1 isoform, and suboptimal JNK activation in response to stress.