Karolina Wojtowicz

The role of aldehyde dehydrogenase (ALDH) in cancer drug resistance.

Chemotherapy in cancer patients is still not satisfactory because of drug resistance. The main mechanism of drug resistance results from the ability of cancer cells to actively expel therapeutic agents via transport proteins of the ABC family. ABCB1 and ABCG2 are the two main proteins responsible for drug resistance in cancers. Recent investigations indicate that aldehyde dehydrogenase (ALDH) can also be involved in drug resistance. Expression of the ABC transporters and ALDH enzymes is observed in normal stem cells, cancer stem cells and drug resistant cancers. Current chemotherapy regimens remove the bulk of the tumour but are usually not effective against cancer stem cells (CSCs) expressing ALDH. As a result, the number of ALDH positive drug resistant CSCs increases after chemotherapy. This indicates that therapies targeting drug resistant CSCs should be developed. A number of therapies targeting CSCs are currently under investigation. These therapies include differentiation therapy using different retinoic acids (RA) as simple agents or in combination with DNA methyltransferase inhibitors (DNMTi) and/or histone deacetylase inhibitors (HDACi). Therapies that target cancer stem cell signaling pathways are also under investigation. A number of natural compounds are effective against cancer stem cells and lead to decreasing numbers of ALDH positive cells and downregulation of the ABC proteins. Combinations of differentiation therapies or therapies targeting CSC signaling pathways with classical cytostatics seem promising. This review discusses the role of ALDH and ABC proteins in the development of drug resistance in cancer and current therapies designed to target CSCs.

MDR gene expression analysis of six drug-resistant ovarian cancer cell lines.

Ovarian cancer is the leading cause of death among gynaecological malignancies. Multiple drug resistance makes cancer cells insensitive to chemotherapy. In this study, we developed six primary ovarian cancer cell lines (W1MR, W1CR, W1DR, W1VR, W1TR, and W1PR) resistant to drugs such as methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel. A chemosensitivity assay MTT test was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and Western blot were also performed to determine mRNA and protein expression of genes involved in chemoresistance. We observed high cross-resistance to doxorubicin, vincristine, and paclitaxel in the cell lines resistant to these agents. We also found a significant correlation between resistance to these drugs and increased expression of P-gp. Two different mechanisms of topotecan resistance were observed in the W1TR and W1PR cell lines. We did not observe any correlation between MRP2 transcript and protein levels. Cell lines resistant to agents used in ovarian cancer treatment remained sensitive to methotrexate. The main mechanisms of drug resistance were due to P-gp expression in the doxorubicin, vincristine, and paclitaxel resistant cell lines and BCRP expression in the topotecan resistant cell line.

Drug transporter expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line

Ovarian cancer is characterized by the higher mortality among gynecological cancers. In results of MDR development during chemotherapy cancer cells become resistant to further treatment. Microarray techniques can provide information about MDR development at gene expression level. ABC and SLC transporters are most important proteins responsible for this phenomenon. In this study changes of ABC and SLC genes expression pattern in drugs resistant sublines of the A2780 ovarian cancer cell line were demonstrated. The cytostatic resistant sublines were generated by culture of A2780 cell line with an increasing concentration of the indicated drugs. As screening methods, we used Affymetrix U219 Human Genome microarrays. Independent t-tests were used to determinate statistical significances of results. Genes that expression levels were higher than assumed threshold (upregulated above threefold and downregulated under -3 fold) were visualized using scatter plot method, selected and listed in table. Between the ABC genes increased expression of seven genes and decreased expression of three genes were observed. Expression of two genes was increased or decreased depending on the cell line. We observed significant (more than tenfold) increase in expression of four ABC genes: ABCA8, ABCB1, ABCB4 and ABCG2 and decreased expression of ABCA3 gene. We also observed changes in expression of 32 SLC genes. Between them we observe increased expression of 17 genes and decreased expression of 15 genes. Expression of four genes was increased or decreased dependent on cell line. The expression of nine SLC genes increased or decreased very significantly (more than tenfold). Five genes were significantly upregulated: SLC2A9, SLC16A3, SLC16A14, SLC38A4 and SLC39A8. Four additional genes were significantly downregulated: SLC2A14, SLC6A15, SLC8A1 and SLC27A2. Expression profiles of these genes give strong arguments for assumption of correlation between expression of ABC and SLC genes and drug resistance phenomenon. Identifying correlations between specific drug transporters and cytostatic drug resistance will require further investigation.

Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line

The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell.

Expression of MDR1 and MDR3 gene products in paclitaxel-, doxorubicin- and vincristine-resistant cell lines.

