Table 1.
Correlation of FRα and RFC immunoreactivities with different diagnostic categories and clinicopathological parameters in ovarian cancer.
Figure 1.
Overexpression of FRα in ovarian cancer.
(A) qPCR analysis of FRα mRNA in ovarian tumors and the corresponding non-tumor counterparts. (B) mRNA (upper panel) and protein (lower panel) expression of FRα in two immortalized ovarian epithelial cell lines, HOSE 6-3 and HOSE-17-1, and nine ovarian cancer cell lines, OVCAR-3, SKOV-3, OVCA 420, OVCA433, OC316, Dov13, ES-2, TOV21G, SW626, as assessed by qPCR and immunoblotting respectively. (C) Immunoreactivity of FRα in serous (a) and mucinous (e) benign ovarian cystadenomas, serous (b) and mucinous (f) borderline ovarian tumors and serous (c), mucinous (g), clear cell (d) and endometrioid (h) ovarian carcinomas. Scale bar = 100 µm. (D) Kaplan-Meier overall (left panel) and disease-free (right panel) survival curves for ovarian cancer patients with high and low levels of FRα (cut off at mean).
Table 2.
Correlation between clinicopathological parameters and mRNA expression of FRα and RFC in ovarian cancers.
Figure 2.
Down-regulation of RFC in ovarian cancers and correlation with prognosis of patients.
(A) qPCR analysis of RFC mRNA in ovarian tumors and the corresponding non-tumor counterparts. (B) mRNA (upper panel) and protein (lower panel) expression of RFC in two immortalized ovarian epithelial cell lines, HOSE 6-3 and HOSE-17-1, and nine ovarian cancer cell lines, SKOV-3, OVCAR-3, OVCA 420, OVCA433, OC316, Dov13, ES-2, TOV21G, SW626, as assessed by qPCR and immunoblotting respectively. (C) Immunoreactivity of RFCin inclusion cyst (a) and serous ovarian carcinomas (b). Scale bar = 100 µm. (D) Kaplan-Meier overall (left panel) and disease-free (right panel) survival curves for ovarian cancer patients with high and low levels of RFC (cut off at mean). (E) Kaplan-Meier overall (left panel) and disease-free (right panel) survival curves for high FRα expressed ovarian cancer patients with high and low levels of RFC (cut off at mean).
Table 3.
Correlation of FRα amplification and RFC promoter methylation with their mRNA expression in ovarian cancers.
Figure 3.
Promoter hypermethylation of RFC gene in ovarian cancers.
(A) The relative mRNA expression of RFC in SKOV-3 and OVCA 420 after 5-aza-dc treatment with indicated concentrations for 72 hours. Each experiment was performed in triplicate. Bars, means of fold change ±SD. *, P<0.05. (B) Representative ovarian cancers (CA) (upper panel) and ovarian cell lines (lower panel) of MSP on RFC methylation status. M, DNA marker; P, positive control; N, negative control; M, methylated alleles; U, unmethylated alleles.
Figure 4.
Folate induced SKOV-3 cell proliferation, migration and invasion and down-regulated E-cadherin through FRα.
(A) Stable knockdown of FRα mRNA and protein in SKOV-3 as detected by qPCR (left panel) and immunoblotting (right panel) respectively. **, P<0.005. (B) Cell proliferation rate of control and shFRα SKOV-3 cells treated with 6, 12 and 60 nM folate at 2, 4, 6 and 8 days displayed as fold change relative to control without folate treatment (0 nM). n = 3; *, P<0.05. (C) In vitro migration (left panel) and invasion assays (right panel) in control and shFRα SKOV-3 cells treated with 0, 12 and 60 nM folate using Transwell membrane without or with Matrigel coating respectively. Upper panels: representative images of migrating or invading SKOV-3 cells. Lower panels: Cell migration or invasion from SKOV-3 presented as percentage of control treated with 0 nM folate; n = 3; *, P<0.05; **, P<0.005. (D) Immunoblotting on FRα and E-cadherin using protein lysates prepared from control and shFRα SKOV-3 (left panel). Relative E-cadherin protein level as analyzed by ImageJ software (US National Institutes of Health); n = 3; *, P<0.05; **, P<0.005 (right panel).
Figure 5.
Ectopic overexpression of RFC in FRα-positive SKOV-3 counteracted folate-mediated cell proliferation, migration and invasion, and down-regulation of E-cadherin.
(A) Cell proliferation rate of SKOV-3 cells with ectopically expressed RFC or control vector (control) treated with 60 nM folate after 5 days displayed as fold change compared to control without folate treatment (0 nM); n = 3; *, P<0.05. (B) In vitro migration (left panel) and invasion assays (right panel) in SKOV-3 cells with ectopically expressed RFC or control vector treated with 0 and 60 nM folate displayed as percentage of control treated with 0 nM folate; n = 3; *, P<0.05; **, P<0.005. (C) Immunoblotting of RFC and E-cadherin using protein lysates prepared from SKOV-3 cells with ectopically expressed RFC or control vector (left panel). Relative E-cadherin protein level as analyzed by ImageJ software (US National Institutes of Health); n = 3; **, P<0.005 (right panel).