Article Figures & Data
Figures
- Figure 1.
Eribulin reduced viable CCS cell number by induction of G2–M cell-cycle arrest and apoptosis. A, Four CCS cell lines, Hewga-CCS, MP-CCS-SY, KAS, and SU-CCS1 were treated with 0–30 nmol/L eribulin for 96 hours and viable cell number estimated by WST-1 assay. The calculated IC50 values are shown in the table. B and C, Cell lines were treated with 10 (B) and 100 (C) nmol/L eribulin for 0 to 24 hours, stained with PI, and analyzed for cell-cycle stage by flow cytometry. D, Cell lines were treated with 0.1 to 1,000 nmol/L eribulin or vehicle for 72 hours, and expression of the apoptosis marker cleaved caspase-3 was evaluated by Western blotting. Data in A are presented as mean ± SD, n = 3.
- Figure 2.
Eribulin inhibited growth, elevated melanin synthesis, and induced vascular remodeling of Hewga-CCS and MP-CCS-SY xenograft tumors. A and B, Hewga-CCS and MP-CCS-SY cells were engrafted in nude mice. Mice were then treated with either eribulin (1 or 3 mg/kg) or vehicle by intravenous injection (5 mice/group). The size of Hewga-CCS (A) and MP-CCS-SY (B) tumors during treatment are shown. C, Fontana–Masson staining for melanin and quantitative evaluation of melanin-positive cell number (% of total cells) in both xenograft tumors of all three treatment groups (five fields counted/group) are shown. D, IHC staining for CD31 and quantification of CD31-positive cell number in both xenograft tumors of all three treatment groups (five fields counted/group) are shown. E, IHC staining for HIF1α in both xenograft tumors from all three treatment groups is shown. F, Tumor tissues from Hewga-CCS and MP-CCS-SY xenografts at the endpoint were collected. Whole-cell proteins were extracted and subjected to Western blot analyses. Scale bars are 100 μm. Data in A, B, C, and D are presented as mean ± SD. *, P < 0.05; **, P < 0.01 vs. the vehicle group.
- Figure 3.
Eribulin upregulated melanin synthesis, TYR activity, and melanocytic differentiation markers in CCS cells. A and B, CCS cells were cultured for 96 hours with 10 nmol/L eribulin or vehicle. The melanin content (A; proportion of total protein relative to control) and TYR activity (B; relative to control) in CCS cells are shown. C, CCS cells were treated with 10 nmol/L eribulin for 0, 24, 48, 72, or 96 hours. EWSR1–ATF1, MITF, TYR, TRP1, and TRP2 protein expression levels were estimated by Western blotting. Data in A and B are presented as mean ± SD, n = 3. **, P < 0.01 vs. the control group.
- Figure 4.
Eribulin induces melanocytic differentiation through inhibition of ERK1/2. A and B, CCS cells were treated with 10 nmol/L eribulin for 0, 24, 48, 72, or 96 hours. MITF (A) and EWSR1–ATF1 (B) mRNA levels in CCS cells were quantified using qRT-PCR (normalized to β-actin). C, Hewga-CCS cells were cultured for 48 or 96 hours in the presence of 10 nmol/L eribulin or vehicle. Cells were then treated with the protein synthesis inhibitor cycloheximide for 0, 3, 6, 12, or 24 hours, and protein lysates were prepared. MITF and β-actin protein expression levels were examined by Western blot analysis. Quantification of relative MITF expression (normalized to β-actin) is shown. D, CCS cells were treated with 10 nmol/L eribulin for 0, 24, 48, 72, or 96 hours. Phosphorylation of ERK1/2 (p-ERK1/2) was assessed by Western blot analysis. E, Hewga-CCS and MP-CCS-SY cells were treated with the selective ERK1/2 inhibitor SCH772984 at 100 nmol/L for 0, 24, 48, 72, or 96 hours and then subjected to Western blot analyses with the indicated antibodies. F, Tumor tissues from Hewga-CCS and MP-CCS-SY xenografts at the endpoint were harvested, and lysates were prepared for Western blot analyses using the indicated antibodies. Data in A, B, and C are presented as mean ± SD, n = 3. *, P < 0.05 vs. the control group.
- Figure 5.
Reoxygenation elevates MITF protein levels through inhibition of ERK1/2 signaling and reduces CCS cell growth. A and B, Hewga-CCS and MP-CCS-SY cells grown under 1% oxygen, 3% oxygen, or atmospheric (21%) oxygen in the absence (A and B, left) or presence (A and B, right) of 100 nmol/L SCH772984. HIF1α, MITF, and p-ERK1/2 protein expression levels were assessed by Western blot analysis. C, MITF mRNA levels in both CCS cells grown under 1%, 3%, or 21% oxygen for 24 hours were quantified using qRT-PCR. D, Cell proliferation in both CCS cells grown under 3%, 5%, or 21% oxygen for 96 hours was estimated by WST-1 assay. E, Wound healing assays were conducted under 3%, 5%, or 21% oxygen. Data in C, D, and E are presented as mean ± SD, n = 3. *, P < 0.05 and **, P < 0.01 vs. the vehicle group.
- Figure 6.
Knockdown of MITF debilitates the antiproliferative effect of eribulin on CCS cells. KAS and MP-CCS-SY cells were transfected with MITF-specific siRNAs or a nontargeting negative control siRNA. A, MITF protein expression levels of both cells 48 hours after MITF knockdown were evaluated by Western blot analysis. B, Proliferation of both cells was measured by WST-1 assay during 1 to 3 days of culture. C, Cells were treated with different concentrations of eribulin for 48 hours and growth inhibition was measured by WST-1 assay. D, Cells were treated with 10 nmol/L eribulin for 48 hours, and expression of cleaved caspase-3 was assessed by Western blot analysis. E, Schematic presentation of the antitumor effects of eribulin on CCS cells. Data in B and C are presented as mean ± SD, n = 3. *, P < 0.05 and **, P < 0.01 versus the control group.
Additional Files
Supplementary Data
- Supplementary Figure S1 - Supplementary Figure S1 shows tumor weights at the end point, body weights during treatment, HE staining and immunohistochemical staining in Hewga-CCS and MP-CCS-SY xenograft tumors of all three treatment groups.
- Supplementary Figure S2 - Supplementary Figure S2 shows measurement of melanin content and WST-1 cell proliferation assay in Hewga-CCS and MP-CCS-SY cells treated with SCH772984.
- Supplementary Figure S3 - Supplementary Figure S3 shows western blot in Hewga-CCS and MP-CCS-SY cells treated with CoCl2 in the absence or presence of SCH772984, and qRT-PCR in both CCS cells treated with CoCl2.
- Supplementary Table S1 - Supplementary Table S1 shows information of antibodies used in this study.
Volume 19, Issue 3
