Romidepsin (FK228) and its analogs directly inhibit phosphatidylinositol 3-kinase activity and potently induce apoptosis as histone deacetylase/phosphatidylinositol 3-kinase dual inhibitors

Abstract

Activation of phosphatidylinositol 3-kinase (PI3K) signaling is involved in carcinogenesis and cancer progression. The PI3K inhibitors are considered candidate drugs for cancer treatment. Here, we describe a drug screening system for novel PI3K inhibitors using Saccharomyces cerevisiae strains with deleterious mutations in the ATP-binding cassette transporter genes, because wild-type S. cerevisiae uses drug efflux pumps for reducing intracellular drug concentrations. By screening the chemical library of the Screening Committee of Anticancer Drugs, we identified the histone deacetylase (HDAC) inhibitor romidepsin (FK228) and its novel analogs. In vitro PI3K activity assays confirmed that these compounds directly inhibit PI3K activity at μM-range concentrations. FK-A5 analog was the most potent inhibitor. Western blotting revealed that these compounds inhibit phosphorylation of protein kinase B and downstream signaling components. Molecular modeling of the PI3K-FK228 complex indicated that FK228 binds to the ATP-binding pocket of PI3K. At μM-range concentrations, FK228 and FK-A5 show potent cytotoxicity, inducing apoptosis even in HDAC inhibitor-resistant cells. Furthermore, HDAC/PI3K dual inhibition by FK228 and FK-A5 at μM-range concentrations potentiates the apoptosis induction, mimicking the effect of combining specific HDAC and PI3K inhibitors. In this study, we showed that FK228 and its analogs directly inhibit PI3K activity and induce apoptosis at μM-range concentrations, similar to HDAC/PI3K dual inhibition. In future, optimizing the potency of FK228 and its analogs against PI3K may contribute to the development of novel HDAC/PI3K dual inhibitors for cancer treatment.

Figure 1

Identification of FK228 and its analogs as candidate phosphatidylinositol 3‐kinase (PI3K) inhibitors using Saccharomyces cerevisiae. (A) YPH499 was cotransformed with the following sets of URA3 and LEU2 marked vectors: top row, pRS316 and pRS315; second row, pSJ01 (p110α) and pRS315; and third row, pSJ01 and pSJ21 (PTEN). Serial 10‐fold dilutions of the transformants were deposited on solid synthetic minimal medium–uracil–leucine (SD‐U‐L) and synthetic galactose medium (SGal)‐U‐L media. (B) YPH499 transformants were incubated in SGal‐U‐L medium, with or without 50 μM LY294002. Then A ₆₀₀ was measured as an indicator of yeast growth. (C,D) Transformants of YPH499 and AD1‐9 expressing p110α were cultured in SGal‐U‐L medium containing various concentrations of LY294002. Sensitivity for LY294002 was compared by detecting the concentration at which growth inhibition was rescued (arrow). (E) Screening results of the chemical library of the Screening Committee of Anticancer Drugs. AD1‐9 transformants expressing p110α were cultured in SGal‐U‐L media containing each compound. A ₆₀₀ values are the mean of experiments carried out in triplicate. Test compounds are arranged according to A ₆₀₀ values in the figure. LY294002, DMSO, and FK228 and its analogs are indicated.

Figure 2

Structures and histone deacetylase (HDAC) inhibitory activity of FK228 and its analogs. The structures of FK228 and the analogs examined in this study and their IC ₅₀ values for HDAC1 and HDAC6 are shown.

Figure 3

Effects of FK228 and its analogs on phosphatidylinositol 3‐kinase (PI3K) activity. (A) Inhibition of PI3K activity was evaluated by mobility shift assay. The read‐out value of a control reaction (complete reaction mixture) was set as a 0% inhibition, and the percentage inhibition of each test solution was calculated at a concentration of 20 μM. Data are the mean of two experiments carried out in duplicate. (B–E) IC ₅₀ values of FK228, FK‐A5, SP‐3, and LY294002 were calculated from concentration versus % inhibition curves by fitting to a four‐parameter logistic curve. (F) Inhibition of PI3K activity of FK228 was evaluated under the conditions of two different ATP concentrations, 50 and 500 μM. Each concentration versus % inhibition curve is shown.

Figure 4

Molecular modeling of the phosphatidylinositol 3‐kinase (PI3K)–FK228 complex. (A) Molecular surface structure of the PI3K–FK228 complex model. Orange, reduced form of FK228; red, p85α; white, p110α. (B) Side view of Fig. 4(A). (C) Enlarged picture around the ligand. A ball and stick model shows the reduced FK228 in the PI3K ribbon and line drawing.

Figure 5

Western blot analysis of protein kinase B (AKT) and its downstream components in cells treated with FK228 or FK‐A5. (A) Evaluation of the experimental time course. PC3 cells were treated with LY294002, FK228, or FK‐A5 at a concentration of 10 μM. (B) Effect of various concentrations of FK228 or FK‐A5 on AKT and its downstream signaling components. PC3 cells were treated with FK228 or FK‐A5 for 3 h.

Figure 6

Antiproliferative effects of FK228, FK‐A5, and SP‐3. HCT116, RKO, and CO115 colorectal cancer cells were treated with: DMSO; 50 μM LY294002 (LY); 2.5 μM suberoylanilide hydroxamic acid (SAHA); a combination of 50 μM LY with 2.5 μM SAHA; FK228, FK‐A5, or SP‐3 at concentrations of 5, 50, 500 nM, 5, and 50 μM; and a combination of 50 μM LY with FK228, FK‐A5, or SP‐3 at concentrations of 5, 50, and 500 nM. The cell viability was assayed 24 h after treatment. The ratio of surviving cells to the controls treated with 1% DMSO was calculated.

Figure 7

Effects of FK228, FK‐A5, and SP‐3 on apoptosis, cell cycle, protein kinase B (AKT) phosphorylation, and histone acetylation. (A,B) HCT116 colorectal cancer cells were treated with: DMSO; 50 μM LY294002 (LY); 2.5 μM suberoylanilide hydroxamic acid (SAHA); a combination of 50 μM LY with 2.5 μM SAHA; FK228 or FK‐A5 at concentrations of 5, 50, 500 nM, and 5 μM; and a combination of 50 μM LY with FK228 or FK‐A5 at concentrations of 5, 50, and 500 nM. The cells were harvested 24 h after treatment. (C,D) Western blot analysis of poly(ADP‐ribose) polymerase (PARP) cleavage, AKT dephosphorylation, and histone acetylation by FK228, FK‐A5, or SP‐3 in HCT116 and CO115 cells cultured as indicated in Fig. 6.