Piotr Stasiak

Elacridar Reverses P-gp-Mediated Drug Resistance in Ovarian Cancer Cells in 2D and 3D Culture Models

Multidrug resistance (MDR) remains a major obstacle in the treatment of ovarian cancer. MDR is often mediated by the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). In this study, we evaluated the ability of elacridar, a dual P-gp and BCRP inhibitor, to overcome MDR in W1, an ovarian cancer cell line sensitive to Paclitaxel (PAC) and its PAC-resistant variants. Cells were cultured under both two-dimensional (2D) and three-dimensional (3D) conditions to account for differences in tumor-like microenvironments. The MDR1 gene and P-gp protein expression were determined for the analyzed model; P-gp activity was measured by flow-cytometry and fluorescent observation, with and without elacridar. The MTT tests were carried out to evaluate how elacridar, combined with chemotherapeutics, affects cell viability. Our results demonstrate that elacridar effectively inhibited transporter activity and increased cellular sensitivity to PAC and DOX. The inhibitory effect was observed in both 2D and 3D cultures, although the re-sensitization effect in 3D conditions was less pronounced, reflecting the complexity of tumor-specific resistance mechanisms. These findings highlight elacridar as a promising compound for reversing MDR in ovarian cancer and emphasize the importance of 3D models in preclinical drug evaluation. Further studies in advanced in vitro and in vivo models are required to assess the potential of elacridar better.

The Role of Elacridar, a P-gp Inhibitor, in the Re-Sensitization of PAC-Resistant Ovarian Cancer Cell Lines to Cytotoxic Drugs in 2D and 3D Cell Culture Models

Chemotherapy resistance is a significant barrier to effective cancer treatment. A key mechanism of resistance at the single-cell level is the overexpression of drug transporters in the ABC family, particularly P-glycoprotein (P-gp), which leads to multidrug resistance (MDR). Inhibitors of these transporters can help re-sensitize cancer cells to chemotherapeutics. This study evaluated elacridar (GG918 and GF120918), a potent third-generation P-gp inhibitor, for its ability to reverse MDR in paclitaxel (PAC)-resistant ovarian cancer cell lines. Sensitive and PAC-resistant cells were cultured in two-dimensional (2D) and three-dimensional (3D) models. MDR1 gene expression was analyzed using Q-PCR, and P-gp protein expression was examined via Western blot and immunofluorescence. Drug sensitivity was evaluated with MTT assays, and P-gp activity was analyzed by flow cytometry and fluorescence microscopy. Elacridar effectively inhibited P-gp activity and increased sensitivity to PAC and doxorubicin (DOX) in 2D cultures but not cisplatin (CIS). In 3D spheroids, P-gp activity inhibition was observed via Calcein-AM staining. However, no re-sensitization to PAC occurred and limited improvement was observed for DOX. These findings suggest that elacridar effectively inhibits P-gp in both 2D and 3D conditions. However, its ability to overcome drug resistance in 3D models is limited, highlighting the complexity of tissue-specific resistance mechanisms.

Elacridar Inhibits BCRP Protein Activity in 2D and 3D Cell Culture Models of Ovarian Cancer and Re-Sensitizes Cells to Cytotoxic Drugs

Chemotherapy resistance is a major obstacle in the treatment of ovarian cancer, often resulting in disease recurrence and poor prognosis for patients. A key contributor to this resistance is the overexpression of ATP-binding cassette (ABC) transporters, including breast cancer resistance protein (BCRP/ABCG2), which actively effluxes chemotherapeutic agents such as topotecan (TOP) or mitoxantrone (MIT), limiting their intracellular accumulation and efficacy. This study investigated the potential of elacridar (GG918), a potent dual P-gp and BCRP inhibitor, to overcome drug resistance in ovarian cancer cell lines. Both TOP-sensitive and TOP-resistant ovarian cancer cells were grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroid models to better mimic the tumor microenvironment. The expression of the ABCG2 gene was quantified via qPCR and BCRP protein levels were assessed by western blotting and immunofluorescence. Drug response was evaluated using MTT viability assays, while BCRP transporter activity was examined using flow cytometry and microscopic assessment of the intracellular retention of BCRP fluorescent substrates (Hoechst 33342 and MIT). In both 2D and 3D cultures, elacridar effectively inhibited BCRP function and significantly enhanced sensitivity to TOP. These findings suggest that elacridar can inhibit BCRP-mediated drug resistance in ovarian cancer cell models.

