Curdione suppresses human uterine leiomyosarcoma cells viability
After treatment with grade concentrations (0-500 μM) curdione for 12, 24, 48 and 72 h, the SK-UT-1 and SK-LMS-1 cells viability were detected by CCK8 assay respectively. As shown in Fig. 2, curdione inhibited the cells viability in a concentration and time dependent manner at concentration higher than 10 μM. The 50% inhibited concentration (IC50) were determined to find the ideal dose for the following experiments. The IC50 and the corresponding 95% confidence intervals of SK-UT-1 and SK-LMS-1 cell are 327.0 (297.7 -362.8) μM and 334.3 (309.9 - 362.5) μM respectively. To ensure more than 85% cells under good condition for following experiments, less than one third of IC50 curdione (100 μM) for proper dose.
Curdione inhibited human uterine leiomyosarcoma cells proliferation
To further confirm the anti-proliferation effect of Curdione on uterine leiomyosarcoma cell, SK-UT-1 and SK-LMS-1 cells were treated with curdione for 24 h, EdU assay and the proliferation marker ki67 were detected by immunofluoresent. As Fig.3 shows, compared with control, curdione decreased the EdU and ki67 positive rate of both cell lines in a concentration dependent manner.
Curdione induces G2/M phase arrest in human uterine leiomyosarcoma cells
There are close relation between proliferation and cell cycle distribution. To explore the effect of curdione on cell cycle progression, flow cytometry analysis was conducted to detect the cycle distribution, western blot were conducted to detect the cell cycle related proteins. As shown in Fig 4 a, compared with control the G2/M population increased after treated with 100 μM Curdione for 24 h, The cell populations in S phase decreased, and no significant changes occur in G0/G1 phase. Which indicated that, curdione induced cell cycle arrest in G2/M phase. The cell cycle related proteins analysis show that, curdione up-regulated the proteins P21 and CyclinB1, while down regulated Cdc2.
Curdione induces apoptosis in human uterine leiomyosarcoma cells
Curdione induced both early and late apoptotic of cells in a concentration-dependent manner. After treatment with curdione or not for 24 h, flow cytometry was performed with Annexin V-FITC/ Propidium Iodide (PI) double staining to quantify apoptosis. As shown in Fig.5, compared with control, the early apoptotic ratio elevated by 2.95-fold (1.9% and 1.6%) and 4.25-fold (5.6% and 6.8%) respectively; the late apoptotic ratio increased from 1.9% to 6.4% in SK-UT-1 cells, and from 1.2% to 4.9%in SK-LMS-1 cells; the total apoptosis ratio increased by 8.2% in SK-UT-1, and 8.9% in SK-LMS-1 cells. Which indicated that, both early and late apoptotic ratio of SK-UT-1 and SK-LMS-1 cells increased with curdione treatment in a dose-dependent manner.
Curdione induces autophagy in human uterine leiomyosarcoma cells
Autophagy is another important mechanism that regulate cell fate. To detect whether autophagy was induced by curdione, the level of autophagic marker proteins LC3-II, Beclin-1 and P62 in both cells were determined by western blotting. As shown in fig 6, curdione upregulated LC3-II and Beclin-1, and downregulated P62 in a dose-dependent manner. To further clarify that autophagy induced by curdione was pro-survival or pro-death, 3-MA (an autophagy inhibitor) was used to detect the influence of cell viability. Before treated with curdione, SK-UT-1 and SK-LMS-1 cells were incubated with autophagy inhibitor 3-MA for 2 h, the results showed that, 3-MA blocked the inhibitory effect of curdione on cell viability and apoptosis of SK-UT-1 and SK-LMS-1 cells significantly.
Curdione induced the crosstalk between apoptosis and autophagy in human uterine leiomyosarcoma cells
Curdione induced apoptosis and autophagy, to explore whether there was crosstalk between apoptosis and autophagy. SK-UT-1 and SK-LMS-1 were pre-treatment with Z-VAD-FMK (apoptosis inhibitor) or 3-MA (autophagy inhibitor) for 2 h, and then incubated with curdione for 24 h, apoptosis ratio were detected by flow cytometry. The apoptosis and autophagy marker proteins were determined by western blot. The results showed that, apoptosis ratio induced by curdione was blocked by Z-VAD-FMK and abolished by 3-MA (Fig.7 a). Western blot results show that, the decreased expression of LC3II, beclin1 and increased expression of p62 induced by curdione were alleviated by Z-VAD-FMK, the up regulation of cleaved caspase 3, 6, 9 induced by curdione were attenuated by 3-MA (Fig.7 b).
Curdione inhibited IDO1 expression in human uterine leiomyosarcoma cells
Western blot were performed to quantify the effect of curdione on IDO1 protein expression in SK-UT-1 and SK-LMS-1 cells. As Fig. 8 shows, Curdione down-regulated IDO1 expression of human uterine leiomyosarcoma cells in a concentration – dependent manner. The above data show that, curdione suppressed the proliferation of SK-UT-1 and SK-LMS-1 cells and down-regulated IDO1 expression. To further detected the relationship between regulation effect on IDO1 expression and anti-proliferation of curdione in both cell lines. Cells were pretreatment with IDO1 inhibitor for 2 h, and then incubated with curdione for 24 h. The results showed that, epacadostat reversed the suppression effect on cell viability induced by curdione in both cell lines. To further investigate this effect, IDO1 knockdown and overexpression were performed. The results showed that, compared with curdione alone group, silence of IDO1 overcame the suppression effect on cell viability induced by curdione. While, IDO1 overexpression enhanced the inhibitory effect induced by curdione.
