Curdione Induces Antiproliferation Effect on Human Uterine Leiomyosarcoma via Targeting IDO1

Abstract

Objectives: Curdione is one of the active ingredients of a traditional Chinese herbal medicine-Curcuma zedoary and established anti-tumor effects. Uterine leiomyosarcoma (uLMS) is a rare gynecological malignancy, with no standard therapeutic regimen at present. The aim of this study was to explore the potential anti-tumor impact of curdione in uLMS and elucidate the underlying mechanisms.

Methods: In vitro functional assays were performed in the SK-UT-1 and SK-LMS-1 cell lines. The in vivo model of uLMS was established by subcutaneously injecting SK-UT-1 cells, and the tumor-bearing mice were intraperitoneally injected with curdione. Tumor weight and volume were measured at specific time points. The biosafety was evaluated by monitoring changes of body weight and the histopathology in the liver and kidney. The expression levels of relevant proteins were analyzed by western blotting and immunohistochemistry.

Results: Curdione decreased the viability and proliferation of uLMS cells in a concentration and time-dependent manner. In addition, the curdione-treated cells exhibited significantly higher rates of apoptosis and autophagic death. Curdione also decreased the tumor weight and volume in the SK-UT-1 xenograft model compared to the untreated control without affecting the body bodyweight or pathological injury of liver and kidney tissues. At the molecular level, the anti-tumor effects of curdione were mediated by indoleamine-2, 3-dioxygenase-1 (IDO1).

Conclusion: Curdione exhibited an anti-uLMS effect in vitro and in vivo; the underlying mechanism involved in IDO1 mediate apoptosis, autophagy, and G2/M phase arrest.

Keywords: Indoleamine-2, 3-dioxygenase-1; apoptosis; autophagy; curdione; uterine leiomyosarcoma.

Figure 1

Effect of curdione on the viability in uLMS cells. The dose and time response of curdione in (A) SK-UT-1 and (B) SK-LMS-1 cells. The viability of cells treated with varying concentrations of curdione for 24 h, or 100 μM curdione for 12, 24, 48 and 72 h were detected by CCK8. Accordingly, IC50 of (C) SK-UT-1 and (D) SK-LMS-1 cells were calculated by non-linear regression using GraphPad Prism7. (E) Chemical structure of curdione. All values were expressed as the mean ± SD, n = 3. ^* P < 0.05, ^** P < 0.01 compared with control.

Figure 2

Effect of curdione on the proliferation of uLMS cells. Immune fluorescence detection of the proliferation effect of curdione in uLMS cells. Cultured SK-UT-1 and SK-LMS-1 cells were treated with 0, 25, 50, and 100 μM curdione for 24h, and stained with (A) EdU and (B) Ki67, the stained cells were observed by fluorescence microscope (×40 magnification). Quantitative analysis of the positive fluorescence density by Image J, Independent experiments were performed three times. Data were presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01 compared with control.

Figure 3

Curdione induces G2/M phase arrest in uLMS cells. (A) Flow cytometry analyzes the cell cycle distribution in SK-UT-1 and SK-LMS-1 cells treated with 0, 25, 50, and 100 μM curdione for 24 h. (B) Western blotting detection of the P21, CylinB1, and Cdc2 expression in SK-UT-1 and SK-LMS-1 cells treated with 0, 25, 50, and 100 μM curdione for 24 h. Independent experiments were performed three times. All data were presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01 compared with control.

Figure 4

Curdione induces caspases-mediate apoptosis in uLMS cells. SK-UT-1 and SK-LMS-1 cells were treated with 0, 25, 50, and 100 μM curdione for 24 h, (A) Flow cytometry with Annexin V-FITC/PI staining was conducted to analyze the apoptosis ratio. (B) Immunofluorescent analysis with TUNEL staining was performed to detected apoptosis using laser confocal microscopy (×40 magnification), Scale bars = 100 μm. (C) Western blotting detection of the pro-and cleaved-caspase 3, 6, 8, and 9 expressions. The relative protein expression level was normalized to that of GAPDH. Independent experiments were repeated three times, all data were present with means ± SD, n = 3. *P < 0.05, **P < 0.01 compared with control.

