Article Figures & Data
Figures
- Figure 1.
The regulation of CLDN4 by C-CPE treatment in EOC cells. A, qRT-PCR analysis of CLDN4 mRNA expression levels in various EOC and normal HOSE cells treated with or without C-CPE (5 μg/mL) for 48 hour. β-Actin was used as an endogenous control to standardize the amount of RNA in each reaction. Data, mean ± SD from triplicate experiments. B, effect of C-CPE on CLDN4 protein expression in EOC cell lines. Whole lysates from the above EOC cell cultures were subjected to Western blot analysis with antibodies to CLDN4 and β-actin (loading control). C, C-CPE downregulated CLDN4 protein expression in a time-dependent manner. SKOV-3 cells were cultured with or without C-CPE (5 μg/mL) for 24, 48, and 72 hours and another 24 hours after removal of C-CPE. Whole cell lysates were subjected to Western blot analysis. The experiments were done in triplicate.
- Figure 2.
A, immunofluorescent staining of CLDN4 in SKOV-3 and RMUG-L spheroids in 3D culture. SKOV-3 and RMUG-L spheroids were cultured in growth factor–reduced Matrigel for 10 days and then treated with or without C-CPE (5 μg/mL) for 72 hours. Top, left, confocal fluorescence microscopy images show redistribution of CLDN4 in a diffused staining pattern and morphologic changes in C-CPE–treated spheroids. In contrast, CLDN4 was retained at the cell–cell border with strong staining and on membrane with weak cytoplasmic reactivity in control spheroids. Nuclei were counterstained with DAPI. Scale bars, 20 μm. Top, right, the phase-contrast images show the representative 3D structures of SKOV-3 and RMUG-L cells. Bottom, the percentage of positive spheroids was determined by counting 30 spheroids in each of 3 independent experiments. Spheroids were scored positive when the cells exhibited apparent CLDN4 relocalization and morphologic changes. *, P < 0.001 versus control. B, immunofluorescent staining of CLDN4 in monolayer culture of OVCA429 cells treated in the presence or absence of C-CPE (5 μg/mL) for indicated time. Scale bars, 40 μm.
- Figure 3.
C-CPE enhanced chemosensitivities of EOC cells in a CLDN4-dependent manner in vitro. A, cytotoxicity of C-CPE. Left, after exposure to increasing concentrations of C-CPE (0.01–15 μg/mL) or medium alone (control) for 48 hours, cell growth and viability of SKOV-3 cell was evaluated by MTT assay. Right, after treatment with or without C-CPE (5 μg/mL) for indicated time, growth curve of SKOV-3 cells was measured by MTT assay. B–D, C-CPE effect on chemosensitivity of EOC cells to low-dose chemotherapy agents. On the basis of the result of Figure 1B, three EOC cell lines expressing CLDN4 protein at varying levels were selected: OVCA429 (highest), SKOV-3 (higher), and TOV112d (deficient) cells. Left, after exposure to Taxol alone at the indicated concentrations or the combination of Taxol and C-CPE (5 μg/mL) for 24 hours, cell viability was compared in each cell lines by MTT assay, respectively. Right, after exposure to Carboplatin alone at the indicated concentrations or the combination of Carboplatin and C-CPE (5 μg/mL) for 24 hours, cell viabilities were compared in each cell lines by MTT assay, respectively. Data, mean ± SD from triplicate experiments. *, P < 0.05; **, P < 0.001. TX, Taxol; CP, Carboplatin.
- Figure 4.
The combination therapy of C-CPE and Taxol attenuates EOC xenograft growth in vivo. Female SCID mice were inoculated s.c. with 5 × 106 SKOV-3 cells. Four weeks later, the mice harboring large tumor burden were divided randomly into 3 groups and administered i.p. with Taxol alone (20 mg/kg), Taxol combined with C-CPE (0.1 mg/kg), or vehicle (PBS) twice a week for 4 weeks. A, growth curves of tumors were presented as the mean volume normalized to the start volume. *, the combination of C-CPE and Taxol led to a significant tumor suppression compared with vehicle or Taxol alone (P < 0.05). Data, mean ± SD from the mice in each group. B, immunostaining of Ki67 and TUNEL to evaluate cell proliferation and apoptosis in EOC xenografts. After 4 weeks of treatment, the combination therapy markedly attenuates tumor cell proliferation and accelerates apoptosis relative to the use of single-agent Taxol. The graph represents the percentage of apoptotic or Ki-67 positive nuclei per high field from pooled tumors from 5 mice per group. *, P < 0.05; **, P < 0.001. Scale bar, 50 μm.
Tables
- Table 1.
Representative list of upregulated and downregulated genes in C-CPE–treated SKOV-3 cells versus control cells
Gene symble Name Functions Fold change NDUFB9 NADH dehydrogenase (ubiquinone) 1 beta subcomplex Metabolism 8 TNFRSF21 Tumor necrosis factor receptor superfamily, member 21 Tumor suppressor 8 MMP23B Matrix metalloproteinase 23B Metastasis/adhesion 7 RAB27B RAB27B, member RAS oncogene family Oncogene 7 QPCTL Glutaminyl-peptide cyclotransferase-like Metabolism 7 UBQLN1 Ubiquilin 1 Ubiquitin pathway 6 UBE2J1 Ubiquitin-conjugating enzyme E2, J1 Ubiquitin pathway 6 UBE4A Ubiquitination factor E4A Ubiquitin pathway 6 RAB18 member RAS oncogene family Oncogene 6 UCHL1 Ubiquitin carboxyl-terminal esterase L1 Ubiquitin pathway 5 SFN Stratifin Metastasis/growth inhibition 4 CCNB1 Cyclin B1 Cell cycle 4 PGM1 Phosphoglucomutase 1 Metabolism −2 MMP2 Matrix metalloproteinase 2 Metastasis/adhesion −3 SYK Spleen tyrosine kinase Metabolism −4 RAP2A RAP2A, member of RAS oncogene family Oncogene −4 PGM1 Phosphoglucomutase 1 Metabolism −4 MKNK2 MAP kinase interacting serine/threonine kinase 2 Metabolism −5 CD47 CD47 antigen Metastasis/adhesion −5 SULT1C1 Sulfotransferase family, cytosolic, 1C, member 1 Metabolism −5 ACHE Acetylcholinesterase Metabolism −5 RIT1 Ras-like without CAAX 1 Oncogene −6 WISP2 Wnt1 inducible signaling pathway protein 2 Wnt signaling Pathway −11
Supplementary Data
Supplementary Figures S1-S3.
Files in this Data Supplement:
- Supplementary Data - Supplementary Figures S1-S3.
Volume 17, Issue 5
