Apoptosis, cellular division or mitotic catastrophe? Effects of kinase inhibition and DNa damage in lung cancer cells

Abstract

Non-small cell lung cancer (NSCLC) cells are generally characterized by a low response to conventional anti-cancer agents, such as DNA-damaging drugs. Treatments that induce DNA damage relies on cellular signalling resulting in the induction of growth arrest and apoptotic cell death. Deregulation of apoptosis contributes to resistance and failure of death execution may occur even in cases when all components of the apoptotic machinery are present in the cell. One possible reason for the inhibition of cell death is aberrant signalling of kinases, favouring cell survival. The main goal of this project was to understand the function of the apoptotic machinery in human lung cancer cells resistant to anti-cancer treatment and to investigate if the efficiency of the killing of these cells could be modified by using inhibitors of phosphorylation-based signalling. The NSCLC cell line U1810 is resistant to induction of apoptosis by conventional DNA damage-inducing anti-cancer treatments; however these cells are sensitive to the action of the broad range protein kinase inhibitor staurosporine (STS).

The two STS analogues PKC 412 and Ro 31-8220 are more specific protein kinase C (PKC) inhibitors compared to STS. The anti-tumour effects of PKC 412 and Ro 31-8220, when used alone or in combination with DNA-damaging treatments, were investigated. Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to treatment with the DNA-damaging drug etoposide. PKC 412 induced death of a small number of U1810 cells, and sensitized cells to gamma radiation and etoposide. The difference in cell death induction upon treatment with Ro 31-8220 and PKC 412 could not be explained by a difference in the potency of PKC inhibition of the two STS analogues. Interestingly, Ro 31-8220 increased, whereas PKC 412 decreased, activity-related phosphorylation of the protein kinase Akt. Moreover, etoposide increased phosphorylation of Akt, which could be reversed by PKC 412. Inhibitors (wortmannin and LY-294002) of the Akt upstream activator PI3-kinase had a similar effect on U1810 cell as PKC 412. LY-294002, wortmannin and PKC 412 promoted apoptosis in a cell cycle dependent manner. LY-294002 and wortmannin induced apoptosis in late mitosis, whereas PKC 412 treatment provoked apoptosis in early mitosis and also induced formation of multinucleated cells. LY-294002 and wortmannin, in a similar manner as PKC 412, sensitized U1810 as well as H157 NSCLC cells to etoposide. PD 98059, which is an inhibitor of the protein kinase MEK, could not induce apoptosis when used alone in neither of the two NSCLC cell lines. However, in H157 cells PD 98059 enhanced apoptosis induced by treatment with etoposide in combination with wortmannin or LY-294002.

STS, PKC 412, etoposide or etoposide in combination with PKC 412 induced apoptosis at different time points and with different potency in U1810 cells. All four treatments resulted in cellular activation of caspase-3-like activity. Caspases cleave many proteins and are often determining apoptosis. Nevertheless, irrespective of the type of apoptosis-inducing treatment used, an inhibitor of caspase activity could not prevent apoptosis-related nuclear condensation in U1810 cells. Instead, upon all four different treatments, nuclear apoptosis was dependent upon release of Apoptosis inducing factor (AIF) from mitochondria. When etoposide and PKC 412 were combined, there was an increase in the release of AIF from mitochondria of U1810 cells. By using inhibitors of Poly(ADP-ribose) polymerase (PARP) we could show that release of AIF from mitochondria upon treatment with etoposide alone, or in combination with PKC 412, demands PARP activity. Single treatment with PKC 412 induced AIF release and nuclear apoptosis independently of PARP. PKC 412 decreased inhibitory phosphorylation of the protein Bad, a protein which upon activation is known to promote release of mitochondrial proteins.

Collectively, our data suggest that etoposide and PKC 412 complement each other for the induction of apoptosis in NSCLC cells. Both drugs, in different manners, induce upstream mitochondrial events that promote increased release of mitochondrial AIF which determines apoptosis. PKC 412, possibly through inhibition of PI3-kinase downstream signalling, increases death of NSCLC cells through provoking mitotic catastrophe, and possibly also through activation of Bad. Etoposide increases cell death through induction of PARP-dependent release of AIF from mitochondria.