Effects of U0126 and MK2206 on cell growth and re-growth of endometriotic stromal cells grown on substrates of varying stiffness

Abstract

Endometriosis is a common gynecological disorder responsible for infertility and pelvic pain. A complete cure for patients with endometriosis awaits new targets and strategies. Here we show that U0126 (a MEK inhibitor) and MK2206 (an AKT inhibitor) synergistically inhibit cell growth of deep endometriotic stromal cells (DES) grown on polyacrylamide gel substrates (PGS) of varying stiffness (2 or 30 kilopascal [kPa]) or plastic in vitro. No significant differences in cell proliferation were observed among DES, endometrial stromal cells of patients with endometriosis (EES) from the proliferative phase (P), EES-S (secretory phase) and EES-M (menstrual phase) compared to cells grown on a substrate of the same stiffness at both higher (U0126 [30 μM] and MK2206 [9 μM]) and lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined doses. However, cell re-growth of DES after drug discontinuation was higher than that of EES-P and EES-S when cells were grown on rigid substrates at both combined doses. Combination U0126 and MK2206 treatment is more effective than each drug alone in cell growth inhibition of DES. However, further studies are required to investigate the mechanisms underlying high cell survival and proliferation after drug discontinuation for developing target therapies that prevent recurrence.

Figure 1

Comparison of cell proliferation of deep endometriotic stromal cells DES (n = 14), endometrial stromal cells of patients with endometriosis (EES) derived from the proliferative phase (EES-P) (n = 10), EES derived from the secretory phase (EES-S) (n = 6) and EES derived from the menstrual phase (EES-M) (n = 5) grown on PGS of varying stiffness (2 (A) or 30 kPa (B)) or plastic (C) treated with combination U0126 and MK2206. Dose 1: U0126 (15 μM) and MK2206 (4.5 μM). Dose 2: U0126 (30 μM) and MK2206 (9 μM). P: EES-P. S: EES-S. M: EES-M. Numerical values are presented as box and whisker plots showing medians and the smallest and largest data points ≤1.5 × IQR from the 25th and 75th percentiles, respectively.

Figure 2

Effects of combined treatment with U0126 and MK2206 on cell proliferation in DES (A) (n = 14), EES-P (B) (n = 10), EES-S (C) (n = 6), or EES-M (D) (n = 5). Cells were grown on PGS of varying stiffness (2 or 30 kPa) or plastic. Dose 1: U0126 (15 μM) and MK2206 (4.5 μM). Dose 2: U0126 (30 μM) and MK2206 (9 μM). Numerical values are presented as box and whisker plots showing medians and the smallest and largest data points ≤1.5 × IQR from the 25th and 75th percentiles, respectively. *p < 0.05: 2-kPa PGS versus plastic. ^#p < 0.05: 30-kPa PGS versus plastic. ^¶p < 0.05: 2-kPa versus 30-kPa PGS.

Figure 3. Effects of either U0126 (30 μM) alone, MK2206 (9 μM) alone, or combination U0126 (30 μM) and MK2206 (9 μM) on Annexin V-positive cells of DES (n = 12), EES-P (n = 6), EES-S (n = 6), and EES-M (n = 5).

Cells were grown on PGS of varying stiffness (2 or 30 kPa) or plastic. P: EES-P. S: EES-S. M: EES-M. Numerical values are presented as box and whisker plots showing medians and the smallest and largest data points ≤1.5 × IQR from the 25th and 75th percentiles, respectively. *p < 0.05 versus EES-P. ^¶p < 0.05 versus EES-S. ^#p < 0.05 versus EES-M.

Figure 4

(A) Representative photomicrograph of cytochemical staining of senescence-associated beta-galactosidase (SA-βgal) activity in DES. SA-βgal activity in DES treated with either vehicle (DMSO) alone, U0126 (30 μM) alone, MK2206 (9 μM) alone, or combination U0126 (30 μM) and MK2206 (9 μM). Scale bar: 50 μm. (B,C) Effects of either U0126 (30 μM) alone, MK2206 (9 μM) alone, or combination U0126 (30 μM) and MK2206 (9 μM) on mRNA levels of cyclin D1, p53, and p21 in DES (B) and EES-P (C) from the same patients (n = 6). *p < 0.05 versus control. ^#p < 0.05 versus MK2206. Numerical values are presented as box and whisker plots showing medians and the smallest and largest data points ≤1.5 × IQR from the 25th and 75th percentiles, respectively. Levels of cyclin D1, p53, and p21 mRNAs are presented relative to the level of the reference gene, GAPDH. C: control, U: U0126, MK: MK2206, U + MK: U0126 + MK2206.

Figure 5

Comparison of cell proliferation of DES (n = 14), EES-P (n = 10), EES-S (n = 6) and EES-M (n = 5) grown on PGS of varying stiffness (2 (A) or 30 kPa (B)) or plastic (C) after a 72-h discontinuation of U0126 and MK2206. P: EES-P. S: EES-S. M: EES-M. Dose 1: U0126 (15 μM) and MK2206 (4.5 μM). Dose 2: U0126 (30 μM) and MK2206 (9 μM). *p < 0.05 versus DES. ^#p < 0.05 versus EES-M. Numerical values are presented as box and whisker plots showing medians and the smallest and largest data points ≤1.5 × IQR from the 25th and 75th percentiles, respectively.

Figure 6

Cell proliferation in DES (A) (n = 14), EES-P (B) (n = 10), EES-S (C) (n = 6), or EES-M (D) (n = 5) after a 72-h drug discontinuation of combination U0126 and MK2206. Cells were grown on PGS of varying stiffness (2- or 30-kPa PGS) or plastic. Dose 1: U0126 (15 μM) and MK2206 (4.5 μM). Dose 2: U0126 (30 μM) and MK2206 (9 μM). *p < 0.05: 2-kPa PGS versus plastic. ^#p < 0.05: 2-kPa versus 30-kPa PGS.

Figure 7

(A) Representative photomicrograph of LC3A/B expression in DES and EES-M. LC3A/B expression in DES and EES-M treated with either vehicle (DMSO) alone or MK2206 (9 μM) alone. Scale bar: 50 μm. (B) Effects of the combined treatment of U0126 and MK2206 with versus without chloroquine on cell proliferation of DES (n = 6) after a 72-h drug discontinuation. Cells were grown on PGS of varying stiffness (2 or 30 kPa) or plastic. *p < 0.05 with versus without chloroquine. Numerical values are presented as box and whisker plots showing medians and the smallest and largest data points ≤1.5 × IQR from the 25th and 75th percentiles, respectively.