Multiple drug resistance is one of the main reasons for low chemotherapeutic efficiency in cancer patients. The proteins that are most frequently implicated to play a role in this mechanism are transmembrane proteins that are members of the ABC family. The most important ABC protein is MDR1 (ABCB1), which is expressed in over fifty percent of drug-resistant cancers. The phosphatidylcholine transporter, MDR3 (ABCB4), exhibits high homology with MDR1. An increasing body of evidence suggests that MDR3 plays a role in drug resistance. In the present study, we used doxorubicin-, paclitaxel- and vincristine-resistant cancer cell lines. A chemosensitivity assay MTT test was performed to assess drug resistance. Quantitative real-time polymerase chain reaction analyses were performed to determine the mRNA expression levels of the MDR1 and MDR3 genes. We observed dose-dependent responses to doxorubicin, paclitaxel and vincristine in the investigated cell lines. In all of the drug-resistant cell lines that we studied, we observed increased MDR1 and MDR3 transcript levels. In a doxorubicin-resistant variant of the LoVo cell line (LoVoDx), MDR3 was expressed at higher levels than MDR1. We also observed high correlations between MDR3 expression and resistance to doxorubicin and paclitaxel. Our results suggest that MDR3 plays an active and important role in drug resistance in the investigated cell lines.

Increased Expression of Several Collagen Genes is Associated with Drug Resistance in Ovarian Cancer Cell Lines

Ovarian cancer is the most lethal gynaecological cancer. The main reason for the high mortality among ovarian cancer patients is the development of drug resistance. The expression of collagen genes by cancer cells can increase drug resistance by inhibiting the penetration of the drug into the cancer tissue as well as increase apoptosis resistance. In this study, we present data that shows differential expression levels of collagen genes and proteins in cisplatin- (CIS), paclitaxel- (PAC), doxorubicin- (DOX), topotecan- (TOP), vincristine- (VIN) and methotrexate- (MTX) resistant ovarian cancer cell lines. Quantitative real-time polymerase chain reactions were performed to determine the mRNA levels. Protein expression was detected using Western blot and immunocytochemistry assays. In the drug resistant cell lines, we observed the upregulation of eight collagen genes at the mRNA level and based on these expression levels, we divided the collagen genes into the following three groups: 1. Genes with less than a 50-fold increase in expression: COL1A1, COL5A2, COL12A1 and COL17A1. 2. Genes with greater than a 50-fold increase in expression: COL1A2, COL15A1 and COL21A1. 3. Gene with a very high level of expression: COL3A1. Expression of collagen (COL) proteins from groups 2 and 3 were also confirmed using immunocytochemistry. Western blot analysis showed very high expression levels of COL3A1 protein, and immunocytochemistry analysis showed the presence of extracellular COL3A1 in the W1TR cell line. The cells mainly responsible for the extracellular COL3A1 production are aldehyde dehydrogenase-1A1 (ALDH1A1) positive cells. All correlations between the types of cytostatic drugs and the expression levels of different COL genes were studied, and our results suggest that the expression of fibrillar collagens may be involved in the TOP and PAC resistance of the ovarian cancer cells. The expression pattern of COL genes provide a preliminary view into the role of these proteins in cytostatic drug resistance of cancer cells. The exact role of these COL genes in drug resistance requires further investigation.

Inhibition of protein glycosylation reverses the MDR phenotype of cancer cell lines

BACKGROUND Multidrug resistance proteins are one of the most important factors that cause chemotherapy resistance, which in turn reduces therapeutic efficacy and survival for cancer patients. Tunicamycin is one of the most well-known inhibitors of N-glycosylation and is considered a powerful adjunct that can increase the effectiveness of many drugs. Tunicamycin blocks the first step of P-gp (glycoprotein P) and BCRP (breast cancer resistance protein) N-glycosylation, which is a very important modification for the activity and cellular localisation of these proteins. METHODS The effects of tunicamycin on ovarian and colorectal cancer cells were examined in multiple cell lines. The primary ovarian cancer cell line W1 and the established ovarian cancer cell line A2780 were compared against their drug-resistant derivatives W1TR/W1PR (TR: topotecan resistant; PR: paclitaxel resistant) and A2780T1 (topotecan resistant), respectively. We also compared the colorectal cancer cell line LoVo against its doxorubicin-resistant derivative LoVo/Dx. Cell viability was determined by the MTT assay. The glycopeptides were subjected to deglycosylation using the endoglycosidase PNGase F. A2780T1, LoVo/Dx and W1PR cells were treated with the protein degradation inhibitors MG132 and BMA. Protein expression was detected by western blot and immunocytochemistry. RESULTS In this study, we showed via the MTT assay that tunicamycin significantly decreased the viability of cancer cell lines that were co-treated with a chemotherapeutic drug. Western blot analysis showed that, in LoVo/Dx and W1PR cells, tunicamycin treatment resulted in the expression of a 70kDa P-gp protein instead of the mature 170kDa P-gp. Treatment with MG132 or BMA fully suppressed the effect of tunicamycin in the case of W1PR cells only. In tunicamycin-treated W1TR cells, the size of the BCRP protein did not differ from that of its native unglycosylated form. In tunicamycin-treated A2780T1 cells, BCRP expression was completely inhibited, but pre-treatment with MG132 or BMA suppressed the effect of tunicamycin. Immunocytochemistry analysis indicated that tunicamycin only affected the translocation of P-gp but not that of BCRP. After treatment, we observed higher P-gp expression in the cytoplasm than at the cell membrane. CONCLUSIONS Our results indicated that tunicamycin may enhance the effect of chemotherapy by interfering with the localisation and function of transporter proteins that are responsible for multidrug resistance.

