Tamoxifen resistance induction results in mitoxantrone resistance in MCF-7 breast cancer cells via upregulation of ABCG2 expression

doi:10.21203/rs.3.rs-2627530/v1

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Research Article

Tamoxifen resistance induction results in mitoxantrone resistance in MCF-7 breast cancer cells via upregulation of ABCG2 expression

https://doi.org/10.21203/rs.3.rs-2627530/v1

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published 13 Jun, 2023

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One of the important barriers in the treatment of breast cancer is the development of tamoxifen resistance. Different mechanisms underlying tamoxifen resistance were identified. In this thesis, we aimed to assess the effect of tamoxifen resistance induction on ABCG2 gene/protein expression level and function in the parent MCF-7 breast cancer cell line and its tamoxifen-resistant (MCF-7/TAMR) cell line.

The ABCG2 mRNA and protein expression were compared in MCF-7 and its tamoxifen-resistant derivative MCF-7/TAMR cells using Real-time PCR and western blot methods, respectively. For investigating the effect of tamoxifen-induced resistance on cross-resistance to other chemotherapy drugs such as mitoxantrone (as a well-known ABCG2 substrate), an MTT assay was used. Flow cytometry was applied to compare ABCG2 pump function between two cell lines using mitoxantrone accumulation assay. ABCG2 mRNA expression was also analyzed in tamoxifen-sensitive (TAM-S) (N=9) and tamoxifen-resistant (TAM-R) (N=9) breast tumor tissues.

Our results indicated that the levels of ABCG2 mRNA, protein, and activity were significantly higher in MCF-7/TAMR cells compared to tamoxifen sensitive cell line and mitoxantrone was less toxic in the resistant cell line. Data further discovered that tamoxifen-resistant cells with high ABCG2 activation had a poor response when given rescue mitoxantrone chemotherapy. ABCG2 was also upregulated in tissue samples obtained from tamoxifen-resistant ER+ breast cancer patients compared to tamoxifen-sensitive patients. Increased expression of the ABCG2 mRNA and protein is a phenomenon that occurs after the emergence of resistance to tamoxifen and can cause cross-resistance to mitoxantrone in MCF-7 breast cancer cells.

ABCG2

Breast cancer

Mitoxantrone resistance

Tamoxifen resistance

Breast cancer is the most prevalent malignancy in women, with annually over 2.0 million newly diagnosed cases and 0.6 million deaths (Sung et al., 2021). Apart from surgery, ER + breast cancer patients receive anti-hormonal therapies, including selective estrogen receptor (ER) modulators (SERMs; tamoxifen), selective ER down-regulators (SERDs; fulvestrant), and aromatase inhibitors (AI; anastrozole) (Osborne, 1998). About 75% of breast cancer patients with estrogen receptors in their tumor cells use tamoxifen (TAM) as adjuvant endocrine therapy (Cejalvo et al., 2017). However, primary and secondary tamoxifen resistance occurs in 30 to 50% of patients treated for ER + breast cancer (Lønning, 2007, Abdel-Hafiz, 2017). Mechanisms involved in tamoxifen resistance are loss/modification in ER expression, changes in co-regulatory proteins, and the alteration in the signaling mechanisms involved in cell survival, proliferation, and apoptosis (Viedma-Rodríguez et al., 2014). Another proposed mechanism is the increased efflux of tamoxifen by ATP binding cassette (ABC) transporters located in the cell membrane of the cancer cells (Sugimoto et al., 2003).

In our preliminary studies on tamoxifen-resistant cell lines (MCF-7/TAMR) developed from parental MCF-7 cells, we found an alteration in the mRNA expression of some of the well-known clinically meaningful ABC transporters. One of these modified transporters was ABCG2, which has increased expression in our tamoxifen-resistant (TAMR) breast cancer cell model. This increased level of ABCG2 expression is further confirmed by our findings that ABCG2 expression was upregulated in tissue samples from TAM-resistant breast cancer patients compared to TAM-responsive patients. These findings raised the question of whether ABCG2 overexpression is at least partly responsible for developing tamoxifen resistance in breast cancer cells. To address this question, we compared the tamoxifen sensitivity of ABCG2 overexpressing cell line (~ 100 fold), MCF-7/MX, with parental MCF-7 and MCF-7/TAMR cells to see whether the higher expression level of ABCG2 is associated with tamoxifen resistance or not.