COX2 is necessary for curdione-decreased IDO1 expression in human uterine leiomyosarcoma cells
To explore whether IDO1 mediate the suppression effect of curdione is dependent on COX2. NS398, an antagonism of COX2, significantly reversed the inhibitory effect of curdione on IDO1 expression in SK-UT-1 and SK-LMS-1 cells. To further confirm that, COX2 mediates curdione down-regulated IDO1 expression, transiently transfection technology approaches to manipulate COX2 expression in both cell lines were designed. Silence or overexpression of COX2 in mRNA and protein levels were confirmed by qRT-PCR and western blot analysis, knockdown COX2 reversed the inhibitory effect of curdione on IDO1 expression, while the inhibitory effect significantly enhanced by the over expression of COX2 in both cell lines. These findings collectively indicated that, dependent on COX2, curdione suppressed IDO1 expression in SK-UT-1 and SK-LMS-1 cells.
PKCδ/GSK3β/β-catenin pathway mediate curdione decrease IDO1 expression in uLMS cells
To further illustrate the mechanism involved in COX2-mediated induction of IDO1 expression in uLMS. The effects of curdione on the activation of PKCδ, GSK3β and β-catenin in human uterine leiomyosarcoma cells were detected. As shown in Fig 10, curdione promoted the PKCδ, GSK3β and β-cantein dephosphorylation significantly in a concentration-dependent manner. COX2 antagonist NS398 blocked the dephosphorylation of PKCδ, GSK3β and β-catenin pathway. To further confirm this mechanism, SK-UT-1 and SK-LMS-1 cells were transfected with either COX2-siRNA or COX2-pCMV6, and then treated with curdione for 24 h. The results show that, the PKCδ, GSK3β and β-catenin pathway activation were attenuated by COX2-siRNA and enhanced by the overexpression of COX2. The PKCδ, GSK3β and β-catenin pathway activity and the inhibitory effect of curdione on IDO1 expression were abolished by PKCδ inhibitor and GSK3β inhibitor. Which indicated that, the depression effect of curdione on IDO1 expression is mediated by the dephosphorylation of the PKCδ, GSK3β and β-catenin pathways in uLMS cells.
Curdione inhibited cell proliferation by induced cell cycle arrest in G2/M, apoptosis and autophagy in human uterine leiomyosarcoma cells
To confirm whether downregulation IDO1 is involved in the anti-proliferation of curdione in uLMS cells and mediated by the PKCδ/GSK3β/βcatenin dependent COX-2 pathway, SK-UT-1 and SK-LMS-1 cells were pretreatment with rottlerin or LY2090314 or NS398 for 2 h, and then incubated with curdione for 24 h. As shown in Fig. 11a, the anti-proliferation effect of curdione was attenuate by selective GSK3β inhibitor Rottlerin and β catenin inhibitor LY2090314, and COX-2 inhibitor NS-398 blocked the anti-proliferation effect of curdione. Fig. 11b shows that, the upregulation of cleaved-caspase 3, LC3, Beclin1, Cdc2, CyclinB1 and down regulation of P62 induced by curdione were abolished by IDO1 inhibitor epacadostat, GSK3β inhibitor Rottlerin, β catenin inhibitor LY2090314 and COX-2 inhibitor NS-398. The above results collectively indicated that IDO1 mediated PKCδ/GSK3β/βcatenin pathway dependent on COX-2 is involved in the anti-proliferative effect of curdione on human uterine leimyosarcoma cells.
Curdione suppress the growth of uterine leiomyosarcoma in vivo
To further explore the anti-uterine leiomyosarcoma effect of curdione in vivo, we established a xenograft tumor model by subcutaneous injection with SK-UT-1 cells into the right flanks of BALB/c nude mice. Tumor xenograft mice were intraperitoneal injection with 100 mg/kg/day or 200 mg/kg/day curdione or not for 21 days. The body weight, histopathological examination, tumor volume and weight were measured to evaluate the effect of curdione on the uterine leiomyosarcoma in vivo. Fig. 12 a-e showed that, compared with control, curdione reduced tumor volume and weight markedly. While, no significant body weight loss were detected in curdione treated xenograft mice. The histopathological results showed that, no obvious histopathological lesion were detected in liver and kidney tissue, which meaning curdione induced no liver and kidney injuries in vivo (Fig. 8 f). Above all, Curdione exhibited anti-uterine leiomyosarcoma efficacy with few toxicity in vivo.
Curdione induces apoptosis and autophagy in SK-UT-1 xenografts
To determine the mechanism involved in the inhibitory effect of curdione on human uterine leiomyosarcoma in vivo. The tissues protein expression of IDO1, COX2, the cleaved caspase-3, LC3-II, Becline1, P62, PKCδ, GSK3β, β-catenin were detected by western blot and immunohistochemistry assay. Consistent with the results in vitro, curdione up-regulated the protein expression of IDO1, COX2, the cleaved caspase-3, LC3-II, dephosphorylation of PKCδ, GSK3β, β-catenin and down-regulated ki67, p62 in tumor tissues (Fig. 13 a). The immunohistochemistry assay showed the same changes of protein levels observed in western blot (Fig. 13 b). The above results showed that, echoing to the results in vitro, curdione inhibited the expression of IDO1, COX2 and the phosphorylation of PKCδ/GSK3β/β-catenin pathway in vivo. In conclusion, acting on COX2 mediate IDO1 expression via PKCδ, GSK3β, β-catenin pathway, curdione suppress the growth of human uterine leiomyosarcoma in tumor xenografts.