2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion

Abstract

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.

Keywords: 2HE2; ERK; drug resistance; ovarian cancer; platinum.

Figure 1

2-methoxyestradiol restores platinum drug sensitivity to A2780CisR co-cultured with MS-5. (A) A2780CisR cells co-cultured with MS-5 and treated with RJY13 (5 μM) and combined with 10 μM of pharmaceutical inhibitors for 24 h. Cell lysates were used to monitor levels of cleaved PARP using PARP ELISA kit [15]. Pharmaceutical inhibitors including imatinib, inhibitor of Abl kinase [34], ceritinib, inhibitor of Alk kinase [35], Bay-61-3606 inhibitor of Syk kinase [36], tyrphostin AG-1296 inhibitor of PDGFR kinase [37], PD98059 inhibitor of MEK kinase [38], SB202190 inhibitor of p38 MAPK [39], gefitinib inhibitor of EGFR kinase [40], Y-27632 ATP-competitive inhibitor of ROCK-I and ROCK-II kinases [41], GNF-5 allosteric inhibitor of Abl kinase [42], ZM39923 inhibitor of JAK1/3 kinase [43], LY 294002 broad-spectrum inhibitor of PI3K kinase [44], ibrutinib inhibitor of Btk kinase [45], 2-methoxyestradiol endogenous metabolite of 17β-estradiol [46], and axitinib inhibitor of VEGFR kinases [47]. The chemical structure of RJY13 is also shown. (B) A2780CisR cells were treated with RJY13 (5 μM) in monoculture or in co-culture with MS-5 in the presence of 10 μM of Y-27632, 2-hydroyestadiol, a prodrug of 2ME2, 2ME2, LY 294002, and BAY 87-2243 selective hypoxia-inducible factor-1 (HIF-1) inhibitor [30]. The chemical structure of 2ME2 is also shown. Levels of cleaved PARP were monitored by immunoblotting. (C) A2780CisR cells were treated with RJY13 (5 μM) in monoculture or in co-culture with MS-5 in the presence of 10 μM of Y-27632, 2-hydroyestadiol, 2ME2, LY 294002. In addition, A2780CisR cells were treated with 50 mM of CoCl₂. α-Tubulin was used as a loading control. * p < 0.01 and ** p < 0.001. The experiment was repeated twice with comparable outcomes.

Figure 1

2-methoxyestradiol restores platinum drug sensitivity to A2780CisR co-cultured with MS-5. (A) A2780CisR cells co-cultured with MS-5 and treated with RJY13 (5 μM) and combined with 10 μM of pharmaceutical inhibitors for 24 h. Cell lysates were used to monitor levels of cleaved PARP using PARP ELISA kit [15]. Pharmaceutical inhibitors including imatinib, inhibitor of Abl kinase [34], ceritinib, inhibitor of Alk kinase [35], Bay-61-3606 inhibitor of Syk kinase [36], tyrphostin AG-1296 inhibitor of PDGFR kinase [37], PD98059 inhibitor of MEK kinase [38], SB202190 inhibitor of p38 MAPK [39], gefitinib inhibitor of EGFR kinase [40], Y-27632 ATP-competitive inhibitor of ROCK-I and ROCK-II kinases [41], GNF-5 allosteric inhibitor of Abl kinase [42], ZM39923 inhibitor of JAK1/3 kinase [43], LY 294002 broad-spectrum inhibitor of PI3K kinase [44], ibrutinib inhibitor of Btk kinase [45], 2-methoxyestradiol endogenous metabolite of 17β-estradiol [46], and axitinib inhibitor of VEGFR kinases [47]. The chemical structure of RJY13 is also shown. (B) A2780CisR cells were treated with RJY13 (5 μM) in monoculture or in co-culture with MS-5 in the presence of 10 μM of Y-27632, 2-hydroyestadiol, a prodrug of 2ME2, 2ME2, LY 294002, and BAY 87-2243 selective hypoxia-inducible factor-1 (HIF-1) inhibitor [30]. The chemical structure of 2ME2 is also shown. Levels of cleaved PARP were monitored by immunoblotting. (C) A2780CisR cells were treated with RJY13 (5 μM) in monoculture or in co-culture with MS-5 in the presence of 10 μM of Y-27632, 2-hydroyestadiol, 2ME2, LY 294002. In addition, A2780CisR cells were treated with 50 mM of CoCl₂. α-Tubulin was used as a loading control. * p < 0.01 and ** p < 0.001. The experiment was repeated twice with comparable outcomes.

