Human ovarian cancer stem cells (OCSCs) are one of the main factors affecting ovarian cancer cell metastasis, recurrence, prognosis and tolerance to chemotherapy drugs. However, the mechanisms of OCSC proliferation and invasion are not clear. Recent studies suggest that anisomycin can inhibit the proliferative and invasive ability of various tumor cells by increasing the production of the toxic amyloid β (Aβ1-42) peptides from the amyloid precursor protein (APP). We explored whether anisomycin could also suppress human OCSC proliferation and invasion. The CD44+/CD117+ OCSCs were enriched from human clinical ovarian tumor tissues. OCSCs treated with anisomycin showed reduced proliferation compared to controls. Moreover, anisomycin significantly suppressed the invasive capacity of OCSCs
Human ovarian cancer stem cells (OCSCs) are one of the main factors affecting ovarian cancer metastasis, recurrence, prognosis and tolerance to chemotherapy drugs (
A recent study showed that inhibition of microRNA-20a expression increases APP expression and suppresses ovarian cancer cell proliferation and invasion (
Another important factor regulating Aβ1-42 production is the expression of the long non-coding RNA BACE1-antisense transcript (lncRNA BACE1-AS), which is upregulated in patients with Alzheimer's disease. The lncRNA can stabilize BACE1 mRNA and generate additional BACE1 through a feed-forward mechanism, thereby stimulating Aβ1-42 production (
While anisomycin has been shown to have antiproliferative ability in Jurkat T cells (
All samples were collected from the inpatient clinic of Shanghai Pudong New Area Gongli Hospital (Shanghai, China) between June 2013 and December 2014. All human materials were obtained according to consent regulation and approved by the Ethics Review Committee of the World Health Organization of Collaborating Center of Research in Human Production authorized by Shanghai Municipal Government. Briefly, 4
Total RNA from each cell was isolated using TRIzol reagent (Invitrogen), according to the manufacturer's protocol. The RNA samples were treated with DNase I (Sigma-Aldrich, St. Louis, MO, USA), quantified and reverse-transcribed into cDNA using the ReverTra Ace-α First Strand cDNA Synthesis kit (Toyobo). Quantitative RT-PCR was conducted using a RealPlex4 real-time PCR detection system from Eppendorf Co., Ltd. (Germany), with SYBR-Green Real-Time PCR Master Mix (Toyobo) as the detection dye. Amplification was performed over 40 cycles with denaturation at 95°C for 15 sec and annealing at 58°C for 45 sec. Target cDNA was quantified using the relative quantification method. A comparative threshold cycle (Ct) was used to determine gene expression relative to a control (calibrator) and steady-state mRNA levels are reported as an n-fold difference relative to the calibrator. For each sample, the maker gene Ct value was normalized using the formula Ct = Ct_genes-Ct_18sRNA. To determine relative expression levels, the following formula was used: ΔΔCt = ΔCt_all_ groups-ΔCt_blank control_group. The values used to plot relative expression of markers was calculated using the expression 2−ΔΔCt. The mRNA levels were calibrated based on levels of 18s rRNA. The cDNA of each gene was amplified using primers as previously described (
Northern blotting experiments were performed as previously described (
Cells were seeded (2×103/well) in 96-well plates and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2, until 85% confluent. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); Sigma Chemicals reagent (5 mg/ml) was added to the maintenance cell medium at different time points, and incubated at 37°C for an additional 4 h. The reaction was terminated with the addition of 150
Total protein extracts were resolved by 12% SDS-PAGE and transferred on polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking, the PVDF membranes were washed four times for 15 min with Tris-buffered saline with Tween-20 (TBST) at room temperature and incubated with primary antibody (
All steps were performed as previously described in 6-well plates (
All steps were performed as previously described (
Apoptosis was evaluated using an Annexin V-FITC/PI staining kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions and as previously described (
The cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking, the cells were incubated first with primary antibody (
OCSCs were divided into two groups: a negative control group (OCSCs pretreated with DMSO), which were treated with DMSO for 72 h; and an experimental group (OCSCs pretreated with anisomycin), which were treated with anisomycin (at its half maximal inhibitory concentration, IC50=8.5
Each experiment was performed as least three times and data are shown as the mean ± SE where applicable. Differences were evaluated using Mann-Whitney test. The probability p<0.05 was considered to indicate a statistically significant result.
