Paraoxonase-2 (PON2) protects oral squamous cell cancer cells against irradiation-induced apoptosis

Abstract

Purpose: Patients with oral squamous cell carcinomas (OSCC) often receive radiotherapy to preferentially induce apoptosis of cancer cells through generation of overwhelming DNA damage. This is amplified by generation of reactive oxygen species (ROS), thereby causing oxidative stress and cell death. However, tumors resist through different mechanisms, including upregulation of anti-apoptotic factors and enhanced ROS resistance. We recently reported that the antioxidative enzyme PON2 significantly enhances cellular stress resistance by attenuating mitochondrial ROS-mediated apoptosis. Further, PON2 is often upregulated in cancer. This prompted us to investigate its yet unknown role in the protection of OSCC against irradiation-induced cell death.

Methods: PON2 expression was determined after 7 Gy singular irradiation in four OSCC cell lines (PCI-13, PCI-52, SCC-4, SCC-68) accompanied by the detection of caspase 3/7 activity. A direct role of PON2 was tested by siRNA-mediated knockdown. In vivo PON2 expression was tested in five patients with oral carcinoma and compared with healthy mucosa for the evaluation of clinical significance.

Results: PON2 is variably expressed in OSCC in vitro and in vivo. Compared with the other cell lines, SCC-4 cells showed twofold more basal PON2 (p ≤ 0.05) and the lowest caspase 3/7 activity after singular irradiation (p ≤ 0.05). Contrarily, irradiation led to 1.2-fold induction of PON2 in PCI-13 with no effect on SCC-4 (≤0.05), suggesting that PON2 levels reflect the cells' irradiation sensitivity. In agreement, PON2 knockdown resulted in significant higher apoptosis rates (p ≤ 0.05).

Conclusion: Our findings give first evidence that upregulation of PON2 may protect OSCC against irradiation-induced apoptosis.

Fig. 1

Western blot analysis of OSCC cells was used in this study. PON2 protein (30 µg) was separated on a 12 % SDS–polyacrylamide gel and blotted to nitrocellulose membrane. PON2 was detected by standard procedures as described above, using primary polyclonal anti-PON2 antibody and primary monoclonal antibody against α-tubulin. Molecular weight in kiloDalton (kDa)

Fig. 2

Quantitative evaluation of basal PON2 protein expression in vitro: Relative PON2 expression levels normalized to tubulin were determined in OSCC cell lines by chemiluminescence, using Quantity-one software. SCC-4 cells presented with the highest level of basal PON2 protein. Symbols represent mean ± SEM. (n = 7); statistical significance was evaluated using one-way ANOVA (*p ≤ 0.05, **p ≤ 0.005)

Fig. 3

Quantitative analysis of basal PON2 protein expression in vivo: Relative PON2 expression normalized to tubulin was determined from tumor tissue of patients with OSCC (n = 5) by chemiluminescence, using Quantity-one software. Expression levels were determined by comparison with healthy mucosa of the appropriate patient. Similar to the in vitro results shown in Fig. 2, the in vivo analysis revealed a variable but regular PON2 expression in OSCC

Fig. 4

Induction of PON2 protein in the indicated OSCC cell lines after singular irradiation with 7 Gy: The graph shows quantitative analysis of the Western blot analysis using chemiluminescence: SCC-4 cells showed the lowest inductive effect after irradiation. Symbols represent mean ± SEM (n = 4). Significant difference was calculated using one-way ANOVA (*p ≤ 0.05)

Fig. 5

High endogenous PON2 expression level correlates with low irradiation-induced caspase-3 activation: Indicated cells were left untreated or irradiated singularly with 7 Gy. Caspase 3/7 activity was measured 24, 48 and 72 h after irradiation, as shown in a–d: activity of caspase 3/7 was significantly higher in all cell lines 48 and 72 h after irradiation, compared with the non-radiated controls. e SCC-4 cells with a twofold PON2 expression presented the lowest caspase 3/7 activity 72 h after irradiation compared with the other cell lines. a–d Symbols represent mean ± SEM. Statistical significance was calculated using two-way ANOVA (*p ≤ 0.05, **p ≤ 0.001, p ≤ 0.0001). e Statistical differences were calculated using one-way ANOVA (*p ≤ 0.05)

Fig. 6

Temporary PON2 knockdown results in significantly elevated caspase 3/7 activity: Increased cell death rates were assessed at the latest 72 h after singular irradiation, compared with the non-radiated controls and control RNAi-treated cells in all cell lines (a–d). Statistical significance was calculated using two-way ANOVA (*p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.0001). e Western blot of SCC-4 cells 72 h after PON2 knockdown. SCC-4 cells were left untreated or were treated with 50 nmol/L unspecific (control RNAi) or PON2-specific RNAi, prior to irradiation. Three days later, extracts (30 µg each) were subject to Western blotting with anti-PON2 and anti-α-tubulin antibodies. The diminishing PON2 signal 72 h after treatment proves sufficient PON2 knockdown