On the other hand, when patients develop resistance to endocrine therapy, chemotherapy might be an alternative treatment (Cardoso et al., 2018, Goetz et al., 2019). However, these endocrine therapy-resistant patients may also show a poor response to different chemotherapy drugs; a phenomenon called multidrug resistance (MDR). One of the main mechanisms of MDR is the increased efflux of anticancer agents due to the overexpression of ABC transporters. The upregulation of the ABCG2 gene has been found in different human resistance solid tumor tissues such as ovary, colon, breast, and gastric cancers (Robey et al., 2009, Hu et al., 2017, Wang et al., 2017, Chung et al., 2019). ABCG2 upregulation in the breast cancer cell models was also associated with resistance to chemotherapy drugs such as mitoxantrone (Ross et al., 1999) and anthracyclines (Coley, 2008) used in hormone-insensitive or advanced breast cancer treatments. Considering these findings, as a second hypothesis, we also investigated the possible role of ABCG2 overexpression as an outcome of tamoxifen resistance in developing cross-resistance to mitoxantrone as a well-known ABCG2 substrate. For this purpose, the sensitivity of the tamoxifen-resistant breast cancer cell lines, MCF-7/TAMR, toward mitoxantrone was compared to both MCF-7 and MCF-7/MX (resistant to mitoxantrone) cells.

Cell culture and treatments

Human MCF-7 breast carcinoma cell lines, and the tamoxifen-resistant
cell line (MCF-7/TAMR) (generously provided by Dr. Norbert Nass, Otto-von-Guericke University, Magdeburg, Germany.) were cultured in phenol red-free-RPMI 1640 medium ( Bio Idea, Iran, Cat. No: BI-1008-01) with glutamate containing 10% charcoal-stripped steroid-depleted FBS and, 100U/ml penicillin/100μg/ml streptomycin in the presence of 5% CO2 at 37°C. MCF-7/TAMR cells were developed from parental MCF-7 cells by continuous exposure to 10 nM tamoxifen (Sigma-Aldrich, St. Louis, MO, USA, H7904) for six months. ABCG2-overexpressing MCF-7/MX cells (generously provided by Dr. Erasmus Schneider, Wadsworth Center, New York State Department of Health, USA) were also cultured in RPMI 1640 medium with 10% FBS (Biochrom AG, Berlin, Germany) in the presence of 5% CO2 at 37°C. 0.1 μg/ml MX (NanoAlvand, Tehran, Iran) was also added to the MCF-7/MX cell line to preserve the multidrug-resistant (MDR) phenotype.

MTT cell viability assay

Cells (3.0×10³ cells/well) were cultivated in 96-well plates, and two days after seeding; different treatments were applied for 72 hs. Following the completion of each treatment, 10 μL of a 5 mg/ml MTT solution was poured into each well, and they were incubated for 3 h. Then 100 µl Dimethyl sulfoxide (DMSO) was effused to solubilize the crystals of formazan, and the absorbance was detected at 570 nm and 630 nm (Background absorbance) on the ELISA plate reader (Biotek®). At least three replicates were assessed for each concentration, and the viability was calculated as the percentage of control. The results were reported as means ± SD of three experiments independently.

Tamoxifen treatment: To validate the resistance of MCF-7/TAMR cells to tamoxifen, MCF-7 and MCF-7/TAMR were exposed to the medium containing increasing concentrations of 4OH-TAM (1 – 2*10⁴ nM) for 72 hs, and then MTT assay was performed.

To explore the potential role of ABCG2 in tamoxifen resistance phenotype, the sensitivity of the ABCG2 overexpressing MCF-7/MX cells was also compared with MCF-7 and MCF-7/TAMR cells toward TAM at 500 nM (the concentration in which the MCF-7/TAMR and their parental MCF-7 cells showed the maximum difference in the viability) for 72 hs using MTT method.

Mitoxantrone treatment: To evaluate the potential role of TAM resistance in developing cross-resistance to MX via ABCG2 upregulation, the sensitivity of MCF-7, MCF-7/TAMR, and MCF-7/MX cell lines to mitoxantrone (1, 5, 10 and 50 μM) was analyzed by MTT assay following a 72 h-treatment time.

