Differential outcome of JNK inhibition in prolifrating and differentiated adult muscle - derived stem cells after chemotherapeutic treatment.

Audrone Kalvelyte, Vilnius University Institute of Biochemistry, Vilnius, Lithuania

Audrone Kalvelyte, Aurimas Stulpinas, Natalija Krestnikova, Ausra Imbrasaite, Daiva Baltriukiene and Kristina Baltrunaite

Nowadays, the combination of conventional chemotherapeutic drugs with inhibitors of intracellular signaling molecules is a promising strategy for cancer treatment. It is known that conventional anticancer drugs, as well as signal molecule-targeted inhibitors themselves may affect both normal and malignant cells of organism. Therefore, in order to protect stem cells from chemotherapeutic damage it is of great importance to determine the impact of apoptosis-regulating signaling pathways underlying sensitivity of adult stem cells. Among MAP kinases it is c-Jun NH2-terminal protein kinase that is activated in response to different kinds of stress. The importance of JNK activation in deciding cell fate in response to anticancer treatment makes JNK signaling targeting an attractive therapeutic strategy. This research was aimed to dissect the role of stress kinase JNK in apoptosis regulation in adult muscle-derived stem cells (MDSC) and their differentiated counterparts upon exposure to cisplatin and other DNA damaging anticancer drugs. MDSC cell lines Myo have unlimited proliferative potential in vitro and are able to differentiate into myogenic, osteogenic, adipogenic and neurogenic lineages. We found that the role of JNK in regulating MDSC apoptosis changes from proapoptotic in proliferating cells to antiapoptotic in differentiated cells. The combination of cisplatin with pan-JNK inhibitor SP600125, the efficiency of which was confirmed by decreased phosphorylation of c-Jun, promoted apoptosis in differentiated Myo cell population, but reduced cell death in population of undifferentiated, proliferating cells. JNK acts as antiapoptotic molecule in osteo-, adipo-, and neurogenically differentiated Myo cells, along with previously described myogenically differentiated cells. Different durations of MAPKs phosphorylation were associated with their opposing functions in cell apoptosis regulation. Our results contradict the notion that prolonged activation of JNK is related to its proapototic role. Gradual and prolonged increase of JNK and its target transcription factor c-Jun phosphorylation were found both in proliferating and differentiated cells exposed to cisplatin, daunorubicin and doxorubicin. However, the different localization of phosphorylated JNK was observed after cisplatin treatment in proliferating and differentiated Myo cells, nuclear or cytoplasmic, respectively. Data revealed that cisplatin induced apoptosis in Myo cells both through extrinsic and intrinsic, mitochondrial, pathways. The major regulators of mitochondrial apoptosis pathway, proapoptotic Bax and antiapoptotic Bcl-2, proteins were regulated in JNK-dependent manner in the direction that correlates with JNK role in apoptosis. JNK inhibition in differentiated cells exposed to cisplatin resulted in decrease of antiapoptotic Bcl-2 protein level and increase of proapoptotic Bax, and vice versa in proliferating Myo cells. Protective action of JNK may involve the cross-talk with other antiapoptotic signaling pathways. In this study cisplatin and SP600125 co-treatment resulted in decrease of Akt phosphorylation in differentiated but not in proliferating cells, showing that JNK is involved in positive regulation of Akt activity in differentiated cells. Thus, JNK differentially regulates Akt in proliferating and differentiated Myo cells. Therefore, when applying appropriate drug therapy, special attention should be paid to the dynamic nature of stem cells, especially to the cell response dependence on differentiation status.