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. 2019 Sep 26:2019:7475295.
doi: 10.1155/2019/7475295. eCollection 2019.

IL-2 Induces Transient Arrest in the G1 Phase to Protect Cervical Cancer Cells from Entering Apoptosis

María Del Carmen Lagunas-Cruz  1 , Arturo Valle-Mendiola  1 , Jonathan Trejo-Huerta  1 , Leticia Rocha-Zavaleta  2 , María de Lourdes Mora-García  1 , Adriana Gutiérrez-Hoya  1   3 , Benny Weiss-Steider  1 , Isabel Soto-Cruz  1
Affiliations

IL-2 Induces Transient Arrest in the G1 Phase to Protect Cervical Cancer Cells from Entering Apoptosis

María Del Carmen Lagunas-Cruz et al. J Oncol. .

Abstract

Interleukin 2 (IL-2) has been used for the treatment of different types of cancer that express the IL-2 receptor (IL-2R). However, the effect of IL-2 on cervical cancer cells is unknown. IL-2R is present in normal cells of the immune system but not in the healthy cervix. We report that IL-2R is expressed in cervical cancer cells. IL-2 decreases cervical cancer cell proliferation via transient arrest of the G1 phase, which does not result in apoptosis or senescence. IL-2 upregulates the expression of p53 and p21 and downregulates cyclin D. In addition, we report the resistance of cervical cancer cells to treatments that induce apoptosis in HeLa and INBL cells. When arrested cells were treated with cisplatin, the cytokine protected cells from apoptosis induced by cisplatin. The effects of IL-2 on the cell cycle do not induce cellular senescence or activate the proapoptotic protein Bax. The cell arrest induced by IL-2 is conferring protection to cells against apoptosis.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Presence of the alpha, beta, and gamma subunits (IL-2R) in the cervical cancer cell lines HeLa and INBL. The expression of the IL-2R subunits was determined using specific antibodies for the alpha, beta, and gamma subunits by flow cytometry on a FACSAria II cytometer (BD, USA). The histograms corresponding to the HeLa cell line (a) and the INBL cell line (b) are shown. We defined the gate for positive events such that all negative controls (isotypes) represented 1% of events. Then, this gate was applied to all the conditions. Representative flow cytometry plots of one out of three independent experiments. (c) The graph representation of the basal expression compared to the expression after the stimulation with IL-2 is shown. The expression of the IL-2R subunits is statistically significant. ∗P < 0.05, ∗∗∗P < 0.001. Results are the median of three independent experiments.
Figure 2
Figure 2
IL-2 induces a decrease in cell proliferation. Cells were incubated in the presence or absence of 10, 50, and 100 IU/ml of IL-2 for 24 and 48 hours. (a) In the HeLa cell line, a significant decrease in cell proliferation was observed after 48 hours of incubation with 100 IU/ml of IL-2. (b) In the INBL cell line, there is a significant decrease in cell proliferation at 48 hours of incubation with 100 IU/ml of IL-2. ∗P < 0.05. Results are the median of five independent experiments.
Figure 3
Figure 3
Effect of IL-2 on cervical cancer cell apoptosis. HeLa (a) and INBL (b) cells were incubated in the presence or absence of IL-2 for 48 hours. Cells were fixed and incubated in the presence of annexin-V and evaluated by flow cytometry. No significant apoptosis was observed in either cell line. Puromycin-induced apoptosis was used as a positive control (c). Dot plots show apoptosis percentage after propidium iodide and FITC-Annexin V flow cytometry analysis on a FACSAria II cytometer (d). Representative flow cytometry plots of one out of three independent apoptosis assays.
Figure 4
Figure 4
Effect of IL-2 on the expression of Bax. HeLa and INBL cells were incubated with or without 100 IU/ml of IL-2 for 24 and 48 hours. RNA was obtained, and RT-PCR was performed. The expression ratio of Bax in HeLa cells (a) and INBL cells (b) is shown. Statistical analysis was performed with a Student's t-test of paired nonparametric data. It was performed using the Statistical Package Graphpad Prism 5.0. ∗P < 0.05.
Figure 5
Figure 5
Effect of IL-2 on cell-cycle phases. HeLa and INBL cells were incubated with and without 100 IU/ml of IL-2 for 48 hours to determine cell-cycle phases. (a, b) A significant arrest in G1 phase was observed after IL-2 treatment for 48 hours in INBL cell line. After inducing G1 arrest by treating cells with 100 IU of IL-2 for 48 hours, the medium containing IL-2 was removed, and fresh culture medium supplemented with 10% FBS was added. The cells were cultured further for 24 and 48 hrs. (c, d) HeLa and INBL cells reentry to the cell cycle. ∗P < 0.05.
Figure 6
Figure 6
IL-2 does not induce senescence in HeLa and INBL cells. HeLa and INBL cells were treated with 100 IU/ml of IL-2 for 96 hours, and beta-galactosidase activity characteristic of senescent cells was analysed (blue color) using a commercial senescence detection kit. A representative image of three independent experiments is shown.
Figure 7
Figure 7
Effect of IL-2 on the p53, p21, and cyclin D expression in cervical cancer cells. HeLa and INBL cells were incubated in the presence or absence of 100 IU/ml of IL-2 for 24 and 48 hours. RNA was obtained, RT-PCR was performed, and the products were separated by electrophoresis in an agarose gel. The results show the bands and the expression ratio for p53 (a), p21 (b), and cyclin D (c). (d) Histogram and graph representation of p53 acetylation. Statistical analysis was performed with a Student's t-test of paired, nonparametric data. It was performed using the statistical package Graphpad Prism 5.0. ∗P < 0.05.
Figure 8
Figure 8
IL-2 protects G1-arrested cervical cancer cells from entering apoptosis. Cells were incubated in the presence or absence of 100 IU/ml of IL-2 for 48 hours. Then, 5 μg/ml of cisplatin was added to the cell cultures, and the mixtures were incubated for 24 and 48 hours. Apoptosis was evaluated using the PE Annexin-V kit apoptosis detection kit. The percentage of apoptotic cells was evaluated by flow cytometry. Percentage of cell death in HeLa cells (a) and INBL cells (b) is shown. Statistical analysis was performed with a Student's t-test of paired, nonparametric data. It was performed using the statistical package Graphpad Prism 5.0. ∗P < 0.05, ∗∗P < 0.001, and ∗∗∗P < 0.0001.
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