Figure 5

Curdione induces autophagic death in uLMS cells. (A) Curdione induced autophagy. Beclin1, LC3, and P62 expression in SK-UT-1 and SK-LMS-1 cells treated with 0, 25, 50, and 100 μM curdione for 24 h were determined by Western blotting. (B) The autophagy induced by curdione was pro-death. The viability of SK-UT-1 and SK-LMS-1 cells treated with 3-MA (2 mM) for 2 h following with curdione (100 μM) for 24 h was detected by CCK8. Statistical analysis data were present with means ± SD, n = 3. *P < 0.05, **P < 0.01 compared with control; ^# P < 0.05; ^## P < 0.01 compared with curdione alone group.

Figure 6

IDO1 knockdown reversed the anti-proliferation effect of curdione in uLMS cells. (A) Curdione down-regulated IDO1 expression in a dose-dependent manner. IDO1 expression of SK-UT-1 and SK-LMS-1 cells treated with 0, 25, 50, 100 μM curdione for 24 h were analyzed by Western blotting. (B) Curdione down-regulated IDO1 expression in a time-dependent manner. IDO1 expression of uLMS cells treated with 100 μM curdione for 24, 48, 72 h was analyzed by Western blotting. (C) mRNA levels of IDO1 in uLMs cells after transfections with IDO1-siRNA was detected by RT-PCR, (D) Protein levels of IDO1 in uLMs cells after transfections with IDO1-siRNA was detected by Western blotting. (E) Epacadostat attenuated the suppressive effect on uLMS cells. The viability of uLMS cells pre-treated with IDO1 inhibitor epacadostat (25 nM) following with curdione was detected by CCK8. (F) IDO1-siRNA reversed the suppressive effect on uLMS cells. The viability of uLMS cells transfected with IDO1-siRNA was detected by CCK8. All data were present with means ± SD, n = 3. *P < 0.05, **P < 0.01 compared with control; ^# P < 0.05, ^## P < 0.01 compared with curdione group.

Figure 7

IDO1 mediates apoptosis, autophagy, and G2/M phase arrest induced by curdione in uLMS cells. Western blotting detection of (A) cleaved caspase 3, 6, 9; (B) Beclin1, LC3, and P62; (C) P21, CylinB1, and Cdc2 expression in SK-UT-1 and SK-LMS-1 cells pre-treated with epacadostat (25 nM) for 2 h following with curdione for 24 h. All data were presened with mean ± SD, n = 3. *P < 0.05; **P < 0.01 compared with control; ^# P < 0.05, ^## P < 0.01 compared with curdione alone group.

Figure 8

Anti-growth effect of curdione in the SK-UT-1 xenograft tumor model. SK-UT-1 xenograft tumor models were established, and randomly divided into three groups (n = 5), and then injected intraperitoneally (i.p.) with 100 or 200 mg/kg/d curdione, or same volume saline. Tumor volume and body weight were measured every three days. 21 days later, all mice were sacrificed. Representative images of (A) tumor-bearing nude mice and (B) subcutaneous dissection of tumor tissue were photographed, (C) tumor weight, (D) tumor volume, and (E) body weight was determined and recorded. (F) The pathological injury of liver and kidney tissues was assessed by the H&E staining (×200 magnification). Scale bars = 100 μm. All data were presented with mean ± SD, n = 3. *P < 0.05, **P < 0.01 compared with control.

Figure 9

IDO1 mediates the suppressive effect of curdione in the SK-UT-1 xenograft tumor model. (A) Western blotting detection of protein expression of IDO1, cleaved caspase-3, Beclin1, LC3, p62 in tumor tissue. (B) Immunohistochemistry staining of Ki67, IDO1, LC3, p62, cleaved caspase-3, and TUNEL in tumor tissue (×200 magnification), Scale bars = 100 μm. (C) Expression scores of Ki67, IDO1, LC3, p62, cleaved caspase-3, and TUNEL was calculated according to immunostaining intensity and positive expression rate. All statistical analysis data were present with mean ± SD, n = 3. *P < 0.05, **P < 0.01 compared with control.