Furoxan-piplartine hybrids as effective NO donors and ROS inducers in PC3 cancer cells: design, synthesis, and biological evaluation.

Conjugation of the naturally occurring product piplartine (PPT, 1), which is a potent cytotoxic compound and ROS inducer, with a diphenyl sulfonyl-substituted furoxan moiety (namely, 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide), an important type of NO donor, an ether linker of different chain lengths is described, characterized and screened for the anticancer potential. The cytotoxicity of the new hybrids was evaluated on a panel of human cancer cell lines (MCF-7, PC3 and OVCAR-3) and two non-cancer human cells (MCF10A and PNT2). In general, the synthesized hybrids were more cytotoxic and selective compared to their furoxan precursors 4-6 and PPT in the above cancer cells. Particularly, PC3 cells are the most sensitive to hybrids 7 and 9 (IC values of 240 nM and 50 nM, respectively), while a lower potency was found for the prostate normal cells (IC = 17.8 μM and 14.1 μM, respectively), corresponding to selectivity indices of 75 and 280, respectively. NO generation by the PPT-furoxan compounds in PC3 cells was confirmed using the Griess reaction. Furthermore, the cell growth inhibitory effect of 9 was significantly attenuated by the NO scavenger carboxy-PTIO. The intracellular ROS generation by 7 and 9 was also verified, and different assays showed that co-treatment with the antioxidant -acetyl-l-cysteine (NAC) provided protection against PPT-induced ROS generation. Further mechanistic studies revealed that 7 and 9 had strong cytotoxicity to induce apoptosis in PC3 cells, being mediated, at least in part, by the NO-release and increase in ROS production. Notably, the ability of 9 to induce apoptosis was stronger than that of 7, which may be attributed to higher levels of NO released by 9. Compounds 7 and 9 modulated the expression profiles of critical regulators of cell cycle, such as (p21), , and (cyclin D1), as well as induced DNA damage. Overall, tethering the furoxan NO-releasing moiety to the cytotoxic natural product PPT had significant impact on the potential anticancer activity and selectivity of the novel hybrid drug candidates, especially 9, as a result of synergistic effects of both furoxan and PPT's ability to release NO, generate ROS, induce DNA damage, and trigger apoptosis.