Abstract
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, via 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 (IC50 values of 240 nM and 50 nM, respectively), while a lower potency was found for the prostate normal cells (IC50 = 17.8 μM and 14.1 μM, respectively), corresponding to selectivity indices of ca. 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 N-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 CDKN1A (p21), c-MYC, and CCND1 (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.