Pro-apoptotic activity of novel synthetic isoxazole derivatives exhibiting inhibitory activity against tumor cell growth in vitro

Abstract

In order to develop potential anticancer agents stimulating apoptosis, novel 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives have been synthetized. The original structures of geldanamycin and radicicol, which are known natural heat shock protein (HSP) inhibitors, were deeply modified because both of them exhibit several drawbacks, such as poor solubility, hepatotoxicity, intrinsic chemical instability or deprivation of the in vivo activity. This novel class of synthetic compounds containing the isoxazole nucleus exhibited potent and selective inhibition of HSP90 in previous studies. Biological assays (focusing on in vitro antiproliferative effects and pro-apoptotic activity) in human erythroleukemic K562 cells (as a model system referring to tumor cells grown in suspension), glioblastoma U251-MG and glioblastoma temozolomide (TMZ)-resistant T98G cell lines (two model systems referring to tumor cells grown attached to the flask), were performed. Almost all isoxazole derivatives demonstrated significant antiproliferative and pro-apoptotic activities, showing induction of both early and late apoptosis of K562 cells. Different effects were observed on the glioma U251-MG and T98G cells, depending on the structure of the analogues. Antiproliferative and pro-apoptotic activities in K562 cells were associated with the activation of the erythroid differentiation program. The present study demonstrated that 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives should be considered for in vivo studies focusing on the development of anticancer drugs acting, at least partially, via activation of apoptosis.

Keywords: K562 cells; T98G cells; U251-MG cells; apoptosis; cancer; caspases; glioblastoma; heat shock protein inhibitors.

Figure 1.

Chemical structures of compounds 1–8 (3,4-isoxazolediamides) analyzed in the present study.

Figure 2.

Chemical structures of compounds 9–13 (4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridines) analyzed in the present study.

Figure 3.

Effects of synthetic isoxazole derivatives on apoptosis of K562 cells. (A) Control untreated K562 cells. Representative examples of the effects of compounds (B) 4, (C) 7, (D) 8, (E) 9 and (F) 11, according to the Annexin-V assay performed on K562 cells treated for 72 h with two different concentrations of each isoxazole derivative. The complete set of data is shown in Table II.

Figure 4.

Effects of synthetic isoxazole derivatives on apoptosis of U251-MG cells. (A) Control untreated U251-MG cells. Representative examples of the effects of compounds (B) 2, (C) 3, (D) 4, (E) 5 and (F) 11, according the Annexin-V assay performed on U251-MG cells treated for 72 h with two different concentrations of each isoxazole derivative. The complete set of data is shown in Table III.

Figure 5.

Effects of synthetic isoxazole derivatives on apoptosis of T98G cells. (A) Control untreated T98G cells. Representative examples of the effects of compounds (B) 3, (C) 4, (D) 5 and (E) 9 in the Annexin-V assay performed on T98G cells treated for 72 h with two different concentrations of each isoxazole derivative. The complete set of data is shown in Table IV.

Figure 6.

Percentage of apoptotic cells analyzed using the caspase 3/7 kit assay in T98G cells treated for 72 h with 3,4-isoxazolediamides and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridines derivatives 3–11. Results are presented as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. control untreated cells. (−), control untreated cells.

Figure 7.

Effects of synthetic isoxazole derivatives on erythroid differentiation of K562 cells. (A) Example of benzidine-positive K562 cells induced by compound 2 (blue cells). Magnification, ×40. (B) Percentage of induction of erythroid differentiation (% of benzidine-positive cells) in K562 cells treated with isoxazole derivatives and MTH (30 nM) used as positive control. Results are presented as the mean±SEM (n=3). **P<0.01 vs. control untreated cells. (−), control untreated cells; MTH, mithramycin.