Abstract
Cardiac glycosides (CGs), prescribed to treat cardiovascular alterations, display potent anti-cancer activities. Despite their well-established target, the sodium/potassium (Na+/K+)-ATPase, downstream mechanisms remain poorly elucidated. UNBS1450 is a hemi-synthetic cardenolide derived from 2″-oxovorusharin extracted from the plant Calotropis procera, which is effective against various cancer cell types with an excellent differential toxicity. By comparing adherent and non-adherent cancer cell types, we validated Mcl-1 as a general and early target of UNBS1450. A panel of CGs including cardenolides ouabain, digitoxin and digoxin as well as bufadienolides cinobufagin and proscillaridin A allowed us to generalize our findings. Our results show that Mcl-1, but not Bcl-xL nor Bcl-2, is rapidly downregulated prior to induction of apoptosis. From a mechanistic point of view, we exclude an effect on transcription and demonstrate involvement of a pathway affecting protein stability and requiring the proteasome in the early CG-induced Mcl-1 downregulation, without the involvement of caspases or the BH3-only protein NOXA. Strategies aiming at preventing UNBS1450-induced Mcl-1 downregulation by overexpression of a mutated, non-ubiquitinable form of the protein or the use of the proteasome inhibitor MG132 inhibited the compound's ability to induce apoptosis. Altogether our results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.
Highlights
Epidemiological studies correlated a reduced incidence of specific cancer types with the regular intake of selected Cardiac glycosides (CGs).[1,2] Preclinical in vitro and in vivo research demonstrated the ability of CGs to impact cell proliferation and survival of a variety of cancer cell models[3] at low nanomolar concentrations
CGs possess a steroid core structure and are sub-divided in two families depending on the chemical group on the D-ring at position 17: cardenolides (UNBS1450, ouabain, digitoxin and digoxin) contain a butyrolactone group, whereas bufadienolides exhibit an α-pyrone group
We prevously described an early downregulation of Mcl-1 without affecting Bcl-2 levels in line with results obtained with ouabain,[22] digitoxin[23] or selected bufadienolides.[9,24]
Summary
Epidemiological studies correlated a reduced incidence of specific cancer types with the regular intake of selected CGs.[1,2] Preclinical in vitro and in vivo research demonstrated the ability of CGs to impact cell proliferation and survival of a variety of cancer cell models[3] at low nanomolar concentrations. CGs affect cancer cell models typically resistant to canonical cytocidal agents.[4,5,6] combinational treatments triggered synergistic effects.[7,8,9]. The altered expression levels of anti-apoptotic members of the Bcl-2 (B-cell lymphoma 2) protein family is a common feature in cancer.[15] Most cancer cell models overexpress one or more of the three major proteins: Bcl-2, Bcl-xL (B-cell lymphoma-extra-large) and Mcl-1 (myeloid cell leukemia-1). Discovered as crucial modulators of apoptosis, anti-apoptotic Bcl-2 proteins emerged more recently as important modulators of other essential cancer processes, including cell cycle, autophagy or cell metabolism.[16] This aspect underlines the importance to target these proteins to further improve existing anti-cancer therapies. Mcl-1 became one of the most investigated members of the Bcl-2 family but the poor availability of specific Bcl-2 family inhibitors targeting this protein hindered improved treatment protocols especially of Mcl-1 overexpressing and chemoresistant cancer types.[17,18]
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