Antihormonal agents as a strategy to improve the effect of chemo-radiation in cervical cancer: in vitro and in vivo study

Abstract

Background: Over the past few years, the concurrent use of cisplatin-based chemotherapy and radiation therapy has dramatically improved the local response and increased overall survival in early-stage cervical cancer. However, for the advanced stages of the disease this standard treatment has proved insufficient. We investigated the capacity of Mifepristone and ICI 182,780, which are anti-progestin and anti-estrogen drugs, respectively, to act as chemo-radiosensitizing agents in cervical cancer cells and cervix xenografts.

Methods: The effect of chemo-radiation alone or combined with Mifepristone or ICI 182,780 was evaluated in HeLa cells and with tumor growth in cervix xenografts. After concomitant chemo-radiotherapy, the effect of each of these antihormonal agents on apoptosis (determined by Annexing V assay) and the cell cycle phases were determined by flow cytometry. The expression of angiogenic factor VEGF in tumor samples was determined using quantitative RT-PCR analysis of VEGF gene expression.

Results: Compared to radiation alone or radiation/cisplatin therapy, there was significantly higher cytotoxicity and a greater antitumoral effect with the combined application of radiation/cisplatin and Mifepristone or ICI 182,780. Analyses of the apoptosis and cell cycle demonstrated changes only with ICI, not with Mifepristone, when was applied in combination with radiation/cisplatin. The analysis of VEGF mRNA expression levels in tumors at the end of the study demonstrated a significant inhibition, compared to radiation only or the radiation/cisplatin treatment, after concurrent chemo-radiotherapy and each one of the antihormonal drugs.

Conclusion: Mifepristone and ICI 182,780 may be potentially promising chemo-radiosensitizing compounds to be used in combination with ionizing irradiation and cisplatin in the treatment of patients with advanced cervical cancer.

Figure 1

Comparison of the clonogenic cell survival of HeLa cells after individual treatments. The cells were treated with 0.75 Gy γ-irradiation, 0.33 μM cisplatin, MF and ICI (10 μM). Data are represented as the mean ± SD of three independent experiments. (*) Indicates a significant difference (p < 0.05) between the MF and IC groups vs control group.

Figure 2

Clonogenic cell survival on HeLa cells after combined treatments. A) Combinations with Mifepristone. B) Combinations with ICI. All clonogenic assays were repeated in duplicate in at least three independent experiments. Values represent the mean ± SD.

Figure 3

Analysis of the cell cycle and apoptosis in HeLa cells after treatments. Cell-cycle distribution was expressed as the percentage of surviving cells compared to the number of cells in the untreated control at 24 h (A), 48 h (B) and 72 h (C). Values represent the mean ± SD. *p < 0.05 (comparing MF or ICI combined with cisplatin/radiation treatment to the treatment with only cisplatin/radiation). Apoptosis was measured by surface AnV staining and flow cytometry, and it was considered positive for AnV+/PI- cells (D). Values represent the mean ± SD *p < 0.05 (comparing MF or ICI combined with radiation/cisplatin to the treatment with only radiation/cisplatin).

Figure 4

Efficacy of antihormonal agents on HeLa cell tumors treated with cisplatin or radiotherapy. HeLa cells were implanted s.c. in both hind limbs of nude mice. Treatment was initiated when the tumors reached 150 mm³. Mifepristone (A) or ICI (B) were administered in combination with radiation, cisplatin, and radiation/cisplatin. B) ICI was also administered in combination with radiation, cisplatin, and radiation/cisplatin. As controls, tumor growth was determined for mice treated only with the vehicle. Data are presented as the mean ± SEM of five to six animals. (*) indicates a significant difference (p < 0.01) between the triple combination groups vs control group; (#) and (†) represent a significant difference vs the dual treatments (p < 0.05) and individual treatments (p < 0.01) respectively.

Figure 5

Relative increase in tumor growth at the end of the study with respect to the initial volume. *p < 0.05 comparing initial vs final.

Figure 6

Percentage of change in body weight for mice treated with cisplatin or radiotherapy, as well as for the combinations with each of the two antihormonal agents. There was no significant difference between groups. Data are presented as the mean ± SEM of five to six animals.

Figure 7

Quantitative Real-Time PCR analysis of VEGF-A relative expression in HeLa xenografts after each treatment. Analysis was done on whole lysated tumors removed at week 10. (*) Indicates a significant difference (p < 0.05) between the triple combination groups vs control group.