br Celastrol inhibits the migration of
3.3. Celastrol inhibits the migration of ovarian cancer cells
We used the wound-healing and transwell assay to research the eﬀect of celastrol (0, 0.25, 0.5, 1 μM) on the migration of A2780, OVCAR3 and SKOV3 after treatment for 24 h. Our results verified that
the migration distance covered by 6-diazo-5-oxo-L-nor-Leucine treated with celastrol clearly decreased compared with those in the control groups (Fig. 3A). The rate of migration was evaluated and was shown to be significantly reduced after treatment with celastrol (Fig. 3B). Consistent with this, the transwell migration assay showed the number of cells that had migrated to the lower chamber was significantly reduced by celastrol in a con-centration dependent manner (Fig. 3C and D). We also found that the migration protein of p-FAK and MMP9 was downregulated (Fig. 3E). Therefore, higher celastrol concentration is associated with a corre-sponding decrease in cell migration.
3.4. Eﬀects of celastrol on cancer stem cell marker expression
SKOV3 cells were much more sensitive to celastrol than A2780 and OVCAR3; therefore, SKOV3 cells were chosen to research expression
patterns. The expression of the CSC marker CD44 and the portion of the CD44highCD24low cell prominently declined after treatment with ce-
lastrol in a dose-dependent manner (Fig. 4A and B). We also carried out quantitative real-time PCR to measure the expression levels of several classic stem cell markers Nanog, Oct4 and Klf4. Stem cell marker ex-pression was markedly reduced by celastrol (Fig. 4C). The results sug-gest that celastrol can depress the properties of cancer stem cells.
3.5. Eﬀects of celastrol on cancer stem cell properties of SKOV3 cells
Colony formation and sphere formation assay was performed to observe long-term eﬃcacy of celastrol on SKOV3 cells and to explore how celastrol treatment aﬀected cancer stem cell properties. The
SKOV3 cell colonies were significantly reduced after celastrol treatment (Fig. 5A and B). Furthermore, we evaluated the sphere forming capacity after adding celastrol to SKOV3 cells in a serum free non-adherent medium. The results revealed that the sphere size and number in SKOV3 cells markedly diminished in a dose-dependent manner after celastrol treatment (Fig. 5C and D). These results were consistent with findings shown in Fig. 4 and revealed that celastrol attenuated stem cell characteristics in SKOV3 cells.
3.6. Celastrol inhibits the expression of Pin1 and blocks multiple cancer driving pathways
To elucidate the molecular mechanisms by which celastrol aﬀects ovarian cancer cells, Pin1 expression was analyzed. After treatment with various concentrations of celastrol for 24 h, the mRNA and protein expression of Pin1 was significantly downregulated in a dose-dependent manner (Fig. 6A and B). To further support the notion that celastrol exerts potent anticancer activity against ovarian cancer by inhibiting Pin1, we examined the eﬀects of celastrol on a set of oncoproteins, which are substrates for Pin1 and whose protein stability is maintained by Pin1. We found that the expression of Akt, p-STAT3, p-P38, p-JNK, and P65 were all reduced (Fig. 6A and B) by celastrol treatment. As demonstrated in Fig. 6C, the concentration of IL-6 that can promote cancer progression was significantly reduced in a dose-dependent manner when the SKOV3 cells were treated with celastrol. The results demonstrate that celastrol may exert its eﬃcacy against ovarian cancer mainly through inhibiting Pin1 and thereby blocking multiple cancer pathways simultaneously.
Fig. 5. Eﬀect of celastrol on cancer stem cell properties of SKOV3 cell. Single cell suspension seeded in triplicate at 300 cells per well in six-well plates and treated with diﬀerent concentrations of celastrol (0, 0.25, 0.5, and 1 μΜ). Visible colony cells were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet. Plates were photographed and colonies counted manually (A and B). Scale bar, 200 µm. SKOV3 cells were plated at a density of 5 × 103 cells per well in ultra-low attachment six well plates and treated with diﬀerent concentrations of celastrol (0, 0.25, 0.5, and 1 μΜ) after plating the cells for 24 h. Cells were grown for 10 days. The generated spheroids were photographed using an inverted microscope and counted (C and D). Images shown are representative of three independent experiments. ★P < 0.05, ◆P < 0.01, ▲P < 0.001.
Celastrol is of particular interest because of its superior anti-cancer capabilities against a variety of cancer types, however, the mechanism for its anti-cancer eﬀect are not fully understood in ovarian cancer. In the present study, we demonstrated that celastrol could significantly suppress the viability and migration and induced apoptosis and cell cycle arrest at G2/M phase with obvious down-regulation of Cyclin D1, CDK2, CDK4, Bcl-2 and increasing of Caspase-3, Caspase-8, Bax in dose-dependent manner. Celastrol suppressed the expression of Pin1 and then blocked the expression of Akt, STAT3, P38, JNK, P65 and IL-6. Celastrol treatment inhibited the expression of stem cell marker CD44,