We examined a total of seven ovarian cancer patients with undifferentiated and mixed epithelial ovarian carcinoma. The characteristics of all ovarian patients are summarized in
Cell viability (MTT) assays revealed no significant difference in the viability of anisomycin (8.5
Our qRT-PCR analysis showed that the expression of lncRNA BACE1-AS in the anisomycin-treated OCSCs was significantly elevated compared to the DMSO treated OCSCs or untreated OCSCs after 72 h (
In terms of protein expression, our western blot analysis confirmed that expression of amyloid-β-related proteins (BACE1 and Aβ1-42) were significantly increased in the anisomycin treated OCSCs compared to the DMSO treated or untreated OCSCs after 72 h (
All mice inoculated with anisomycin or DMSO pretreated OCSCs developed tumors ~7 weeks after injection. Although both experimental groups eventually developed tumors, tumors formed by anisomycin pretreated OCSCs grew more slowly than those derived from DMSO pretreated OCSCs (
To evaluate whether anisomycin regulates proliferation and invasion of OCSCs by inhibiting lncRNA BACE1-AS expression, we used a siRNA targeting BACE1-AS. OCSCs were transfected with either siRNA-BACE1-AS or siRNA-Mock and then treated with anisomycin for 72 h. The MTT assay indicated that the survival ratio of the anisomycin treated siRNA-BACE1-AS transfected OCSCs group was significantly higher than the anisomycin treated siRNA-Mock transfected OCSCs group or the anisomycin treated control group (
Anisomycin is a small molecule that has previously shown efficacy in inhibiting proliferation of a variety of tumor cells. In the present study, we found anisomycin (at IC50 dose of 8.5
Similar to our results, previous studies have shown that anisomycin can upregulate APP, BACE1 and PS1 expression by reducing methyltransferase activity in SH-SY5Y cells (
We propose that anisomycin increases lncRNA BACE1-AS expression, which in turn, stabilizes BACE1 mRNA and increases BACE1 expression through a feed-forward mechanism. As BACE1 is crucial for APP processing and Aβ1-42 production, which are toxic to cells, anisomycin could therefore result in OCSC apoptosis (
We also found that xenografts formed by anisomycin pretreated OCSCs were smaller and experienced reduced proliferation compared to those formed by DMSO pretreated OCSCs. This suggests that increasing lncRNA BACE1-AS expression (e.g., with anisomycin) is a novel strategy for reducing OCSC proliferation and invasion, and holds potential for future ovarian cancer treatment.
In conclusion, we showed that anisomycin suppresses proliferation and invasion of human OCSCs by increasing lncRNA BACE1-AS levels. In turn, lncRNA BACE1-AS can increase BACE1 mRNA stability, enhance APP processing, and elevate levels of the Aβ1-42 peptide, which are toxic to tumor cells. Therefore, our results suggest a novel mechanism for the antiproliferative or anti-invasive activity of anisomycin, and indicate that targeting lncRNA BACE1-AS expression could be a potential treatment for ovarian cancer.
The present study was supported by grant from the National Natural Science Foundation of China (no. 81202811), the Project funded by China Postdoctoral Science Foundation (nos. 2014M550250 and 2015T80455), the Shanghai Municipal Health Bureau Fund (no. 20124320), the Key Discipline Construction Project of Pudong Health Bureau of Shanghai (WZx2014-03), and Discipline Leader Project of Pudong Bureau of Shanghai (PWRd2014-02), and the Shanghai Pudong District Science and Technology Innovation Project (no. PKJ2015-Y52).
Anisomycin suppresses human OCSC proliferation and invasion. (A) MTT assays showing a significant decrease in proliferation of OCSCs over time when cells are treated with anisomycin; **p<0.01 vs. untreated group; *p<0.05 vs. untreated group; #p>0.05 vs. untreated group; n=3. (B) Flow cytometric assay showing that the percentage of cells in early apoptosis (Annexin V-FITC+/PI−) in anisomycin-treated OCSCs is elevated significantly compared to the untreated OCSCs. (C) Transwell migration invasion assay showing significantly fewer invading anisomycin-treated OCSCs/well than DMSO-treated OCSCs/well or untreated OCSCs group/well; *p<0.05 vs. untreated group; #p>0.05 vs. untreated group; n=3. (D) Soft agar colony formation assay indicating that anisomycin-treated OCSCs create substantially fewer colonies when plated at low density than DMSO-treated OCSCs or untreated OCSCs; *p<0.05 vs. untreated group; #p>0.05 vs. untreated group; n=3.
Anisomycin stimulated expression of lncRNA BACE1-AS and apoptosis-related genes in OCSCs. (A) The qRT-PCR assay shows elevated expression of lncRNA BACE1-AS in the anisomycin-treated OCSCs (red) compared to the DMSO treated OCSCs (blue) or untreated OCSCs (green) after 72 h of treatment; the mRNA levels were calibrated based on levels of 18s rRNA; **p<0.01 vs. untreated group; n=3. (B) The qRT-PCR reveals elevated expression of Bace1, APP, Bcl2 and Bax mRNAs in the anisomycin-treated OCSCs (red) compared to the DMSO treated OCSCs (blue) or the untreated OCSCs (green) after 72 h of treatment. However, the mRNA expression of Ki67 is decreased in the anisomycin-treated OCSCs compared to the DMSO treated or untreated OCSCs after 72 h of treatment. The mRNA levels were calibrated based on levels of 18s rRNA; **p<0.01 vs. untreated group; *p<0.05 vs. untreated group; #p>0.05 vs. untreated group; n=3.