Microscopic examination of the cell morphologies

MCF-7 and MCF-7/TAMR cells were seeded at a density of 3 ×10⁵ cells per well of 6-well plates. Following overnight incubation, the cells were treated with tamoxifen (500 nM) for 72 h and microscopic images were captured by a CCD camera connected to an inverted phase-contrast microscope (INVERSO-TC100, Medline Scientific Limited, United Kingdom) for visualization of the appearance changes of the cells following the treatments.

Tissue Samples

All data for the process of sample preparation was explained in our earlier study (Jahangiri et al., 2018). Briefly, Iran National Tumor Bank reviewed surgical pathology histories of patients with breast cancer who experienced breast surgery and lymph node separation from 2005 to 2014. All breast cancer patients with assessed clinicopathological information were chosen. Then, ER+ breast cancer patients who have been prescribed tamoxifen in their last line of therapy were chosen. Likewise, ER-breast cancer patients were excluded. Patients who used neo-adjuvant or extra hormone therapies before the principal management were not in this study. Selected breast cancer patients received tamoxifen for 6 months-5 years or more. Eighteen patients for this study were selected. Afterward, these patients stayed in equally two groups: tamoxifen-resistant (TAMR) and tamoxifen-sensitive (TAMS). In the TAMR ones, patients presented recurrence signs after 6 months or less through TAM treatment. Symptoms included relapse of breast cancer tissue and metastasis to other tissue organs including lung, bone, liver, or death. Breast cancer patients without recurrence symptoms were included in the TAMS group. Tissues were kept at -80 °C for further molecular experiments.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RT-qPCR method was used to analyze the relative ABCG2 mRNA expression in MCF-7, MCF-7/TAMR, and MCF-7/MX cells and also in patients’ tissue samples. 72 hTotal RNA was extracted from the cells and tissue samples using a RiboEx^TM Kit (GeneAll ®, Seoul, South Korea) according to the company’s instructions. To assess the concentration and purity of the isolated RNAs, the NanoDrop 2000C device (Thermo Scientific, USA) was utilized. The absorbance ratio at 260/230 and 260/280 of each extracted RNA was about 1.8-2.2. First-stranded cDNA synthesis (reverse transcription) was carried out using oligo-dT primers on 5 µg of extracted RNA using a cDNA Synthesis Kit (Yekta Tajhiz Azma, Iran). Real-time PCR was performed using the ABI Prism® 7700 sequence detector system by YTA SYBR Green qPCR Master Mix (yekta Tajhiz Azma, Iran). The sequences of primer pairs were reported in Table 1. Dissociation curve analysis confirmed the amplification of the particular products for each primer set. Standard curves of reference (β-actin) and target (ABCG2) genes were set. It was previously revealed that β-actin is a convenient and stable control endogenous gene in breast cells and particularly is invariant for TAMR and TAMS breast tissue samples (Guo et al., 2013). Relative quantification was carried out using the 2^-ΔΔCt method as the target/reference gene ratio of the samples divided by the target/reference gene ratio of the control samples using the VS2.3 StepOne ^TM Software. The thermal condition used for amplification was an initial denaturation at 95°C for 4 min, 40 amplification cycles at 95°C for 5 secs, 60°C for 30 secs, and then 72°C for 30 sec.

Table 1- Lists of primers in RT-qPCR analysis (Mosaffa et al., 2009)

Gene

Sequences

ABCG2

Forward: 5'- TATCAATGGGATCATGAAACCTGG-3'

Reverse: 5'-GCGGTGCTCCATTTATCAGAAC-3'

β-actin

Forward: 5'-TCATGAAGTGTGACGTGGACATC-3'

Reverse: 5'-CAGGAGGAGCAATGATCTTGATCT-3'

Flow-cytometry analysis of mitoxantrone accumulation as an indicator of ABCG2 activity

Mitoxantrone accumulation was assessed in MCF-7, MCF-7/TAMR, and MCF-7/MX cells using the flow cytometry method (Valinezhad Sani et al., 2021). Cells were washed with phosphate-buffered saline (PBS) after trypsinization. To allow mitoxantrone uptake as a well-known substrate of the ABCG2 pump, cells (~0.5×10⁶ cells/ml) were incubated in phenol red-free-RPMI 1640 with 10% charcoal-stripped FBS (complete medium) with 3µM MX for 30 min at 37°C. Following accumulation of MX within the cells, the medium was deleted, and after washing with cold PBS, the cells were, re-suspended in a complete medium without MX, and intercellular mitoxantrone fluorescence was detected using a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) via FL3 channel. Normalized geometric-mean fluorescence intensities (Δgeo-MFI) of histograms of these cells which are correlated inversely with ABCG2 activity were reported. Delta geo-MFI was calculated by subtracting the geo-MFI of the untreated cells (resulted from autofluorescence) from the geo-MFI of the MX-treated cells.

