STAT3 inhibitor, cucurbitacin I, is a novel therapeutic agent for osteosarcoma

Abstract

The development of clinical agents remains a costly and time-consuming process. Although identification of new uses of existing drugs has been recognized as a more efficient approach for drug discovery than development of novel drugs, little screening of drugs that might be used for a rare malignant tumor such as osteosarcoma (OS) has been performed. In this study, we attempted to identify new molecular targeted agents for OS by employing Screening Committee of Anticancer Drugs (SCADS) kits. To screen compounds for OS treatment, their effect on cell viability of the OS cell lines 143B, MG63, HOS, SAOS-2, and HUO9 were evaluated. Candidate drugs were narrowed down based on a global anti-proliferative effect against these five OS cell lines. After excluding cytotoxic compounds and compounds unsuitable for in vivo administration, cucurbitacin I was extracted. Cucurbitacin I has been found to have cytotoxic and anti-proliferative properties against several tumors through inhibition of signal transducer and activator of transcription 3 (STAT3) activation. Cucurbitacin I dose- and time-dependently inhibited the proliferation of all five OS cell lines. Following cucurbitacin I treatment, STAT3 was inactivated and analysis of Mcl-1, cleaved PARP and caspase-3 indicated apoptosis induction. Expression of cell cycle regulator proteins, such as phospho-cyclin D1, c-Myc and survivin, were suppressed. Finally, cucurbitacin I potently inhibited the tumor growth of human OS 143B cells in nude mice. Our in vitro and in vivo results suggest that STAT3 inhibition by cucurbitacin I will be an effective and new approach for the treatment of OS.

Figure 1

Initial screening of the SCADS library for molecules that inhibit the cell viability of osteosarcoma cell lines. Five OS cell lines were treated for 24 h with compounds from the SCADS library and cell viability was evaluated. Compounds which had a broad effect on the five OS cell lines were selected by color imaging. Each column corresponds to an OS cell line (HUO9, 143B, HOS, Saos2 or MG63). White areas indicate >50% reduction in cell viability compared to the control, gray areas indicate a reduction of 20–50%, and black areas indicate a reduction of 0–20%. The compounds used in the assay/examined were cucurbitacin I, AKT inhibitor IV, SU11652, sunitinib and pazopanib.

Figure 2

Effect of the selected compounds on cell viability of monolayer and 3D cultures of OS cell lines. (A) Analysis of the dose-dependent effect of the five selected compounds on the viability of five OS cell lines in monolayer culture. (B) Analysis of the dose-dependent effect of the five selected compounds on the viability of five OS cell lines in 3D collagen culture. (C) Time-dependent inhibition of the proliferation of five OS cell lines in monolayer culture by 10 μM of cucurbitacin I.

Figure 2

Effect of the selected compounds on cell viability of monolayer and 3D cultures of OS cell lines. (A) Analysis of the dose-dependent effect of the five selected compounds on the viability of five OS cell lines in monolayer culture. (B) Analysis of the dose-dependent effect of the five selected compounds on the viability of five OS cell lines in 3D collagen culture. (C) Time-dependent inhibition of the proliferation of five OS cell lines in monolayer culture by 10 μM of cucurbitacin I.

Figure 2

Effect of the selected compounds on cell viability of monolayer and 3D cultures of OS cell lines. (A) Analysis of the dose-dependent effect of the five selected compounds on the viability of five OS cell lines in monolayer culture. (B) Analysis of the dose-dependent effect of the five selected compounds on the viability of five OS cell lines in 3D collagen culture. (C) Time-dependent inhibition of the proliferation of five OS cell lines in monolayer culture by 10 μM of cucurbitacin I.

Figure 3

Quantification of apoptotic cell populations. Depending on fluorescence intensity of Annexin V (x-axis) and propidium iodide (PI, y-axis), the populations can be distinguished into double-negative (healthy) cells, Annexin V-positive (early apoptotic cells) and double-positive (late apoptotic and necroptotic) cells.