Figure 2

2HE2 restored platinum sensitivity to OC cells independent of phospho-ERK1/2. (A) A2780CisR cells were treated with 2HE2 (3 and 10 μM) and dasatinib (5 μM) alone or in combination with RJY13 (5 μM) and levels of cleaved PARP as well as phospho ERK1/2 were monitored by immunoblot. (B) A2780CisR cells co-cultured with MS-5 were treated with 2HE2 (3 and 10 μM) and dasatinib (5 μM) alone or in combination with RJY13 (5 μM) and levels of cleaved PARP as well as phosphoERK1/2 were monitored by immunoblot. GAPDH was used as a loading control. (C) pERK1/2 levels relative to levels of GAPDH were calculated and blotted. The experiment was repeated twice with comparable outcomes.

Figure 2

2HE2 restored platinum sensitivity to OC cells independent of phospho-ERK1/2. (A) A2780CisR cells were treated with 2HE2 (3 and 10 μM) and dasatinib (5 μM) alone or in combination with RJY13 (5 μM) and levels of cleaved PARP as well as phospho ERK1/2 were monitored by immunoblot. (B) A2780CisR cells co-cultured with MS-5 were treated with 2HE2 (3 and 10 μM) and dasatinib (5 μM) alone or in combination with RJY13 (5 μM) and levels of cleaved PARP as well as phosphoERK1/2 were monitored by immunoblot. GAPDH was used as a loading control. (C) pERK1/2 levels relative to levels of GAPDH were calculated and blotted. The experiment was repeated twice with comparable outcomes.

Figure 3

The role of ERK1/2 activity in mediating 2HE2 activity in restoring platinum sensitivity to OC cells. (A). A2780CisR cells treated with RJY13 (5 μM) grown as a monolayer or in direct co-culture with MSC-5. (B). A2780CisR cells treated with RJY13 (5 μM) grown in direct co-culture with MS-5 cells and exposed to 2HE2 (10 μM) alone or in combination with cobimetinib (2 μM) for 24 h. (C). A2780CisR cells treated with RJY13 (5 μM) grown in direct co-culture with MS-5 cells and exposed to dasatinib (5 μM) alone or in combination with cobimetinib (2 μM) for 24 h. Cell lysates in A-C were used to monitor levels of cleaved PARP using the PARP ELISA kit [15]. (D). Immunoblot of A2780CisR cells grown as a monolayer or in direct co-culture with MSC-5 and exposed to RJY13 (5 μM) combined with 2HE2 (10 μM) or fisetin (10 μM) alone or in combination with cobimetinib (2 μM). Filters were probed with anti-cPARP, pERK1/2, and GAPDH that was used as a loading control. ** p < 0.01 and *** p < 0.001.

Figure 4

Platinum intracellular concentration in OC cells. A2780CisR, A2780, and MS-5 cells were exposed to RJY13 (5 μM) for 6 h as described in materials and methods. Cells were collected, mineralized with 65% HNO3, and then completely dried. Dry platinum-containing material was measured at m/z 214 by an ICP-OES instrument (Agilent Technologies, Santa Clara, CA, USA). (A). Cells growing as monolayers were exposed to RJY13 and levels of platinum were measured. (B). A2780cisR grown as monolayers or in direct co-culture with MS-5 were exposed to RJY13 in combination with 2HE2 (10 μM), dasatinib (5 μM), or fisetin (10 μM). * p < 0.05 and ** p < 0.01.

Figure 5

Effect of 2HE2 and dasatinib on expression levels of platinum transporters. (A) A2780CisR samples grown as monolayers or in direct co-culture with MS-5 were exposed to RJY13 (5 μM) for 24 h in the presence of 2HE2 (10 μM) and dasatinib (5 μM) as indicated. Total RNA was isolated and RT-PCR was performed as described in materials and methods. In this experiment, primers for hABCG2, hABCC1, hABCB1, hCTR2, hATP7B, hGAPDH, and mGAPDH were used as described in materials and methods. PCR products were separated at 1% agarose. Molecular weight DNA is also shown (M) and a sample without any temple (NT) served as negative control. (B) Transporter levels relative to levels of GAPDH were calculated and blotted. PCR products using mGAPDH are shown in Supplemental Figure S2. This experiment was repeated 3 times with similar results. * p < 0.05 and ** p < 0.01.

Figure 5

Effect of 2HE2 and dasatinib on expression levels of platinum transporters. (A) A2780CisR samples grown as monolayers or in direct co-culture with MS-5 were exposed to RJY13 (5 μM) for 24 h in the presence of 2HE2 (10 μM) and dasatinib (5 μM) as indicated. Total RNA was isolated and RT-PCR was performed as described in materials and methods. In this experiment, primers for hABCG2, hABCC1, hABCB1, hCTR2, hATP7B, hGAPDH, and mGAPDH were used as described in materials and methods. PCR products were separated at 1% agarose. Molecular weight DNA is also shown (M) and a sample without any temple (NT) served as negative control. (B) Transporter levels relative to levels of GAPDH were calculated and blotted. PCR products using mGAPDH are shown in Supplemental Figure S2. This experiment was repeated 3 times with similar results. * p < 0.05 and ** p < 0.01.