Western Blot analysis of ABCG2 protein expression

Western blot technique was utilized to evaluate the levels of ABCG2 protein expression in MCF-7, MCF-7/TAMR, and MCF-7/MX cells. The protein concentration was measured according to the Lowry protein extraction method. Equal amounts of proteins (60µg) were separated in SDS polyacrylamide gel (6%). The isolated proteins were transferred to nitrocellulose membranes (Bio-Rad, Hemel Hempstead, UK) electrophoretically. After blocking the membranes, the proteins were immunoblotted specifically with definite antibodies. Special primary antibodies against ABCG2 ) ab130244, Abcam, 1:100) and β-actin) ab8226, Abcam, 1:1000( interacted with secondary m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotechnology Co, Shanghai, 1:3000). Protein bands were then become visible by enhanced chemiluminescence (ECL) reagent (Pierce Rockford, IL, USA) and Geldoc Alliance 4.7 (UK) device.

Flow-cytometry analysis of ABCG2 protein expression level

ABCG2 protein expression in MCF-7, MCF-7/TAMR, and MCF-7/MX was analyzed utilizing the technique defined by Minderman et al. with slight modifications (Minderman et al., 2002). Cells were fixed in formaldehyde 3.7% w/w (10% v/v) for 10 min at room temperature (RT) and kept in 1ml cold 90% methanol for 10 min. To prevent cell clumping, methanol was added very slowly dropwise to the cells while they were vigorously vortexed. After that, cells were washed twice with washing buffer (PBST; PBS containing 0.01% Tween) and further blocked with indicated bovine serum albumin (BSA) 10% w/v for 1h at RT. Following blocking, cells were incubated with 50 μl of the primary monoclonal antibody Anti- ABCG2 solution (ab3380, Abcam, 1:100) on ice for 60 min. Then, cells were washed with cold washing buffer to remove the unbound primary antibody and incubated with 50μl of the fluorescein isothiocyanate (FITC)-linked anti-mouse IgG antibody (sc-2078, Santa Cruz Biotechnology Co, Shanghai, 1:100) for 20 min on ice in the dark environment. Following washing, cells were resuspended in cold PBST and kept in a dark environment until flow cytometric examination using the FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). Samples were gated on forward scatter (FC) versus side scatter (SC) to exclude clumps and cell debris. Green fluorescence from the Linked FITC was detected using the FL1 channel, with a 530/30 nm band-pass filter, and the Δgeo-MFI of these cells as a measure of relative ABCG2 protein expression was calculated by subtracting the geo-MFI of the unstained cells (resulting from autofluorescence) from the geo-MFI of the FITC stained cells was reported.

Statistics

Results were reported as mean ± SD of at least three experiments independently. Data of cell viability experiments were investigated by two-way analysis of variance (ANOVA) and Tukey^, Post-test with Graphpad Prism 6 software. One-way ANOVA was also used to evaluate protein and gene expression in three groups comparison. TAMR and TAMS tissues and cells were compared using an independent t-test. Significance was measured in P-values < 0.05.

Tamoxifen cytotoxicity in MCF-7 and MCF-7/TAMR cancer cells

As shown in (Fig. 1), the effects of tamoxifen on the cell viability of TAM-sensitive and resistant breast cancer cells were evaluated by MTT assay. As expected, MCF-7/TAMR cells showed lower sensitivity toward the cytotoxic effects of TAM at concentrations from 50 nM to 500 nM compared to MCF-7 parental cells.

The effects of tamoxifen treatment on the morphology of the cells

Normally, MCF-7 cells grew in tightly packed cobblestone-like clusters, and MCF-7/TAMR cells were cultivated as loosely packed spindle-like cells. Following treatment with 500 nM tamoxifen, morphological changes appeared in MCF-7 cells including reduction of cell size, rounding up, nuclear fragmentation and condensation, and formation of apoptotic bodies which was indicative of cytotoxic effects of TAM through induction of apoptosis in these cells. Such morphological changes were not observed in TAM-treated MCF-7/TAMR cells (Fig. 2).