Inhibition of ALDH1A1 activity decreases expression of drug transporters and reduces chemotherapy resistance in ovarian cancer cell lines.

The high mortality of ovarian cancer patients results from the failure of treatment caused by the inherent or acquired chemotherapy drug resistance. It was reported that overexpression of aldehyde dehydrogenase A1 (ALDH1A1) in cancer cells can be responsible for the development of drug resistance. To add the high expression of the drug transporter proteins the ALDHA1 is considered as a molecular target in cancer therapy. Therefore, we analysed drug-resistant ovarian cancer cell lines according to ALDHA1 expression and the association with drug resistance. The expression of ALDH1A1, P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) was determined using a microarray and confirmed by Q-PCR, western blot and fluorescence analysis. ALDH1A1 activity was determined using an Aldefluor assay. The impact of all-trans retinoic acid (ATRA) and diethylaminobenzaldehyde (DEAB) on chemotherapy resistance was assessed by the MTT chemosensitivity assay. The most abundant expression of ALDH1A1 was noted in paclitaxel- and topotecan-resistant cell lines where two populations of ALDH-positive and ALDH-negative cells could be observed. Those cell lines also revealed the overexpression of P-gp and BCRP respectively, and were able to form spheres in non-adherent conditions. Pre-treatment with ATRA and DEAB reduced chemotherapy resistance in both cell lines. ATRA treatment led to downregulation of the ALDH1A1, P-gp and BCRP proteins. DEAB treatment led to downregulation of the P-gp protein and BCRP transcript and protein. Our results indicate that ALDH1A1-positive cancer cells can be responsible for drug resistance development in ovarian cancer. Developing more specific ALDH1A1 inhibitors can increase chemotherapy effectiveness in ovarian cancer.

New and Old Genes Associated with Primary and Established Responses to Cisplatin and Topotecan Treatment in Ovarian Cancer Cell Lines

Low efficiency of chemotherapy in ovarian cancer results from the development of drug resistance. Cisplatin (CIS) and topotecan (TOP) are drugs used in chemotherapy of this cancer. We analyzed the development of CIS and TOP resistance in ovarian cancer cell lines. Incubation of drug sensitive cell lines (W1 and A2780) with cytostatic drugs was used to determine the primary response to CIS and TOP. Quantitative polymerase chain reaction (Q-PCR) was performed to measure the expression levels of the genes. We observed decreased expression of the MCTP1 gene in all resistant cell lines. We observed overexpression of the S100A3 and HERC5 genes in TOP-resistant cell lines. Increased expression of the S100A3 gene was also observed in CIS-resistant A2780 sublines. Overexpression of the C4orf18 gene was observed in CIS- and TOP-resistant A2780 sublines. A short time of exposure to CIS led to increased expression of the ABCC2 gene in the W1 and A2780 cell lines and increased expression of the C4orf18 gene in the A2780 cell line. A short time of exposure to TOP led to increased expression of the S100A3 and HERC5 genes in both sensitive cell lines, increased expression of the C4orf18 gene in the A2780 cell line and downregulation of the MCTP1 gene in the W1 cell line. Our results suggest that changes in expression of the MCTP1, S100A3 and C4orf18 genes may be related to both CIS and TOP resistance. Increased expression of the HERC5 gene seems to be important only in TOP resistance.

The significance of lumican expression in ovarian cancer drug-resistant cell lines

Purpose The aim of the present study is to determine the expression of LUM in drug-resistant ovarian cancer cell lines. Methods Doxorubicin- (DOX), topotecan- (TOP) and vincristine- (VIN) resistant variants of the W1 ovarian cancer cell line were used in this study. We used quantitative real-time polymerase chain reactions to determine LUM mRNA levels. Protein expression was detected using Western blot and immunocytochemistry assays. Protein glycosylation was investigated using PGNase F digestion. Immunohistochemistry assays were used to determine protein expression in ovarian cancer patients. Results We observed increased expression of LUM in drug-resistant cell lines at both the mRNA and the protein level. The most abundant LUM expression was observed in TOP-resistant cell line. We observed LUM bands that corresponded to different molecular masses, and the most abundant LUM form was the secreted form, which had a mass of 50 kDa. Double immunofluorescence analysis showed co-expression of LUM and COL3A1 as well as the presence of extracellular COL3A1 in the TOP-resistant cell line. Finally, we detected the LUM protein in ovarian cancer tissue. Conclusion The expression of LUM in cytostatic-resistant cell lines suggests its role in drug resistance. The co-expression of LUM and COL3A1 indicates the significance of LUM in collagen fibre assembly. Expression in ovarian cancer tissue suggests that LUM can play a role in ovarian cancer pathogenesis in ways similar to other cancers.