Induction of a non-apoptotic mode of cell death associated with autophagic characteristics with steroidal maleic anhydrides and 7β-hydroxycholesterol on glioma cells
Résumé
Steroidal maleic anhydrides were prepared in one step: lithocholic, chenodeoxicholic,
deoxicholic, ursocholic, and hyodeoxicholic acid derivatives. Their capability to induce cell
death was studied on C6 rat glioma cells, and 7β-hydroxycholesterol was used as positive
cytotoxic control. The highest cytotoxicity was observed with lithocholic and
chenodeoxicholic acid derivatives (23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5β-
cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-
cholane (compound 1b), respectively), which induce a non-apoptotic mode of cell death
associated with mitochondrial membrane potential loss and reactive oxygen species
overproduction. No cells with condensed and/or fragmented nuclei, no PARP degradation and
no cleaved-caspase-3, which are apoptotic criteria, were observed. Similar effects were found
with 7β-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1b
was more cytotoxic than compound 1a and 7β-hydroxycholesterol. Compound 1b and 7β-
hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b,
and 7β-hydroxycholesterol favour the formation of large acidic vacuoles revealed by staining
with acridine orange and monodansylcadaverine evocating autophagic vacuoles; they also
induce an increased ratio of [LC3-II / LC3-I], and modify the expression of mTOR, Beclin-1,
Atg12, and Atg5-Atg12 which is are autophagic criteria. The ratio [LC3-II / LC3-I] is also
strongly modified by bafilomycin acting on the autophagic flux. Rapamycin, an autophagic
inducer, and 3-methyladenine, an autophagic inhibitor, reduce and increase 7β-
hydroxycholesterol-induced cell death, respectively, supporting that 7β-hydroxycholesterol
induces survival autophagy. Alpha-tocopherol also strongly attenuates 7β-hydroxycholesterol-
induced cell death. However, rapamycin, 3-methyladenine, and α-tocopherol have no effect
on compounds 1a and 1b-induced cell death. It is concluded that these compounds trigger a
non apoptotic mode of cell death, involving the mitochondria and associated with several
characteristics of autophagy.
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