br Western blot analysis br Equal quantities of extract
2.11. Western blot analysis
Equal quantities of extract (25 μg of total protein) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. After blocking, the membranes were sequentially incubated with primary and secondary 474-25-9 diluted in 5% bovine serum albumin. Horseradish peroxidase (HRP)-labeled immunoglobulin (IgG) was used as the secondary antibodies for detection. Antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The expression of the proteins was studied using AlphaImager 2200 (Bio-Techne, Minneapolis, MN, USA).
Table 1
List of gene specific primers for PCR.
Nanog
AAGGCCTCAGCACCTACCTA
TGCACCAGGTCTGAGTGTTC
Klf4
ATGCTCACCCCACCTTCTTC
TTCTCACCTGTGTGGGTTCG
Oct-4
GGTGGAGGAAGCTGACAACA
ATCTGCTGCAGTGTGGGTTT
CD24
AACAGCCAGTCTCTTCGTGG
AGACGCCATTTGGATTGGGT
CD44
CCCATTCGACAACAGGGACA
TGGGGTGTGAGATTGGGTTG
Caspase-3
CCTGGTTCATCCAGTCGCTT
TCTGTTGCCACCTTTCGGTT
Caspase-8
CAGAGCCTGAGAGAGCGATG
AGGCTGAGGCATCTGTTTCC
Caspase-9
CAAGAGTGGCTCCTGGTACG
CGAAACAGCATTAGCGACCC
Bax
ACTGAAGCGACTGATGTCCC
CAAAGATGGTCACGGTCTGC
Bcl-2
GAACTGGGGGAGGATTGTGG
GCCGGTTCAGGTACTCAGTC
CDK2
GGCACGTACGGAGTTGTGTA
ACCCTCAGTCTCAGTGTCCA
CDK4
CAGATGGCACTTACACCCGT
CAACTGGTCGGCTTCAGAGT
Cyclin D1
CAGATCATCCGCAAACACGC
AAGTTGTTGGGGCTCCTCAG
Pin1
AGCTCAGGCCGAGTGTACTA
CTCCTTGGTCCGGGTGATC
Akt
GGACAAGGACGGGCACATTA
CGACCGCACATCATCTCGTA
P65
CCTATAGAAGAGCAGCGTGGG
AGATCTTGAGCTCGGCAGTG
STAT3
AGCAGCACCTTCAGGATGTC
GCATCTTCTGCCTGGTCACT
P38
GTGGCCACTAGGTGGTACAG
CTCGGCCATTATGCATCCCA
JNK
TAAAGCCAGTCAGGCAAGGG
TGGTGGAGCTTCTGCTTCAG
Actin
GGTCACCAGGGCTGCTTTTA
GGATCTCGCTCCTGGAAGATG
Cytokines in culture medium were detected using a BD Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit (BD Bioscience, San Jose, CA, USA). The kit simultaneously measures Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Tumor Necrosis Factor (TNF), Interferon-γ (IFN-γ), and Interleukin-17A (IL-17A) protein levels in a single sample. The operations were per-formed according to the manufacturer's instructions. Samples were measured on Flow Cytometer (BD FACSCanto, San Jose, CA, USA).
2.13. Statistical analysis
All data were expressed as the mean ± S.D. and analyzed using SPSS 17.0 software. All experiments were repeated at least three times. Statistically significant differences between groups were determined using Student's t-tests. P values < 0.05 were regarded as statistically significant.
3. Results
3.1. Celastrol inhibits the proliferation and induces G2/M cell cycle arrest in ovarian cancer cells
The MTT assay showed that the proliferation of A2780, OVCAR3,
and SKOV3 was suppressed by celastrol in a dose-dependent and time-
dependent manner. The suppression of SKOV3 cell proliferation was a
higher than that of the other two cell types (Fig. 1A). Cell cycle analysis
showed that compared with the untreated group, celastrol treatment
significantly increased the G2/M phase from 9.44 ± 1.77% to
30.47 ± 4.99%, and 31.97 ± 5.51% in SKOV3 cells at doses of 0.25, 0.5 and 1 μM, respectively (Fig. 1B and C). Celastrol reduced the po-pulation in S Phase, but had no significant influence on the G0/G1 phase. The results revealed that celastrol caused G2/M phase arrest in a dose-dependent manner in A2780, OVCAR3 and SKOV3 cells. To some extent, SKOV3 cells were much more sensitive to celastrol than A2780 and OVCAR3. To further illuminate the mechanism of cell cycle arrest induced by celastrol, the SKOV3 cells treated with celastrol for 24 h showed that the expression of CDK2, CDK4 and Cyclin D1 was down-regulated greatly using RT-qPCR (Fig. 1D) and by western blot (Fig. 1E).
3.2. Celastrol induces apoptosis in ovarian cancer cells
To study whether celastrol was able to induce apoptosis in A2780, OVCAR3 and SKOV3 cells, the apoptosis assay was performed. As the concentration of celastrol increased, both early and late numbers of apoptotic cells increased. The greatest effect was registered in SKOV3 cells with an increase from 4.2% to 10.0% in early apoptotic cells and 3–27.4% in late apoptotic cells (Fig. 2A and B). These results confirmed that the anti-cancer activity of celastrol in ovarian cancer cells is related to the induction of apoptosis. To further illuminate the mechanism of apoptosis induced by celastrol, the SKOV3 cell was treated with celas-trol for 24 h and the RT-qPCR (Fig. 2C) and western blot (Fig. 2D) re-sults showed that the levels of Caspase-3, Caspase-8, and Bax were significantly increased and the expression of Bcl-2 was downregulated greatly by treatment with celastrol.