Overcoming multidrug resistance by activating unfolded protein response of the endoplasmic reticulum in cisplatin-resistant A2780/CisR ovarian cancer cells

Abstract

Cisplatin is a widely used anti-cancer agent. However, the effectiveness of cisplatin has been limited by the commonly developed drug resistance. This study aimed to investigate the potential effects of endoplasmic reticulum (ER) stress to overcome drug resistance using the cisplatin-resistant A2780/CisR ovarian cancer cell model. The synthetic chalcone derivative (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (named DPP23) is an ER stress inducer. We found that DPP23 triggered apoptosis in both parental cisplatinsensitive A2780 and cisplatin-resistant A2780/CisR ovarian cancer cells due to activation of reactive oxygen species (ROS)-mediated unfolded protein response (UPR) pathway in the endoplasmic reticulum. This result suggests that ROSmediated UPR activation is potential in overcoming drug resistance. DPP23 can be used as a target pharmacophore for the development of novel chemotherapeutic agents capable of overcoming drug resistance in cancer cells, particularly ovarian cancer cells. [BMB Reports 2020; 53(2): 88-93].

Fig. 1

Effect of DPP23 on the activation of caspase-7 in cisplatin-resistant ovarian cancer cells. (A) Chemical structures of cisplatin and DPP23. (B, C) A2780 and A2780/CisR cells treated with (B) 1 µg/ml cisplatin or (C) 10 µM DPP23 for different time periods. Immunoblot analysis was performed using specific antibodies against cleaved caspase-7 and poly (ADP-ribose) polymerase (PARP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The band intensities of cleaved caspase-7 and PARP were measured relative to GAPDH levels using ImageJ software. The data are presented as means ± SD (n = 3). NS, not significant; *P < 0.001; according to Sidak's multiple comparisons test.

Fig. 2

Effect of DPP23 on the activation of caspase-7 in multidrug-resistant uterine sarcoma cells. MES-SA and MES-SA/DX5 cells were treated with (A) cisplatin at different concentrations or (B) 10 µM DPP23 for various time periods. Immunoblot analysis was performed using specific antibodies against cleaved caspase-7 and poly (ADP-ribose) polymerase (PARP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The band intensities of cleaved caspase-7 and PARP were measured relative to GAPDH levels using ImageJ software. The data are presented as means ± SD (n = 3). NS, not significant; **P < 0.001; according to Sidak's multiple comparisons test.

Fig. 3

Effect of DPP23 on reactive oxygen species (ROS)-mediated apoptosis. (A) A2780 and A2780/CisR cells were incubated with 10 µM DCF-DA for 60 min, followed by 10 µM DPP23 for 3 h. DCF fluorescence was detected using an EVOSf1^Ⓡ fluorescence microscope. Scale bar, 400 mm. (B) DCF fluorescence was measured using a FACSCalibur flow cytometer. A2780 and A2780/CisR cells were treated with 10 µM DCF-DA for 60 min, followed by 10 µM DPP23 for 0, 3, 6, and 10 h. DCF fluorescence was measured using a FACSCalibur flow cytometer. (C) A2780 and A2780/CisR cells were treated with 10 µM DPP23 in the absence or presence of 2- or 4-µM N-acetyl cysteine (NAC). Whole cell lysates were prepared and immunoblot analysis was performed using specific antibodies against cleaved caspase-7 and poly (ADP-ribose) polymerase (PARP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The band intensities of cleaved caspase-7 and PARP were measured relative to GAPDH levels using ImageJ software. The data are presented as means ± SD (n = 3). ***P < 0.001; according to Sidak's multiple comparisons test. (D) A2780/CisR cells were treated with vehicle (DMSO) or 10 µM DPP23 in the absence or presence of 4 µM N-acetyl cysteine (NAC). Cells were fixed with ethanol and stained with propidium iodide (PI). PI fluorescence was measured using a NucleoCounter NC-3000. M1, sub-G1 phase; M2, G1 phase; M3, S phase; M4, G2/M phase.

Fig. 4

Effect of DPP23 on the activation of the unfolded protein response (UPR) pathway. (A) A2780 and A2780/CisR cells were treated with 10 µM DPP23 for various time periods. Whole cell lysates were prepared and immunoblot analysis was performed using antibodies against ER stress sensor proteins, GRP78 and IRE1α. GAPDH was used as an internal control. The band intensities of GRP78 and IRE1α were measured relative to GAPDH levels using ImageJ software. The data are presented as means ± SD (n = 3). NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; according to Sidak's multiple comparisons test. (B) Schematic representation of unspliced XBP-1 (uXBP-1) and spliced XBP-1 (sXBP-1) mRNA. The arrow-head indicates the PstI restriction site. Numbers denote nucleotide (nt) positions from the transcription start site. The internal intron site from nt 531 to nt 556 is indicated as a black box. (C) A2780 and A2780/CisR cells were treated with 10 µM DPP23 for various time periods. After total RNA isolation, XBP-1 mRNA was amplified from nt 277 to nt 817 by RT-PCR. Sizes of uXBP-1 (279 bp and 261 bp) and sXBP-1 (540 bp) mRNAs amplicons are indicated. GAPDH mRNA was amplified as the internal control. (D) A2780 and A2780/CisR cells were treated with 10 µM DPP23 for various time periods. Whole cell lysates were subjected to immunoblotting using antibodies against XBP-1. GAPDH was used as an internal control. The band intensities of GRP78 and IRE1α were measured relative to GAPDH levels using ImageJ software. The data are presented as means ± SD (n = 3). NS, not significant; *P < 0.05; ***P < 0.001; according to Sidak's multiple comparisons test. (E) A2780 and A2780/CisR cells were treated with 10 µM DPP23 for various time periods. Whole cell lysates were prepared and immunoblot analysis was performed using an antibody against CHOP and ATF4. GAPDH was used as an internal control. The band intensities of CHOP were measured relative to GAPDH levels using ImageJ software. The data are presented as means ± SD (n = 3). ***P < 0.001; according to Sidak's multiple comparisons test.