TAK1 inhibitor 5Z-7-oxozeaenol sensitizes cervical cancer to doxorubicin-induced apoptosis

Abstract

Aberrant activation of nuclear factor-κB (NF-κB) allows cancer cells to escape chemotherapy-induced cell death and acts as one of the major mechanisms of acquired chemoresistance in cervical cancer. TAK1, a crucial mediator that upregulates NF-κB activation in response to cellular genotoxic stress, is required for tumor cell viability and survival. Herein, we examined whether TAK1 inhibition is a potential therapeutic strategy for treating cervical cancer. We found that TAK1 inhibitor 5Z-7-oxozeaenol significantly augmented the cytotoxic effects of Dox in a panel of cervical cancer cell lines. Treatment with 5Z-7-oxozeaenol hindered Dox-induced NF-κB activation and promoted Dox-induced apoptosis in cervical cancer cells. Moreover, 5Z-7-oxozeaenol showed similar effects in both positive and negative human papillomavirus-infected cervical cancer cells. Taken together, our results provide evidence that TAK1 inhibition significantly sensitizes cervical cancer cells to chemotherapy-induced cell death and supports the use of TAK1 inhibitor with current chemotherapies in the clinic for patients with refractory cervical cancer.

Keywords: 5Z-7-oxozeaenol; TAK1 inhibitor; cervical cancer; chemotherapy; doxorubicin.

Figure 1. 5Z-7-oxozeaenol suppressescell proliferation of cervical cancer cells

(A) Five cervical cancer cell lines were seeded into 96-well plates at a concentration of 1 × 10⁴ perwell and treated with indicated concentrations of 5Z-7 or DMSO. Cell viability was assessed by MTT assays after 72 h of exposure to drug. Data was represented as the mean standard deviation (SD) with P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***) (ANOVA) as indicated. IC50 values of 5Z-7 in cervical cancer cell lines were listed. (B) Morphological changes of five different cervical cancer cell lines treated with increasing concentrations (0 μM, 2 μM, 5 μM, 10 μM) of 5Z-7 were shown.

Figure 2. 5Z-7-oxozeaenol enhances Dox-induced growth inhibitory effect on cervical cancer cells

(A) HeLa, C-33-A, Ca Ski, ME-180 and SiHa were treated with Dox at the indicated concentrations with or without 2 μM 5Z-7 for 48 h. The cell viability was then measured by CCK-8 assay. All values are expressed as the mean standard deviation (SD). P values < 0.05 (*), < 0.01 (**), or < 0.001 (***) were indicated. (B) Five cervical cancer cell lines wereseeded in 12-well plates at 2 × 10³ per well, and then incubated with Dox at the indicated concentrations with or without 2 μM 5Z-7 for 72 h. The cell colonies were fixed, stained and photographed.

Figure 3. 5Z-7-oxozeaenol inhibits anchorage-independent growth on two cervical cancer cells

(A–B) Soft agar assays of HeLa (A) and C-33-A (B) cells treating with increasing concentrations (0 μM, 0.01 μM, 0.05 μM, 0.1 μM) of Dox and with or without 0.5 μM 5Z-7 were conducted. Cell colonies were stained and photographed after 14 days. (C–D) The colony numbers of HeLa (C) and C-33-A (D) cells were calculated. All values are expressed as the mean standard deviation (SD). P values < 0.05 (*), < 0.01 (**), or < 0.001 (***) were indicated.

Figure 4. 5Z-7-oxozeaenol promotes Dox-induced apoptosis

(A–E) HeLa (A) and C-33-A (B), Ca Ski (C), ME-180 (D), SiHa (E) cells were treated with Dox at the indicated time points (0, 6, 12 h) with or without 5Z-7 and protein extracts were subjected to SDS-PAGE and immunoblotted for anti-PARP, and anti-Caspase 3 antibodies. β-Actin was detected as a loading control for all whole cell extracts.

Figure 5. 5Z-7-oxozeaenol inhibits Dox-induced NF-κB, JNK and p38 activation

(A–B) HeLa (A) and C-33-A (B) cells were treated with Dox at the indicated time points (0, 2, 4, 6 h) with or without 5Z-7 and protein extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. β-Actin was detected as a loading control for all whole cell extracts.