Thioredoxin reductase-1 mediates curcumin-induced radiosensitization of squamous carcinoma cells

Abstract

Curcumin, a plant polyphenol, is a widely studied chemopreventive agent with demonstrated antitumor activities in preclinical studies and low toxicity profiles in multiple clinical trials against human malignancies. We previously showed that curcumin radiosensitizes cervical tumor cells without increasing the cytotoxic effects of radiation on normal human fibroblasts. Here we report that an inhibitory activity of curcumin on the antioxidant enzyme thioredoxin reductase-1 (TxnRd1) is required for curcumin-mediated radiosensitization of squamous carcinoma cells. Stable knockdown of TxnRd1 in both HeLa and FaDu cells nearly abolished curcumin-mediated radiosensitization. TxnRd1 knockdown cells showed decreased radiation-induced reactive oxygen species and sustained extracellular signal-regulated kinase 1/2 activation, which we previously showed was required for curcumin-mediated radiosensitization. Conversely, overexpressing catalytically active TxnRd1 in HEK293 cells, with low basal levels of TxnRd1, increased their sensitivity to curcumin alone and to the combination of curcumin and ionizing radiation. These results show the critical role of TxnRd1 in curcumin-mediated radiosensitization and suggest that TxnRd1 levels in tumors could have clinical value as a predictor of response to curcumin and radiotherapy.

Figures

Figure 1. TxnRd1 protein and activity levels in cells with different transformation status correlate with response to curcumin

MSK-Leuk1, human keratinocytes and three squamous carcinoma cell lines: HeLa (cervical), FaDu and SCC-1 (Head & Neck) were analyzed for (A) TxnRd1 protein levels by immunoblot analysis. β-actin was used as a loading control. (B) Basal levels of whole cell TxnRd activity. Activity was measured as nmoles dihydrolipoate formed per mg protein and was normalized to that in MSK-Leuk1 cells. Results represent average of 3 independent experiments (±S.E.) (C) The effect of curcumin on TxnRd activity. FaDu and HeLa cells were treated with 0, 5, 10, 20 and 50 µM curcumin or THC for 8 h and assayed for TxnRd activity. Activity was measured as nmoles dihydrolipoate formed per mg protein and normalized to own cells untreated control. (D) Keratinocytes, MSK-Leuk1, FaDu and HeLa cells were treated with DMSO, 10, 20 or 50 µM curcumin for 8 h. Whole-cell lysates were analyzed for curcumin-induced apoptosis by immunoblot assay using antibodies against cleaved PARP and β-actin.

Figure 2. Stable knockdown of TxnRd1 in HeLa and FaDu squamous carcinoma cell lines

Stable clones were generated from HeLa (left panel) and FaDu (right panel) cells by expressing a non-targeting shRNA (shNT) or an shRNA targeting human TxnRd1 (shTR1). Untransfected cells, shNT and shTR1 clones were analyzed for (A) TxnRd1 mRNA levels by real time RT-PCR analysis. TxnRd1 mRNA levels were normalized to 18s rRNA control and normalized to mRNA levels in untransfected HeLa or FaDu cells. PCR reactions were done in triplicate (±S.E.; *p<0.05). (B) TxnRd1 protein levels assayed by immunoblot analysis. β-actin was used as a loading control. (C) TxnRd enzymatic activity. Results represent the average of 3 independent experiments (±S.E; *p<0.05).

Figure 3. Squamous carcinoma cells with TxnRd1 knockdown are less sensitive to curcumin-mediated radiosensitization

HeLa (A, B) and FaDu (C, D)-shNT control and shTR1 clones were treated with DMSO or 10 µM curcumin for 8 h followed by 0, 2, 4 or 6 Gy doses of IR. Survival was assessed by clonogenic assays. Results represent the averages of 3 independent experiments (± S.E) and data were analyzed by pairwise comparisons. Treatment groups with statistically significant differences are indicated by brackets (* = p

Figure 4. Effect of TxnRd1 knockdown on ROS production and ERK1/2 activation

(A) Increase in ROS in HeLa-shNT or shTR1–3.2 clones was analyzed by flow cytometry using DCF-DA. At least 30,000 events were collected for each treatment condition. Results are representative of three independent experiments. (B) Results from (A) were quantified, represented as percentage increase in fluorescence normalized to shNT unirradiated control (±S.E.; *p<0.05). (C) ERK phosphorylation in response to curcumin treatment. shNT and shTR1-3.2 cells were serum-starved for 24 h. The cells were pretreated with DMSO or 10 µM curcumin for 8 h followed by mock irradiation or irradiation with 6 Gy. Whole cell lysates were analyzed by immunoblot using antibodies against p-ERK1/2 and total-ERK1/2. The results are representative of 3 independent experiments.

Figure 5. TxnRd1 overexpression enhances curcumin-induced toxicity and radiosensitivity in HEK293 cells

(A) Immunoblot analysis of HEK293 cells stably expressing an empty pIRES-neo vector (HEK293-pIRES) or the pIRES vector containing the full length TxnRd1 gene (HEK293-TxnRd1). β-actin was used as a loading control. (B) Basal TxnRd activity in HEK293-pIRES and HEK293-TxnRd1 cells. Results represent the average of 3 independent experiments (±S.E). (C) Cytotoxic effect of curcumin and THC on HEK293-pIRES and HEK293-TxnRd1 cells. Cells were treated with increasing concentrations of curcumin or THC for 8 h. When the untreated cells were confluent, MTT assays were performed. Results represent the averages of 4 independent experiments (±S.E). (D) Effect of curcumin on radiosensitivity of HEK293-pIRES and HEK293-TxnRd1 cells. Cells were treated with DMSO or 10 µM curcumin for 8 h followed by 0, 2, 4 or 6 Gy doses of IR. Long-term survival was assessed by high-density clonogenic survival assay. Results represent the averages of 3 independent experiments (± S.E) and data were analyzed by pairwise comparisons. Treatment groups with statistically significant differences are indicated by brackets (* = p<0.05).