Comparison of ABCG2 mRNA, protein and activity in MCF-7, MCF-7/TAMR, and MCF-7/MX cells

Real-time RT-PCR method was achieved to assess the mRNA expression level of ABCG2 in MCF-7 cells and their resistant MCF-7/TAMR cells. RT-qPCR analysis showed that the levels of ABCG2 mRNA in MCF 7/MX and MCF-7/TAMR cells were approximately 500 and 7 times more than the ABCG2 mRNA levels in MCF-7 cells, respectively (Fig. 3).

As demonstrated in Fig. 4a and 4b, both flow cytometry and western blotting techniques confirmed higher ABCG2 protein expression levels in MCF-7/MX and MCF-7/TAMR compared to MCF-7 cells.

In flow cytometry experiments, higher Δgeo-MFI of MCF-7/MX histogram (~84000) and MCF-7/TAMR (~21000) compared to Δgeo-MFI of MCF-7 cells histogram (~17000) confirmed the higher expression of ABCG2 protein in MCF-7/MX and MCF-7/TAMR cells in comparison to MCF-7 cells. Western blotting also showed about a 5 and a 10-fold increase in the expression of ABCC2 protein level in the MCF-7/ TAMR and MCF-7/MX cells compared to MCF-7 cells.

Since that mitoxantrone has been shown to be an ABCG2 substrate, its accumulation in the cells is correlated inversely with the ABCG2-mediated efflux and activity. As shown in Fig. 5, in accordance with the RT-QPCR and western blot findings, Δgeo-MFI of accumulated MX in MCF-7/MX (~5) and MCF-7/TAMR cells (~16) were both lower than Δgeo-MFI of accumulated MX in MCF-7 cells (~20) which was indicative of higher activity of ABCG2 in MCF-7/TAMR, and MCF-7/MX cells.

Comparison of the TAM sensitivity of the breast cancer cells

To compare the effects of tamoxifen on the viability of the MCF-7/TAMR and MCF-7/MX cancer cells, cells were treated with 500 nM tamoxifen for 72 h, and then the MTT test was done. The results indicated that MCF-7 cells showed the highest sensitivity (35.64 % viability) toward the cytotoxic effects of TAM. MCF-7/MX cells, which overexpress ABCG2 protein more than about 10 folds higher than MCF-7/TAMR cells, were more sensitive to TAM (43.50 % viability) compared to MCF-7/TAMR cells (65.16 % viability) (Fig. 6).

Comparison of the mitoxantrone sensitivity of the cells

To investigate the possible role of ABCG2 overexpression as an outcome of tamoxifen resistance in developing cross-resistance to mitoxantrone as a well-known ABCG2 substrate, an MTT assay was done. Following 72 h treatment with increasing concentrations of MX (0.5, 1, 5, 10, and 50 μM), a decrease in cell viability was observed in all cell lines in a concentration-dependent manner. At 10 to 50 μM concentration of MX, mean cell viability was significantly higher in both MCF-7/MX cells and MCF-7/TAMR compared to MCF-7 cells (Fig. 7).

Comparison of mRNA Expression of ABCG2 between tamoxifen-S and tamoxifen-R Breast cancer Patients

The level of ABCG2 mRNA expression level was assessed by RT-qPCR. ABCG2 was measured in all mentioned breast tumor tissue samples. Fold-change of mRNA expression in the tamoxifen-resistant (TAMR) patients were 4.1 compared to tamoxifen-sensitive (TAMS) patients (Fig. 9). Besides, mRNA mean fold change of each individual was shown to describe a certain change in mRNA expression level in the population of the thesis.

