Study of mechanisms of action of doxorubicin-derived anthracyclins in mammary cells. Involvement of PKC isoforms

ALEJANDRO J. URTREGER; MARÍA LAURA POLO; LEONARD LOTHSTEIN; ELISA D. BAL DE KIER JOFFÉ

San Diego

Congreso; 99th Annual Meeting of the American Associaton for Cancer Research; 2008

American Associaton for Cancer Research (AACR)

AD288 and AD198 are two antibiotics derived from doxorubicin which possess antitumoral capacity. AD288 induces cytotoxic effects through DNA binding and topoisomerase II inhibition. The efficacy of AD198 in breast cancer is not completely proven since its mechanism of action is not completely understood. It is postulated that AD198 exert cytotoxic effects by activating PKCd, a serine-threonine kinase described as proapototic in different cell models. Paradoxically, our group has demonstrated that the overexpression of PKCd in NMuMG mammary cells (NMuMG-PKCd) induced, an increase in its proliferative capacity. In the present work, we want to elucidate the mechanism of action of this drug, and analyze whether the overexpression of PKCd alters the susceptibility of mammary cells to AD198. Both drugs induced a dose-dependent cytotoxic effect in NMuMG cells, however AD198 was more cytotoxic than AD288 only in NMuMG-PKCd cells (IC50 (uM): 9.5 vs. 13.7). Furthermore, only in these cells PKC activation with PMA induced an important increase of AD198 cytotoxicicity. PMA treatment did not affect AD288 cytotoxicity. Next we analyzed the effect of pre-treating the cells with different PKC inhibitors: Bisindolylmaleimide (bis) non specific PKC inhibitor, Go6976 (Go) PKCa and b inhibitor, and Rottlerin (Rot) PKCd inhibitor, in order to define the role of the different PKCs in AD198 and AD288 cytotoxic activity. An increase in control and PKCd transfectants survival was achieved when the cells were treated with AD198 together with Rot (4.9±1.2 and 3.4±0.6 fold over AD198-treated NMuMG-PKCd and control cells respectively, p<0.05). Neither Bis nor Go induced any effect in the survival of cells treated with AD198. No effect on cell survival was observed after treating cells with the different PKC modulators and AD288. Combining different PKC inhibitors pre-treatment, an important increase in the resistance to AD198 was observed only when cells were pre-treated with Go and Rot (12.6±3.5 and 6.6±0.6 times over AD198 treated cells for PKCd and control cells respectively, p<0.05). No drug combination induced any alteration of cell survival when cells were treated with AD288. Interestingly, in all assays NMuMG-PKCd cells showed a higher resistance than control cells to the cytotoxicity induced by both compounds (survival: AD198; 80±10% vs. 41±8% and AD288; 63±9.0% vs. 33±6.2% for PKCd and control cells respectively, p<0.05, MTS assay). In this work we demonstrate that while PKCd seems to be the main target of AD198, the highest cytotoxic inhibition was achieved upon blocking PKCd and classical PKCs together. Since NMuMG-PKCd transfectants were also more resistant to AD288 than control cells, other signaling pathways may be responsible for their resistance to anthracyclins. The further clinical development of AD198 against mammary cancer appears attractive due to its novel and specific mechanism of action.