Monika Świerczewska

The TGFBI gene and protein expression in topotecan resistant ovarian cancer cell lines

The primary limiting factor in achieving cures for patients with cancer, particularly ovarian cancer, is drug resistance. The mechanisms of drug resistance of cancer cells during chemotherapy may include compounds of the extracellular matrix, such as the transforming growth factor-beta-induced protein (TGFBI). In this study, we aimed to analyze the TGFBI gene and protein expression in different sensitive and drug-resistant ovarian cancer cell lines, as well as test if TGFBI can be involved in the response to topotecan (TOP) at the very early stages of treatment. In this study, we conducted a detailed analysis of TGFBI expression in different ovarian cancer cell lines (A2780, A2780TR1, A2780TR2, W1, W1TR, SKOV-3, PEA1, PEA2 and PEO23). The level of TGFBI mRNA (QPCR), intracellular and extracellular protein (Western blot analysis) were assessed in this study. We observed upregulation of TGFBI mRNA in drug-resistant cell lines and estrogen-receptor positive cell lines, which was supported by overexpression of both intracellular and extracellular TGFBI protein. We also showed the TGFBI expression after a short period of treatment of sensitive ovarian cancer cell lines with TOP. The expression of TGFBI in ovarian cancer cell lines suggests its role in the development of drug resistance.

Piperine Targets Different Drug Resistance Mechanisms in Human Ovarian Cancer Cell Lines Leading to Increased Sensitivity to Cytotoxic Drugs

Our goal was to examine the anticancer effects of piperine against the resistant human ovarian cancer cells and to explore the molecular mechanisms responsible for its anticancer effects. Our study used drug-sensitive ovarian cancer cell line W1 and its sublines resistant to paclitaxel (PAC) and topotecan (TOP). We analyzed the cytotoxic effect of piperine and cytostatic drugs using an MTT assay. The impact of piperine on protein expression was determined by immunofluorescence and Western blot. We also examined its effect on cell proliferation and migration. We noticed a different level of piperine resistance between cell lines. Piperine increases the cytotoxic effect of PAC and TOP in drug-resistant cells. We observed an increase in PTPRK expression correlated with decreased pTYR level after piperine treatment and downregulation of P-gp and BCRP expression. We also noted a decrease in COL3A1 and TGFBI expression in investigated cell lines and increased COL3A1 expression in media from W1PR2 cells. The expression of Ki67 protein and cell proliferation rate decreased after piperine treatment. Piperine markedly inhibited W1TR cell migration. Piperine can be considered a potential anticancer agent that can increase chemotherapy effectiveness in cancer patients.

Effect of ALDH1A1 Gene Knockout on Drug Resistance in Paclitaxel and Topotecan Resistant Human Ovarian Cancer Cell Lines in 2D and 3D Model

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.

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.