Figure 4

Analysis of the effect on cell signaling of osteosarcoma cell lines by cucurbitacin I. (A) Western blot analysis of STAT3 expression in the five OS cell lines. β-actin was used as a loading control. (B) The effect of incubation of the five OS cell lines with vehicle (−) or cucurbitacin I (10 μM; +) for 12 h on phosphorylated STAT3 (p-STAT3) was analyzed by western blotting with an anti-p-STAT3 antibody. β-actin was used as a loading control. (C) The effect of incubation of the five OS cell lines with vehicle (−) or cucurbitacin I (10 μM; +) for 24 h on the anti-apoptotic factor, Mcl-1, the apoptotic product, cleaved PARP, cleaved caspase-3 and the signaling molecules phospho-cyclin D1, c-Myc and survivin that regulate proliferation, was analyzed by western blotting. β-actin was used as a loading control. (D) The effect of incubation of the five OS cell lines with vehicle (control) or cucurbitacin I (10 μM) for 24 h on the activation of caspase-3 was analyzed using an ELISA.

Figure 4

Analysis of the effect on cell signaling of osteosarcoma cell lines by cucurbitacin I. (A) Western blot analysis of STAT3 expression in the five OS cell lines. β-actin was used as a loading control. (B) The effect of incubation of the five OS cell lines with vehicle (−) or cucurbitacin I (10 μM; +) for 12 h on phosphorylated STAT3 (p-STAT3) was analyzed by western blotting with an anti-p-STAT3 antibody. β-actin was used as a loading control. (C) The effect of incubation of the five OS cell lines with vehicle (−) or cucurbitacin I (10 μM; +) for 24 h on the anti-apoptotic factor, Mcl-1, the apoptotic product, cleaved PARP, cleaved caspase-3 and the signaling molecules phospho-cyclin D1, c-Myc and survivin that regulate proliferation, was analyzed by western blotting. β-actin was used as a loading control. (D) The effect of incubation of the five OS cell lines with vehicle (control) or cucurbitacin I (10 μM) for 24 h on the activation of caspase-3 was analyzed using an ELISA.

Figure 4

Analysis of the effect on cell signaling of osteosarcoma cell lines by cucurbitacin I. (A) Western blot analysis of STAT3 expression in the five OS cell lines. β-actin was used as a loading control. (B) The effect of incubation of the five OS cell lines with vehicle (−) or cucurbitacin I (10 μM; +) for 12 h on phosphorylated STAT3 (p-STAT3) was analyzed by western blotting with an anti-p-STAT3 antibody. β-actin was used as a loading control. (C) The effect of incubation of the five OS cell lines with vehicle (−) or cucurbitacin I (10 μM; +) for 24 h on the anti-apoptotic factor, Mcl-1, the apoptotic product, cleaved PARP, cleaved caspase-3 and the signaling molecules phospho-cyclin D1, c-Myc and survivin that regulate proliferation, was analyzed by western blotting. β-actin was used as a loading control. (D) The effect of incubation of the five OS cell lines with vehicle (control) or cucurbitacin I (10 μM) for 24 h on the activation of caspase-3 was analyzed using an ELISA.

Figure 5

In vivo antitumor effect of cucurbitacin I. Athymic nude mice with 143B xenografts were treated with vehicle (control), 0.25, 0.5 or 1 mg/kg of cucurbitacin I as described in Materials and methods. (A) The effect on tumor growth was assayed. Data are mean tumor volumes ± standard deviation (SD, error bars) of four mice per group. ^*p<0.01 (0.5 or 1.0 mg/kg vs. control by repeated ANOVA). A significant difference (^#p<0.05; 0.5 and 1.0 mg/kg vs. control at the 2-, 3- and 4-week time-points by one-way ANOVA). (B) The survival rate was analyzed by Kaplan-Meier analysis. A significant difference (^#p<0.01 by log-rank test). (C) Body weight was analyzed at the indicated time-points. A significant difference (^*p<0.05; 0.5 and 1.0 mg/kg vs. control at the 2-, 3- and 4-week time-points by one-way ANOVA).

Figure 6

Immunohistochemical analysis of cucurbitacin I-treated tumors. Representative 143B tumors of athymic nude mice were immunohistochemically analyzed on day 14 after administration of control (A, C, E, G, I, K, M, O and Q) or 1.0 mg/kg cucurbitacin I (B, D, F, H, J, L, N, P and R). (A and B) Hematoxylin-eosin (H&E) staining. (C and D) Immunohistochemical staining of phospho-STAT3. (E and F) p-cyclin D1. (G and H) c-Myc. (I and J) Cleaved PARP. (K and L) Suvivin. (M and N) Cleaved caspase-3. (O and P) TUNEL assay. (Q and R) Negative control.