Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Mar 9;45(3):2296-2308.
doi: 10.3390/cimb45030148.

The Insulin Receptor Substrate 2 Mediates the Action of Insulin on HeLa Cell Migration via the PI3K/Akt Signaling Pathway

Anabel Martínez Báez  1 , Ivone Castro Romero  2 , Lilia Chihu Amparan  1 , Jose Ramos Castañeda  3 , Guadalupe Ayala  1
Affiliations

The Insulin Receptor Substrate 2 Mediates the Action of Insulin on HeLa Cell Migration via the PI3K/Akt Signaling Pathway

Anabel Martínez Báez et al. Curr Issues Mol Biol. .

Abstract

Insulin signaling plays an important role in the development and progression of cancer since it is involved in proliferation and migration processes. It has been shown that the A isoform of the insulin receptor (IR-A) is often overexpressed, and its stimulation induces changes in the expression of the insulin receptor substrates (IRS-1 and IRS-2), which are expressed differently in the different types of cancer. We study the participation of the insulin substrates IRS-1 and IRS-2 in the insulin signaling pathway in response to insulin and their involvement in the proliferation and migration of the cervical cancer cell line. Our results showed that under basal conditions, the IR-A isoform was predominantly expressed. Stimulation of HeLa cells with 50 nM insulin led to the phosphorylation of IR-A, showing a statistically significant increase at 30 min (p ≤ 0.05). Stimulation of HeLa cells with insulin induces PI3K and AKT phosphorylation through the activation of IRS2, but not IRS1. While PI3K reached the highest level at 30 min after treatment (p ≤ 0.05), AKT had the highest levels from 15 min (p ≤ 0.05) and remained constant for 6 h. ERK1 and ERK2 expression was also observed, but only ERK2 was phosphorylated in a time-dependent manner, reaching a maximum peak 5 min after insulin stimulation. Although no effect on cell proliferation was observed, insulin stimulation of HeLa cells markedly promoted cell migration.

Keywords: IRS1; IRS2; PI3K/Akt; cell migration; cervical cancer; insulin receptor.

PubMed Disclaimer

Conflict of interest statement

The authors state that they have no conflict of interest to declare.

Figures

Figure 1
Figure 1
Insulin receptor (IR) isoforms are differentially expressed in HeLa cells. The mRNA levels of IR isoforms and IRS1/2 genes were analyzed in HeLa and MCF7 cell lines. Total RNA was purified, and mRNA levels were analyzed by RT-PCR with specific primers. (A) The 600 and 636 bp fragments correspond to isoforms A and B of the insulin receptor, respectively. Amplified fragments of 763 and 116 bp correspond to IRS-1 and IRS-2, respectively. The amplified fragment of 451bp corresponds to GAPDH (control). (B) Densitometric analysis of IRA, IRB, IRS1, IRS2, and GAPDH mRNA levels. The graph represents the mean ± SEM of three independent experiments (n = 3). ** p < 0.01 compared to IRA.
Figure 2
Figure 2
Effect of insulin on the proliferation of HeLa cells with MTS assay. HeLa cells were treated with different insulin doses at different times of stimulation. The histograms represent the mean value ± standard error of the mean (SEM) of optic density values. The control group is cells without treatment. The graphs represent the mean ± SEM of three independent experiments (n = 6).
Figure 3
Figure 3
Insulin induces the phosphorylation of IR and IRS2 but not IRS1 in HeLa cells. Protein extracts from HeLa cells stimulated with 50 nM insulin were used to evaluate IR, IRS1, phospho-IRS1, IRS2, and phospho IRS2 by WB. (A) Densitometric analysis of phospho-IR and actin protein levels. The first bar is the control group without treatment. One-way ANOVA was performed, followed by the Tukey post hoc test to compare the treated groups against the control group (100%). * p < 0.05. All experiments have been performed in three independent experiments in triplicate, and experimental data were expressed as mean ± standard deviation (SD). (B) Cells were lysed with RIPA. IRS2 protein was immunoprecipitated, and immunoblot analyses were performed to identify the indicated proteins from IP or cell lysates. (C) HeLa cells were stimulated in the absence (control) or presence of 50 nM insulin for the indicated time. MCF7 and MDA-MB-231 cells were used as positive and negative controls, respectively. IRS1 and phospho-IRS1 protein levels were detected by immunoblot analysis. ϐ-Actin was used as a control for protein degradation.
Figure 4
Figure 4
Cervical cancer cells express higher phosphorylation levels of PI3K/Akt1 than ERK1/2 in response to insulin treatment. (A) PI3K, phospho PI3K. (B) Akt1, phospho Akt1. (C) ERK1/2 and phospho ERK1/2 protein levels were analyzed by WB in whole-cell lysates from HeLa cells stimulated with 50 nM insulin. Data are representative of 3 independent experiments. Graphics show densitometric analysis of total levels and phosphorylated protein. The first bar is the control group without treatment. All experiments were performed in three independent experiments in triplicate, and experimental data were expressed as mean ± standard deviation (SD). * p < 0.05 and ** p < 0.01 compared to control (taken as 100%).
Figure 5
Figure 5
Insulin treatment increases migration of HeLa cells. HeLa cells were stimulated with different concentrations of insulin in the presence of mitomycin C and with DMEM medium without SFB; cells were treated with mitomycin C in the control group. Cell migration was evaluated by quantifying the reduction in the open area (a lower percentage of the open area means a higher percentage of cell migration). The histograms represent the mean value ± standard error of the mean (SEM) of the percentage of the open area of three independent experiments in triplicate. One-way ANOVA was performed, followed by the Tukey post hoc test to compare the treated groups against the control group. * p < 0.05; ** p < 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Najjar S. Insulin Action: Molecular Basis of Diabetes. In: Najjar S., editor. eLS. John Wiley & Sons; Hoboken, NJ, USA: 2003. pp. 1–10.
    1. Vella V., Milluzzo A., Scalisi N.M., Vigneri P., Sciacca L. Insulin Receptor Isoforms in Cancer. Int. J. Mol. Sci. 2018;19:3615. doi: 10.3390/ijms19113615. - DOI - PMC - PubMed
    1. Gorgisen G., Gulacar I.M., Ozes O.N. The role of insulin receptor substrate (IRS) proteins in oncogenic transformation. Cell. Mol. Biol. 2017;63:1–5. doi: 10.14715/cmb/2017.63.1.1. - DOI - PubMed
    1. Ma Z., Gibson S.L., Byrne M.A., Zhang J., White M.F., Shaw L.M. Suppression of insulin receptor substrate 1 (IRS-1) promotes mammary tumor metastasis. Mol. Cell. Biol. 2006;26:9338–9351. doi: 10.1128/MCB.01032-06. - DOI - PMC - PubMed
    1. Dearth R.K., Cui X., Kim H.J., Kuiatse I., Lawrence N.A., Zhang X., Divisova J., Britton O.L., Mohsin S., Allred D.C., et al. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2. Mol. Cell. Biol. 2006;26:9302–9314. doi: 10.1128/MCB.00260-06. - DOI - PMC - PubMed

LinkOut - more resources