Figure 1.
Ovarian cancer cell lines tend to express high levels of proinflammatory chemokines and have low p53 activity.
(A) Signature of chemokine ligands and (B) chemokine receptors in human ovarian cancer cell lines. After isolating total RNA from each cell line, PCR array was performed using a customized PCR array plate containing complementary sequences for human chemokine genes. Different colors indicate average cycle threshold with expression ranges from >35 to <25. (C) Protein expression of p53 and Mdm2 in ovarian cancer cell lines. Whole cell lysates were prepared and Western blot was carried out using antibodies specific to p53, Mdm2 and β-actin as loading control. Experiments were performed in duplicate and a representative result is shown. OV, OVCAR-3 cells; SK, SKOV-3 cells; A, A2780 cells; Ca, CaOV-3 cells; TOV, TOV-21G cells.
Figure 2.
Overexpression of p53 downregulates TNF-induced chemokines.
(A) TNF-induced chemokines in SKOV-3 cells. After isolating total RNA, PCR array was performed using a human chemokine PCR array plate. Dotted line indicates 2-fold increase; chemokines with a greater than 2-fold increase are recognized as TNF-induced chemokines. (B) Confirmation of p53 protein expression after transient transfection in SKOV-3 cells. After transfection of empty vector (EM) and p53 expression vector (p53), whole cell lysates were prepared and p53 expression was confirmed by Western blot. β-actin is used as a loading control. (C) Effect of p53 on TNF-induced chemokines. After overnight transfection of vectors, cells were treated with TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Different letters indicate significant differences (P≤0.05) within each chemokine group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE.
Figure 3.
Overexpression of p53 inhibits NF-κB activity.
(A) Nucleotide sequences of promoters for TNF-induced chemokines such as CCL20, CXCL1, 2, 3 and 8. These chemokine promoters contain one NF-κB site at the proximal region, except for CCL20, which has two NF-κB sites at the distal and proximal region. (B) Effect of p53 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p65, cells were treated with TNF (10 ng/ml) for 4 h. (C) Effect of p53 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 (p53 null), OVCAR-3 (p53 mutant) and A2780 (p53 wild-type), cells were treated with TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown; numbers below are relative density values.
Figure 4.
Restoration of p53 delays IκB degradation.
(A) Accumulated effect of p53 on ubiquitylated proteins. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to ubiquitin, p21, IκB, p53 and β-actin (as loading control). Experiments were performed in duplicate and a representative result is shown. (B) Confirmation of p53 activity after transient transfection of p53. ELISA was performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t-test) within each cell line. (C) The effect of p53 on proteasome activity. Assays were performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t-test) within each cell line. (D) Effects of p53 on ubiquitination of IκB. Immunoprecipitated IκB was immunoblotted using ubiquitin antibody. Experiments were performed in duplicate and a representative result is shown.
Table 1.
Effect of p53 on ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) obtained from comparison between empty vector and p53 vector transfected ovarian cancer cells.
Figure 5.
Overexpressed p53 induces Mdm2 whereas does not affect to the binding of NF-κB components and any IKK isoform.
(A) Effect of p53 on Mdm2 expression. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to Mdm2; and β-actin served as loading control. (B) Effects of p53 expression on p53 binding to p65 and IκB. After transient transfection of p53, immunoprecipitated (IP) p53 was immunoblotted (IB) using p65 or IκB antibody. (C) Effect of p53 on expression of various IKK isoforms. After transient transfection of p53, whole cell lysates were prepared and Western blot was carried out using antibodies specific to IKKα, IKKβ, IKKγ, IKKε; β-actin served as loading control. Experiments were performed in duplicate and a representative result is shown.
Figure 6.
Nutlin-3, a p53 stabilizer, downregulates TNF-induced chemokines.
(A) Effect of nutlin-3 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p53, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 4 h. Different letters indicate significant differences (P≤0.05) within each group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE. (B) Effect of nutlin-3 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 cells, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown. (C) Effect of nutlin-3 on TNF-induced chemokines. After overnight transfection of vectors, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Asterisk indicates significant differences (P≤0.05, paired Student's t-test) when compared to the presence of nutlin-3. Experiments were performed in triplicate and all data are shown as mean ± SE.
Figure 7.
Schematic of the proposed mechanisms by which p53 expression regulates proinflammatory chemokines in ovarian cancer.
Chronic inflammation promotes ovarian cancer progression via NF-κB signaling. Wild-type p53 reduces activity of the ubiquitin-proteasome system, resulting in low IκB degradation (blue line). This reduces NF-κB activity, inhibiting proinflammatory chemokine expression and attenuating the proinflammatory tumor microenvironment (blue arrow). On the other hand, p53 increases Mdm2 expression (dark arrow) in a feedback loop to compensate for the reduced activity of the ubiquitin-proteasome system. Loss of p53 observed frequently in advanced ovarian cancer triggers high proinflammatory chemokines by increasing NF-κB signaling which is composed of IκB and p65/p50 followed by a high IκB degradation (red arrow). Enhanced NF-κB activity results in potentiation of the proinflammatory tumor microenvironment for ovarian cancer progression such as peritoneal tumor dissemination and massive ascites. The imbalance between p53 and Mdm2 also contributes to increasing NF-κB signaling via the ubiquitin-proteasome system.