Ovarian cancer rates the highest mortality among all gynecological malignancies. The main reason for high mortality is the development of drug resistance. It can be related to changes in the expression of many drug resistance genes as well as expression of extracellular matrix proteins and cell density in the tumor. We developed a simple two-dimensional and three-dimensional model of drug sensitive A2780 and resistant to cisplatin and paclitaxel variants of ovarian cancer cell line. Using MTT assay, we compared drug resistance in two-dimensional and three-dimensional cell culture conditions. Real-time polymerase chain reaction analysis was used to compare the expression of drug resistance genes. The expression of proteins in spheroids was determined by immunohistochemistry. We observed a moderate increase in cisplatin resistance and a significant increase in paclitaxel resistance between two-dimensional and three-dimensional cell culture conditions. Our findings show that changes in the expression of drug resistance genes may play a crucial role in the drug resistance of cancer cells in traditional cell culture. On the other hand, the drug resistance in spheroids may result from different mechanisms such as cell density in the spheroid, extracellular matrix proteins expression and drug capacity to diffuse into the spheroid.

Ovarian cancer is the most common cause of gynecological cancer death. Cancer Stem Cells (CSCs) characterized by drug transporters and extracellular matrix (ECM) molecules expression are responsible for drug resistance development. The goal of our study was to examine the role of aldehyde dehydrogenase 1A1 (ALDH1A1) expression in paclitaxel (PAC) and topotecan (TOP) resistant ovarian cancer cell lines. In both cell lines, we knocked out the ALDH1A1 gene using the CRISPR/Cas9 technique. Additionally, we derived an ALDH1A1 positive TOP-resistant cell line with ALDH1A1 expression in all cells via clonal selection. The effect of ALDH1A1 gene knockout or clonal selection on the expression of ALDH1A1, drug transporters (P-gp and BCRP), and ECM (COL3A1) was determined by Q-PCR, Western blot and immunofluorescence. Using MTT assay, we compared drug resistance in two-dimensional (2D) and three-dimensional (3D) cell culture conditions. We did not observe any effect of ALDH1A1 gene knockout on MDR1/P-gp expression and drug resistance in the PAC-resistant cell line. The knockout of ALDH1A1 in the TOP-resistant cell line resulted in a moderate decrease of BCRP and COL3A1 expression and weakened TOP resistance. The clonal selection of ALDH1A1 cells resulted in very strong downregulation of BCPR and COL3A1 expression and overexpression of MDR1/P-gp. This finally resulted in decreased resistance to TOP but increased resistance to PAC. All spheroids were more resistant than cells growing as monolayers, but the resistance mechanism differs. The spheroids’ resistance may result from the presence of cell zones with different proliferation paces, the density of the spheroid, ECM expression, and drug capacity to diffuse into the spheroid.

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.

Resistance to chemotherapy is the main obstacle in contemporary ovarian cancer treatment. The aim of this study was the evaluation of expression of HERC5 , IFIH1 , SAMD4 , MCTP1 and SEMA3A mRNA and assessment their role in resistance to cisplatin, paclitaxel, doxorubicin and topotecan in seven ovarian cancer cell lines. MTT assay was used in resistance assessment. Quantitative polymerase chain reaction was performed to measure the expression levels of the genes. We observed different levels of resistance among cell lines. The resistance was not related to the expression of drug transporters genes. The expression of HERC5 and IFIH1 genes was upregulated, and the expression of SEMA3A gene was downregulated. Expression of SAMD4 was upregulated in PEA1, PEA2, and PEO23 cell lines, and expression of MCTP1 was downregulated in A2780, PEA2, and PEO23 cell lines. Upregulation of HERC5 , IFIH1 , and SAMD4 and downregulation of SEMA3A and MCTP1 in TOP-resistant ovarian cancer cell lines may suggest some role of those genes in topotecan resistance development.