Hydrogen sulfide releasing enmein-type diterpenoid derivatives as apoptosis inducers through mitochondria-related pathways

Abstract

In this study, we combined two enemin-type diterpenoid derivatives with two well-established hydrogen sulfide moieties via ester or different anhydride linkers, to search apoptosis inducing drug candidate capable of hydrogen sulfide generating. Therefore, a series of hybrids were synthesized and superior antiproliferative efficacy accompanied with enhanced selectivity was observed under extensive pharmacological evaluations. A standard methylene blue (MB⁺) method was applied to measure the capacity for the hydrogen sulfide generation of all the target derivatives. One particular molecule A1, which contained α-thioctic acid moiety for hydrogen sulfide donating, manifested more potent antiproliferative activity. It exerted inhibitory effects against Bel-7402, SGC-7901 and A549 cell lines with IC₅₀ values of 2.16, 5.07 and 6.98 μM respectively. While it exacted relatively low effects over human normal cell lines L-02 and PBMC with IC₅₀ values of 15.81 μM for the prior and 14.15 μM for the latter, and displayed better selectivity index (SI) than parent diterpenoids. A high dosage of H₂S release was also recorded. Hence, A1 was most suitable for mechanistic exploration on account of both safety and efficacy. The ensuing biological assays revealed central role of apoptosis in A1's mode of action for antiproliferative efficacy, which led to further confirmation of G1 phase cell cycle arrest, mitochondria membrane potential collapse and apoptotic activation in Bel-7402 cells. Further western blot assay on intrinsic mitochondria pathway unlocked intricate interplay among a series of apoptotic related proteins in which Bax, caspase-3 and cytochrome c went through up-regulation, while Bcl-2, Bcl-xL and procaspase-3 undergone down-regulation. In a nutshell, a hydrogen sulfide releasing hybrid A1 was synthesized and antiproliferative evaluation identified it to be a worthy drug candidate for future in depth study.

Keywords: Apoptosis; Enmein-type diterpenoid; Hydrogen sulfide; Mitochondria-related pathway.