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15357163mct180770-sup-205035_3_supp_5318837_p5bqk6.pdf (2.12 MB)

Supplementary materials and Supplementary Figures 1-9 from GnRH-R–Targeted Lytic Peptide Sensitizes BRCA Wild-type Ovarian Cancer to PARP Inhibition

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posted on 2023-04-03, 15:03 authored by Shaolin Ma, Sunila Pradeep, Alejandro Villar-Prados, Yunfei Wen, Emine Bayraktar, Lingegowda S. Mangala, Mark Seungwook Kim, Sherry Y. Wu, Wei Hu, Cristian Rodriguez-Aguayo, Carola Leuschner, Xiaoyan Liang, Prahlad T. Ram, Katharina Schlacher, Robert L. Coleman, Anil K. Sood

Supplementary Materials. Chemical structure and amino acid sequence of EP-100. Supplementary Figure S1. Expression and localization of GnRH-R in ovarian cancer cells. Supplementary Figure S2. The effects of GnRH-R loss on the cytotoxicity of EP-100. Supplementary Figure S3. Effects of doxorubicin, paclitaxel, olaparib, topotecan, and cisplatin on the viability of ovarian cancer cells. Supplementary Figure S4. Combined effects of EP-100 with doxorubicin, paclitaxel, topotecan and cisplatin on ovarian cancer cells. Supplementary Figure S5. Protein expressions as detected by RPPA assay. Supplementary Figure S6. The combined effect of treatment with EP-100 and olaparib in a subcutaneous mice model. Supplementary Figure S7 (related to Supplementary Figure S1 and Supplementary Figure S2). The original figures of the Western Blotting bands for GnRH-R expression. Supplementary Figure S8 (related to Figure 4A). Representative comets from comet assays. Supplementary Figure S9 (related to Figure 4C and D). Representative images from immunofluorescence assay.

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The University of MD Anderson Cancer Center

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CPRIT Research Training Program

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ARTICLE ABSTRACT

EP-100 is a synthetic lytic peptide that specifically targets the gonadotropin-releasing hormone receptor on cancer cells. To extend the utility of EP-100, we aimed to identify effective combination therapies with EP-100 for ovarian cancer and explore potential mechanisms of this combination. A series of in vitro (MTT assay, immunoblot analysis, reverse-phase protein array, comet assay, and immunofluorescence staining) and in vivo experiments were carried out to determine the biological effects of EP-100 alone and in combination with standard-of-care drugs. EP-100 decreased the viability of ovarian cancer cells and reduced tumor growth in orthotopic mouse models. Of five standard drugs tested (cisplatin, paclitaxel, doxorubicin, topotecan, and olaparib), we found that the combination of EP-100 and olaparib was synergistic in ovarian cancer cell lines. Further experiments revealed that combined treatment of EP-100 and olaparib significantly increased the number of nuclear foci of phosphorylated histone H2AX. In addition, the extent of DNA damage was significantly increased after treatment with EP-100 and olaparib in comet assay. We performed reverse-phase protein array analyses and identified that the PI3K/AKT pathway was inhibited by EP-100, which we validated with in vitro experiments. In vivo experiment using the HeyA8 mouse model demonstrated that mice treated with EP-100 and olaparib had lower tumor weights (0.06 ± 0.13 g) than those treated with a vehicle (1.19 ± 1.09 g), EP-100 alone (0.62 ± 0.78 g), or olaparib alone (0.50 ± 0.63 g). Our findings indicate that combining EP-100 with olaparib is a promising therapeutic strategy for ovarian cancer.

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