cAMP-Dependent Signaling and Ovarian Cancer

cAMP-dependent pathway is one of the most significant signaling cascades in healthy and neoplastic ovarian cells. Working through its major effector proteins—PKA and EPAC—it regulates gene expression and many cellular functions. PKA promotes the phosphorylation of cAMP response element-binding protein (CREB) which mediates gene transcription, cell migration, mitochondrial homeostasis, cell proliferation, and death. EPAC, on the other hand, is involved in cell adhesion, binding, differentiation, and interaction between cell junctions. Ovarian cancer growth and metabolism largely depend on changes in the signal processing of the cAMP-PKA-CREB axis, often associated with neoplastic transformation, metastasis, proliferation, and inhibition of apoptosis. In addition, the intracellular level of cAMP also determines the course of other pathways including AKT, ERK, MAPK, and mTOR, that are hypo- or hyperactivated among patients with ovarian neoplasm. With this review, we summarize the current findings on cAMP signaling in the ovary and its association with carcinogenesis, multiplication, metastasis, and survival of cancer cells. Additionally, we indicate that targeting particular stages of cAMP-dependent processes might provide promising therapeutic opportunities for the effective management of patients with ovarian cancer.

The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Doxorubicin and Topotecan Resistant Ovarian Cancer Cell Lines

Epithelial ovarian cancer has the highest mortality among all gynecological malignancies. The main reasons for high mortality are late diagnosis and development of resistance to chemotherapy. Resistance to chemotherapeutic drugs can result from altered expression of drug-resistance genes regulated by miRNA. The main goal of our study was to detect differences in miRNA expression levels in two doxorubicin (DOX)- and two topotecan (TOP)-resistant variants of the A2780 drug-sensitive ovarian cancer cell line by miRNA microarray. The next aim was to recognize miRNAs as factors responsible for the regulation of drug-resistance genes. We observed altered expression of 28 miRNA that may be related to drug resistance. The upregulation of miR-125b-5p and miR-935 and downregulation of miR-218-5p was observed in both DOX-resistant cell lines. In both TOP-resistant cell lines, we noted the overexpression of miR-99a-5p, miR-100-5p, miR-125b-5p, and miR-125b-2-3p and decreased expression of miR-551b-3p, miR-551b-5p, and miR-383-5p. Analysis of the targets suggested that expression of important drug-resistant genes such as the collagen type I alpha 2 chain (COL1A2), protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase—EPHA7, Roundabout Guidance Receptor 2 (ROBO2), myristoylated alanine-rich C-kinase substrate (MARCK), and the ATP-binding cassette subfamily G member 2 (ABCG2) can be regulated by miRNA.

Doxorubicin and topotecan resistance in ovarian cancer: Gene expression and microenvironment analysis in 2D and 3D models

This study explores the mechanisms underlying chemotherapy resistance in ovarian cancer (OC) using doxorubicin (DOX) and topotecan (TOP)-resistant cell lines derived from the drug-sensitive A2780 ovarian cancer cell line. Both two-dimensional (2D) monolayer cell cultures and three-dimensional (3D) spheroid models were employed to examine the differential drug responses in these environments. The results revealed that 3D spheroids demonstrated significantly higher resistance to DOX and TOP than 2D cultures, suggesting a closer mimicry of in vivo tumour conditions. Molecular analyses identified overexpression of essential drug resistance-related genes, including MDR1 and BCRP, and extracellular matrix (ECM) components, such as MYOT and SPP1, which were more pronounced in resistant cell lines. MDR1 and BCRP overexpression contribute to chemotherapy resistance in OC by expelling drugs like DOX and TOP. Targeting these transporters with inhibitors or gene silencing could improve drug efficacy, making them key therapeutic targets to enhance treatment outcomes for drug-resistant OC. The study further showed that EMT-associated markers, including VIM, SNAIL1, and SNAIL2, were upregulated in the 3D spheroids, reflecting a more mesenchymal phenotype. These findings suggest that factors beyond gene expression, such as spheroid architecture, cell-cell interactions, and drug penetration, contribute to the enhanced resistance observed in 3D cultures. These results highlight the importance of 3D cell culture models for a more accurate representation of tumour drug resistance mechanisms in ovarian cancer, providing valuable insights for therapeutic development.