Development of a novel azaspirane that targets the Janus kinase-signal transducer and activator of transcription (STAT) pathway in hepatocellular carcinoma in vitro and in vivo

Abstract

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates genes involved in cell growth, proliferation, and survival, and given its association with many types of cancers, it has recently emerged as a promising target for therapy. In this work, we present the synthesis of N-substituted azaspirane derivatives and their biological evaluation against hepatocellular carcinoma (HCC) cells (IC50 = 7.3 μm), thereby identifying 2-(1-(4-(2-cyanophenyl)1-benzyl-1H-indol-3-yl)-5-(4-methoxy-phenyl)-1-oxa-3-azaspiro(5,5) undecane (CIMO) as a potent inhibitor of the JAK-STAT pathway with selectivity over normal LO2 cells (IC50 > 100 μm). The lead compound, CIMO, suppresses proliferation of HCC cells and achieves this effect by reducing both constitutive and inducible phosphorylation of JAK1, JAK2, and STAT3. Interestingly, CIMO displayed inhibition of Tyr-705 phosphorylation, which is required for nuclear translocation of STAT3, but it has no effect on Ser-727 phosphorylation. CIMO accumulates cancer cells in the sub-G1 phase and decreases STAT3 in the nucleus and thereby causes down-regulation of genes regulated via STAT3. Suppression of STAT3 phosphorylation by CIMO and knockdown of STAT3 mRNA using siRNA transfection displayed a similar effect on the viability of HCC cells. Furthermore, CIMO significantly decreased the tumor development in an orthotopic HCC mouse model through the modulation of phospho-STAT3, Ki-67, and cleaved caspase-3 in tumor tissues. Thus, CIMO represents a chemically novel and biologically in vitro and in vivo validated compound, which targets the JAK-STAT pathway as a potential cancer treatment.

Keywords: Bioinformatics; Cell Migration; Hepatocellular Carcinoma; Janus Kinase (JAK); STAT3.

FIGURE 1.

A, azaspirane scaffold (highlighted with red circle) evolution. 1, atiprimod; 2, staurosporine; 3, lestaurtinib; 4, (2-(2,6-difluorophenyl)-5-(4-methoxyphenyl)-1-oxa-3-azaspiro[5.5]undecane) (DMBO). B, CIMO causes the accumulation of HepG2 cells in the sub-G₁ phase. HepG2 cells (5 × 10⁵/ml) were treated with 10 μmol/liter CIMO for the indicated times, after which the cells were washed, fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry.

FIGURE 2.

A, CIMO suppresses phospho-STAT3 in a dose-dependent manner. HepG2 cells (5 × 10⁵/ml) were treated with the indicated concentrations of CIMO for 6 h, after which whole-cell extract was prepared and resolved on SDS-polyacrylamide gel, electrotransferred onto nitrocellulose membrane, and probed for phospho-STAT3, and the same blot was stripped and reprobed with STAT3 antibody to verify equal protein loading. B, AG490 suppresses phospho-STAT3 in a dose-dependent manner. HepG2 cells (5 × 10⁵/ml) were treated with the indicated concentrations of AG490 for 6 h, after which Western blotting was done as described for A. C, CIMO suppresses phospho-STAT3 levels in a time-dependent manner. HepG2 cells (5 × 10⁵/ml) were treated with 10 μmol/liter CIMO for the indicated times, after which Western blotting was done as described for A. D, CIMO had no effect on phospho-STAT3(Ser-727) and STAT3 protein expression. HepG2 cells (5 × 10⁵/ml) were treated with 10 μmol/liter CIMO for the indicated times, after which Western blotting was done as described for A, and the membrane was probed using antibodies against phospho-STAT3(Ser-727) and STAT3. E, CIMO inhibits the translocation of STAT3 to the nucleus. HepG2 cells (1 × 10⁵/ml) were incubated with or without 10 μmol/liter CIMO for 6 h and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry. The same slides were counterstained for nuclei with Hoechst (50 ng/ml) for 5 min and analyzed under a fluorescence microscope. F, CIMO suppresses phospho-Src, phospho-JAK1, and phospho-JAK2 levels in a time-dependent manner. HepG2 cells (5 × 10⁵/ml) were treated with 10 μmol/liter CIMO, after which whole-cell extracts were prepared, resolved in SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-Src, phospho-JAK1, and phospho-JAK2 antibodies. The same blots were stripped and reprobed with Src, JAK1, and JAK2 antibodies to verify equal protein loading. G, CIMO suppresses STAT3 DNA binding ability in HepG2 cells. HepG2 cells were treated with 10 μmol/liter CIMO for the indicated time, nuclear extracts were prepared, and 5 μg of the nuclear extract protein was used for the ELISA-based DNA-binding assay. *, p < 0.05.

FIGURE 3.

