Elucidation of pro-apoptotic signaling induced by cisplatin

A major clinical problem regarding treatment of malignant tumors with anticancer drugs is inherent or acquired resistance to therapy. One factor contributing to drug resistance of cancer cells is failure to undergo apoptosis. The aim of this thesis was to investigate pro-apoptotic signaling induced by the anti-cancer drug cisplatin in order to contribute to future improvement of cisplatin-based cancer therapy.

The results demonstrate the ability of cisplatin to induce at least two separate proapoptotic pathways, and to affect several cellular organelles. The ERK and the JNK pathways are the two main mitogen activated protein kinase (MAPK) modules of serial kinase activities stimulated by proliferative signaling as well as by cellular stress. The role of ERK in cisplatin-induced apoptosis was studied in four melanoma cell lines. ERK activation was seen 4 h after cisplatin treatment. Inhibition of the pro survival ERK pathway resulted in different responses, from sensitizing to slightly protective. The cellular response to a certain type of treatment can thus not always be predicted.

Cisplatin treatment was found to elicit increased intracellular calcium levels and activation of calpain within 1-3 hours. Involvement of calpain in the apoptotic response was demonstrated using calpain inhibitors which blocked caspase activation and nuclear fragmentation. Moreover, the calpain inhibitor calpeptin was able to block cisplatin-induced cleavage of the proapoptotic Bid. Recombinant Bid was cleaved in vitro by recombinant calpain as well as lysates of cisplatin-treated cells. Apoptosis via the intrinsic (mitochondrial) pathway is known to require activation of the pro-apoptotic Bcl-2 proteins Bak and Bax.

We demonstrate the ability of cisplatin to induce activation of Bak, and suggest a kinase fragment of MEKK1 (deltaMEKK1 ) as mediator of Bak activation, since activation was inhibited in cells expressing a kinase-inactive mutant of MEKK1 (dominant negative MEKK1, dnMEKK1). Involvement of deltaMEKK1 in Bak activation was supported by the ability of a constitutively active kinase mutant of MEKK1 (dominant positive MEKK1, dpMEKKI1) to activate Bak in three cell lines out of four, and to induce apoptosis in two of them. In contrast to dnMEKK1, calpeptin did not affect Bak activation but did block Bid cleavage. On the other hand Bid cleavage was not affected by dnMEKK1. Calpeptin and dnMEKK1 were both able to block nuclear fragmentation and caspase activation by approximately half. When used together, they had an additive inhibitory effect on apoptosis.

Using enucleated cells (cytoplasts) we demonstrated the ability of cisplatin to induce apoptosis in the absence of nuclear DNA. Cisplatin treatment of cytoplasts resulted in calpainmediated Bid cleavage as well as calcium- and calpain-dependent activation of caspase-3. Moreover, calpeptin- sensitive activation of pro-caspase- 12 was found in cisplatin-treated cells and cytoplasts. Caspase-12 is localized at the endoplasmic reticulum (ER) and is activated by ER stress. Cisplatininduced ER stress was supported by increased expression of grp78, an ER chaperone protein.

In summary, we have characterized two novel pro-apoptotic pathways induced by cisplatin and investigated the time course of their activation. One pathway is activated early and involves calciumdependent activation of calpain and calpain-mediated cleavage of Bid. The other pathway involves deltaMEKK1-mediated activation of Bak shortly before onset of caspase-3. We have also identified ER as a new, non-nuclear target of cisplatin, and suggest ER stress as a potent mediator of cisplatin-induced proapoptotic signaling.

List of scientific papers

I. Mandic A, Viktorsson K, Heiden T, Hansson J, Shoshan MC (2001). The MEK1 inhibitor PD98059 sensitizes C8161 melanoma cells to cisplatin-induced apoptosis. Melanoma Res. 11(1): 11-9.
https://pubmed.ncbi.nlm.nih.gov/11254111

II. Mandic A, Viktorsson K, Molin M, Akusjarvi G, Eguchi H, Hayashi SI, Toi M, Hansson J, Linder S, Shoshan MC (2001). Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKK1-dependent manner. Mol Cell Biol. 21(11): 3684-91.
https://pubmed.ncbi.nlm.nih.gov/11340162

III. Mandic A, Viktorsson K, Strandberg L, Heiden T, Hansson J, Linder S, Shoshan MC (2002). Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis. Mol Cell Biol. 22(9): 3003-13.
https://pubmed.ncbi.nlm.nih.gov/11940658

IV. Mandic A, Hansson J, Linder S, Shoshan MC (2002). Cisplatin induces ER stress and nucleus-independent apoptotic signaling. J Biol Chem. Dec 31.
https://pubmed.ncbi.nlm.nih.gov/12509415