Anisomycin stimulates expression of lncRNA BACE1-AS and apoptosis-proteins in OCSCs. (A and B) Western blotting confirms increased expression of BACE1 and Aβ1-42 in anisomycin-treated OCSCs compared to the DMSO OCSCs or the untreated OCSCs after 72 h. However, the expression of Ki67 protein is decreased in anisomycin-treated OCSCs group compared to DMSO-treated or untreated OCSCs. GAPDH was used as a loading control; **p<0.01 vs. untreated group; #p>0.05 vs. untreated group; n=3. (C) Immunofluorescence staining confirming increased BACE1 enzyme and Aβ1-42 expression levels in the anisomycin-treated OCSCs compared to DMSO treated or untreated OCSCs after 72 h.
Anisomycin inhibits OCSC xenograft growth. (A) Images of the excised xenograft tumors from the anisomycin pretreated OCSC mice and the DMSO pretreated OCSC mice; blue circle marks the tumor tissue. (B) Tumor size in the anisomycin pretreated OCSC mice (red) and the DMSO pretreated OCSC mice (blue); **p<0.01 vs. DMSO group; n=7. (C) Tumor weight in the anisomycin pretreated OCSC mice (red) and the DMSO pretreated OCSC mice (blue); **p<0.01 vs. DMSO group; n=7. (D) Representative hematoxylin and eosin (H&E) stained sections of all subcutaneous xenograft tumors, which were categorized as mixed epithelial ovarian carcinoma of moderate or poor differentiation (grade II/III). Immunohistochemical staining indicating that BACE1 and Aβ1-42 protein expression is significantly higher in tumors derived from anisomycin pretreated OCSCs, compared to tumors from DMSO pretreated OCSCs. The expression of the Ki67 protein is significantly decreased in the tumors derived from anisomycin pretreated OCSCs compared to tumors from DMSO pretreated OCSCs. Original magnification, ×200.
siRNA targeting of lncRNA BACE1-AS weakens the inhibitory effects of anisomycin on OCSCs. (A) The MTT assay indicates that the survival ratio of the anisomycin-treated siRNA-BACE1-AS transfected OCSCs (blue) are significantly higher than the anisomycin-treated siRNA-Mock transfected OCSCs (green) or the anisomycin-treated OCSCs (red) after 72 h; *p<0.05 vs. anisomycin-treated group; #p>0.05 vs. anisomycin-treated group; n=3. (B) Western blot analysis shows that the expression of BACE1 is reduced in the anisomycin-treated siRNA-BACE1-AS transfected OCSCs (blue) compared to the anisomycin-treated siRNA-Mock transfected OCSCs (green) or the anisomycin-treated OCSCs (red) after 72 h. GAPDH was used as a loading control; **p<0.01 vs. anisomycin-treated group; #p>0.05 vs. anisomycin-treated group; n=3. (C) Northern blot reveals that the hybridization signal of lncRNA BACE1-AS from the anisomycin-treated OCSCs after 72 h is significantly stronger than from the DMSO treated or untreated OCSCs. (D) The hybridization signal of lncRNA BACE1-AS from the anisomycin-treated siRNA-BACE1-AS transfected OCSCs after 72 h is significantly weaker than in the anisomycin-treated siRNA-Mock transfected OCSCs or the anisomycin-treated OCSCs; the snRNA U6 was used as a loading control.
The source and dilutions of primary antibodies.
Antibodies | Companies | Applications |
---|---|---|
Rabbit anti-human Aβ1-42 | Cell Signaling Technology, Danvers, MA, USA | WB (1:1,000) |
Goat anti-human BACE1 | Santa Cruz Biotechnology, Santa Cruz, CA, USA | WB (1:1,000) |
Rabbit anti-human Ki67 | Santa Cruz Biotechnology, Santa Cruz, CA, USA | WB (1:1,000) |
Rabbit anti-human GAPDH | Cell Signaling Technology, Danvers, MA, USA | WB (1:1,000) |
Donkey anti-goat-Cy3 | Santa Cruz Biotechnology, Santa Cruz, CA, USA | IF (1:100) |
Goat anti-rabbit-FITC | Santa Cruz Biotechnology, Santa Cruz, CA, USA | IF (1:100) |
Characteristics of the patients cohort.
Characteristics | Patients (n=7) |
---|---|
Age median (range) | 38–62 |
≤40 | 2 |
40–60 | 4 |
≥60 | 1 |
Surgical staging | |
I a–c | 1 |
II a–c | 3 |
III a–c | 3 |
IV | – |
Histopathology | |
Serous | – |
Endometrioid | – |
Mucinous | – |
Clear cells | – |
Others (mixed epithelial, undifferentiated) | 7 |
Tumor grade | |
1 | 1 |
2 | 4 |
3 or clear cell | 2 |
Unknown | – |
Treatments | |
Primary surgery | 7 |
Radical surgery | – |
Secondary surgery | – |
Platinum-based chemotherapy | 5 |
Radiotherapy | – |