A, comparative study of cell viability between STAT3-siRNA-transfected and CIMO-treated HepG2 cells. Correspondingly, Western blot analysis was used to assess the levels of phospho-STAT3 and STAT3 in HepG2 cells with siRNA-mediated depletion of STAT3 expression and upon exposure to 4 μm CIMO. Whole-cell extract was prepared and resolved on SDS-polyacrylamide gel, electrotransferred onto nitrocellulose membrane, and probed for phospho-STAT3, and the same blot was stripped and reprobed with STAT3 antibody and β-actin to verify equal protein loading. B, CIMO modulates STAT3-mediated transcription and α2-M promoter activity in HepG2 cells. C, CIMO inhibits IL-6-induced phosphorylation of STAT3, JAK1, and JAK2. Hep3B cells (5 × 10⁵/ml) were treated with 10 μmol/liter CIMO for the indicated times and then stimulated with IL-6 (10 ng/ml) for 15 min. Whole-cell extracts were then prepared, resolved on an SDS-polyacrylamide gel, electrotransferred onto nitrocellulose membrane, and probed with phospho-STAT3, phospho-JAK1, and phospho-JAK2 antibodies. The same blot was stripped and reprobed with STAT3, JAK1, and JAK2 antibody to verify equal protein loading. D and E, CIMO suppresses STAT3-regulated gene products involved in cell proliferation and survival. HepG2 cells (5 × 10⁵/ml) were treated with the 10 μmol/liter CIMO for the indicated time intervals, after which whole-cell extract was prepared, resolved on an SDS-polyacrylamide gel, and electrotransferred onto nitrocellulose membrane, and the membrane was sliced according to molecular weight and probed against Bcl-2, cyclin D1, Survivin, Bak, ICAM-1, Bcl-xL, and Bid. The same blot was stripped and reprobed with β-actin antibody to verify equal protein loading. Error bars, S.E.

FIGURE 4.

A, CIMO activates caspase-3 and induces apoptosis. HepG2 cells (5 × 10⁵/ml) were treated with 10 μmol/liter CIMO for the indicated times, and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blotting against caspase-3 and PARP antibody. The same blot was stripped and reprobed with β-actin antibody to show equal protein loading. B, pervanadate reversed the inhibitory effect of CIMO on phospho-STAT3. HepG2 cells (5 × 10⁵/ml) were treated with the indicated concentrations of pervanadate and 10 μmol/liter CIMO for 4 h, after which whole-cell extracts were prepared, resolved on an SDS-polyacrylamide gel, electrotransferred onto nitrocellulose membrane, and probed for phospho-STAT3 and STAT3. C, inhibitory activity of CIMO on phospho-STAT3 is mediated by protein-tyrosine phosphatase. HepG2 cells (5 × 10⁵/ml) were treated with 10 μmol/liter CIMO for the indicated times; whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blotting against PTP1B, SHPTP1, and SHPTP2 antibody; and the same blot was stripped and reprobed with β-actin antibody to show equal protein loading.

FIGURE 5.

A, CIMO inhibits cell migration. 70 μl of HepG2 cells (5 × 10⁵/ml) were seeded into each compartment of the culture insert, and the insert was removed after 12 h. The width of the wound was measured initially, and cells were incubated with and without CIMO (5 μm, 8 h) and CXCL12 (100 ng/ml, 24 h). B, CIMO inhibits the cell invasion. HepG2 (2 × 10⁵) cells were seeded in the top chamber of BD BioCoat^TM Matrigel^TM. After preincubation with or without 5 μmol/liter CIMO for 8 h, transwell chambers were placed into the wells of a 24-well plate that contained either only basal medium or basal medium with CXCL12 (100 ng/ml) for 24 h. After incubation, the chamber was assessed for cell invasion by staining with crystal violet. Error bars, S.E.

FIGURE 6.

CIMO inhibits the growth of human HCC in vivo. A, representative images of mice from bioluminescent imaging. B, relative tumor burden in athymic mice bearing orthotopically implanted Huh 7-Luc2 tumors treated with vehicles alone (n = 5) or 2 mg/kg (n = 6) or 10 mg/kg (n = 6) CIMO. Points, mean; bars, S.E. *, p < 0.05 (unpaired Student's t test).

FIGURE 7.

Immunohistochemical analysis of phospho-STAT3 (p-STAT3), Ki-67, and caspase-3 showed the inhibition in expression of phospho-STAT3, and Ki-67 and increased levels of cleaved caspase-3 expression in CIMO-treated samples as compared with the control group. The percentage indicates positive staining for the given biomarker. The photographs were taken at a magnification of ×40.

FIGURE 8.

Bioinformatics approach of CIMO interaction toward the kinase domain of JAK2. A, ribbon diagram of the monomer of the JAK2 and its ligand binding site (LBS) of kinase domain (green). B, surface view of JAK2 and the bound CIMO in the ligand binding site region. C, interaction map of ligand binding site domain of JAK2 that interacted with CIMO. The labeled key amino acids are represented as a line model with the carbon atom in black, and other atoms in their parent colors. Shown is the binding of CIMO, whose carbon atom is green, and other atoms with their parent colors.