Microarray-based detection and expression analysis of new genes associated with drug resistance in ovarian cancer cell lines Microarray-based detection and expression analysis of new genes associated with drug resistance in ovarian cancer cell lines

Multiple drug resistance (MDR) of cancer cells is the main reason of intrinsic or acquired insensitivity to chemotherapy in many cancers. In this study we used ovarian cancer model of acquired drug resistance to study development of MDR. We have developed eight drug resistant cell lines from A2780 ovarian cancer cell line: two cell lines resistant to each drug commonly used in ovarian cancer chemotherapy: cisplatin (CIS), paclitaxel (PAC), doxorubicin (DOX) and topotecan (TOP). A chemosensitivity assay - MTT was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and immunofluorescence were also performed to determine mRNA and protein expression of genes/proteins involved in drug resistance (P-gp, BCRP, MRP1, MRP2, MVP). Flow cytometry was used to determine the activity of drug transporters. We could observe cross-resistance between PAC- and DOX-resistant cell lines. Additionally, both PAC-resistant cell lines were cross-resistant to TOP and both TOP-resistant cell lines were cross-resistant to DOX. We observed two different mechanisms of resistance to TOP related to P-gp and BCRP expression and activity. P-gp and BCRP were also involved in DOX resistance. Expression of MRP2 was increased in CIS-resistant cell lines and increased MVP expression was observed in CIS-, PAC- and TOP-, but not in DOX-resistant cell lines. Effectiveness of TOP and DOX in second line of chemotherapy in ovarian cancer can be limited because of their cross-resistance to PAC. Moreover, cross-resistance of PAC-resistant cell line to CIS suggests that such interaction between those drugs might also be probable in clinic.

A major contributor leading to treatment failure of ovarian cancer patients is the drug resistance of cancer cell. CSCs- (cancer stem cells) and ECM (extracellular matrix)-related models of drug resistance are described as independently occurring in cancer cells. Lysyl oxidase (LOX) is another extracellular protein involved in collagen cross-linking and remodeling of extracellular matrix and has been correlated with tumor progression. The expression of LOX, COL1A2, COL3A1, and ALDH1A1 was performed in sensitive (A2780, W1) and resistant to paclitaxel (PAC) (A2780PR1 and W1PR2) and topotecan (TOP) (W1TR) cell lines at the mRNA (real-time PCR analysis) and protein level (Western blot and immunofluorescence analysis). The ALDH1A1 activity was measured with the ALDEFLUOR test and flow cytometry analysis. The protein expression in ovarian cancer tissues was determined by immunohistochemistry. We observed an increased expression of LOX and collagens in PAC and TOP resistant cell lines. Subpopulations of ALDH1A1 positive and negative cells were also noted for examined cell lines. Additionally, the coexpression of LOX with ALDH1A1 and COL1A2 with ALDH1A1 was observed. The expression of LOX, collagens, and ALDH1A1 was also detected in ovarian cancer lesions. In our study LOX, ALDH1A1 and collagens were found to be coordinately expressed by cells resistant to PAC (LOX, ALDH1A1, and COL1A2) or to TOP (LOX and ALDH1A1). This represents the study where molecules related with CSCs (ALDH1A1) and ECM (LOX, collagens) models of drug resistance are described as occurring simultaneously in ovarian cancer cells treated with PAC and TOP.

Background: Low effectiveness of chemotherapy in ovarian cancer results from development of drug resistance. Topotecan is a drug used as second-line chemotherapy for this cancer type. We analyzed development of topotecan resistance in ovarian cancer cell lines. Materials and Methods: A chemosensitivity assay, MTT test, was performed to assess drug resistance. Quantitative polymerase chain reaction (Q-PCR) assays were performed to determine ABCB1, ABCG2, ALDH1A1, IFIH1, SAMD4 and EPHA3 gene expression. Results: We observed dose-dependent responses to topotecan. In all topotecan-resistant cell lines an overexpression of ABCG2, IFIH1 and SAMD4 genes was observed. Expression of ABCB1 gene was observed in one cell line. Expression of ALDH1A1 was up-regulated in A2780 and down-regulated in SKOV-3-resistant cell lines. Short-time exposure led to similar patterns of gene expression for the investigated genes. Conclusion: Expression of ABCG2 and ABCB1 genes plays the most important role in topotecan resistance. The role of other investigated genes seems to be complementary.

Uterine artery Doppler is becoming a routine part of pregnancy surveillance in high-risk pregnancies. Which blood flow velocity waveform index to measure is debated and the 'notch' in early diastole is not widely accepted, as it is a subjective measure. The aim of the present study was to evaluate the different indices in the prediction of adverse outcome of pregnancies suspected for intrauterine fetal growth restriction (IUGR). Uterine artery blood flow was recorded in 217 pregnancies admitted for Doppler ultrasound surveillance due to suspected IUGR. The median gestational age at examination was 38 weeks (range 25-42 weeks). Only cases having bilateral uterine artery notching were included in the evaluation. The uterine artery Doppler spectrum was analyzed for different indices, including evaluation of notch and end-diastolic velocities. Umbilical artery Doppler velocimetry was also performed. The outcome variables chosen were: a small-for-gestational-age (SGA) newborn, preterm birth, and abdominal delivery. ROC-curve calculations were used to compare the different indices. The uterine artery blood velocity pulsatility index (PI) and resistance indices (RI) were the best predictors of adverse outcome of pregnancy. Apart from premature birth, the systolic/end-diastolic ratio was less predictive of adverse outcome. The indices including only diastolic blood velocities were the least predictive of adverse outcome. The group with notch velocity above end-diastolic velocity was compared with those having notch velocity below the end-diastolic velocity. No difference in outcome was seen between the two groups. RI and PI as measures of third trimester utero-placental vascular impedance are the best predictors of adverse outcome of IUGR-suspected pregnancies.

Because of the deep involvement of granulosa cells in the processes surrounding the cycles of menstruation and reproduction, there is a great need for a deeper understanding of the ways in which they function during the various stages of those cycles. One of the main ways in which the granulosa cells influence the numerous sex associated processes is hormonal interaction. Expression of steroid sex hormones influences a range of both primary and secondary sexual characteristics, as well as regulate the processes of oogenesis, folliculogenesis, ovulation, and pregnancy. Understanding of the exact molecular mechanisms underlying those processes could not only provide us with deep insight into the regulation of the reproductive cycle, but also create new clinical advantages in detection and treatment of various diseases associated with sex hormone abnormalities. We have used the microarray approach validated by RT-qPCR, to analyze the patterns of gene expression in primary cultures of human granulosa cells at days 1, 7, 15, and 30 of said cultures. We have especially focused on genes belonging to ontology groups associated with steroid biosynthesis and metabolism, namely “Regulation of steroid biosynthesis process” and “Regulation of steroid metabolic process”. Eleven genes have been chosen, as they exhibited major change under a culture condition. Out of those, ten genes, namely STAR, SCAP, POR, SREBF1, GFI1, SEC14L2, STARD4, INSIG1, DHCR7, and IL1B, belong to both groups. Patterns of expression of those genes were analyzed, along with brief description of their functions. That analysis helped us achieve a better understanding of the exact molecular processes underlying steroid biosynthesis and metabolism in human granulosa cells.