Tamoxifen treatment has been used as endocrine therapy for ER + breast cancer in post-menopausal and pre-menopausal women for latest 40 years. There are about 30–50% of ER + breast cancer patients are not treated by endocrine therapies because of the resistance. Evolving knowledge on the molecular mechanisms of tamoxifen resistance in vitro models provides new insights into the design of more clinically efficient regimens to overcome the resistance, which is of great interest for scientific research. To study these mechanisms, breast cancer cell line models become resistant to tamoxifen experimentally in the labs, which have been used widely for decades. There are different methods to induce resistance to a drug in a cell (McDermott et al., 2014). The method of the long exposure of ER + breast cancer MCF-7 cells to the low concentrations of tamoxifen, to induce resistance in these cells has been shown efficient and reasonable in the way of developing these cell models, as clinical tamoxifen resistance is also mainly induced by long-term drug administration to the ER + breast cancer patients (Knowlden et al., 2003, Hiscox et al., 2006, Yamaguchi et al., 2017). While we were assessing MCF-7/TAMR cell lines (also developed from MCF-7 cells by using the mentioned method) for studying tamoxifen resistance, we found that ABCG2 mRNA (about 7 fold), protein expression levels (about 5 fold), and activity (1.2 fold) in MCF-7TAMR cells have increased significantly following long exposure to tamoxifen compared to parental cells. We also investigated the mRNA expression level of ABCG2 in ER + TAMS and TAMR breast cancer patients to evaluate the corroboration of our in vitro findings with the clinical data. ABCG2 was also overexpressed in TAMR patients in comparison with TAMS ones. To explore if ABCG2 is involved in tamoxifen resistance, we analyzed the tamoxifen sensitivity of another cell line resistant to the chemotherapy drug mitoxantrone through the very high expression of ABCG2 mRNA (about 500 fold), called MCF-7/MX. MTT viability assay showed although the survival rate of MCF-7/MX at exposure to 500 nM of tamoxifen was higher than MCF-7, its viability was less than MCF-7/TAMR cells in this concentration. We concluded that tamoxifen is not a substrate of ABCG2 because the MCF-7/MX cell line that enormously expresses ABCG2 was more sensitive to tamoxifen than to MCF7/TAMR cells. Teft et al. reported that endoxifen and 4-OH-tamoxifen are substrates of the ABCB1 (Pgp1) transporter. However, the relationship between genetic variation in the ABCB1 gene and tamoxifen efficiency is unknown (Teft et al., 2011).

As mitoxantrone is a well-known substrate of the ABCG2 pump, we also examined if the resistance to tamoxifen in our cell model can lead to the development of cross-resistance to this chemotherapy drug, which can be an alternative therapy for tamoxifen-resistant patients. For this purpose, MCF-7, MCF-7/TAMR, and MCF-7/MX cells were exposed to different concentrations of mitoxantrone. Viability data revealed while the MCF-7/MX was the most resistant cell line as expected, MCF-7/TAMR cells showed higher resistance toward mitoxantrone compared with parental cells, MCF-7. It seems that ABCG2 has no role in the development of tamoxifen resistance. Instead, its overexpression is an outcome of the establishment of resistance to tamoxifen and is involved in acquiring MDR phenotype and cross-resistance to mitoxantrone. In the study by Zhu et al., breast cancer cell lines resistant to tamoxifen drug were also resistant to DNA-damaging chemotherapy adriamycin and cisplatin, but not to paclitaxel (Zhu et al., 2018). Furthermore, in another study, inhibiting peptidyl arginine deiminase 2 (PAD2) not only partially returned the response of MCF-7/TAMR cells to tamoxifen but also more efficiently improved the efficacy of the chemotherapy drug docetaxel on MCF-7/TAMR cell lines both in vivo and in vivo (Li et al., 2019).

In summary, our study indicated that developing gradual resistance to tamoxifen-induced ABCG2 mRNA, protein expression level, and function in ER + breast cancer MCF-7 cells. The higher mRNA expression was also detected in breast cancer tissues obtained from the tamoxifen-resistant patients compared to the sensitive ones. Developing resistance to tamoxifen also resulted in cross-resistance to the chemotherapy drug mitoxantrone in MCF-7 breast cancer cells.

MDR Multidrug resistance

Geo-MFI Geometric-mean fluorescence intensity

MX Mitoxantrone

TAM Tamoxifen

DMSO Dimethyl sulfoxide

RT-qPCR Reverse transcription- Quantitative Real-time polymerase chain reaction

ABCG2 ATP binding cassette subfamily G member 2

ER Estrogen receptor

Ethical Approval

All techniques accomplished in this thesis were permitted by the ethics committee at Mashhad University of Medical Sciences, Mashhad, Iran, and gained ethical code IR.MUMS.MEDICAL.REC.1398.600.

Competing interests

The authors declare no competing interests.

Authors' contributions

A. D. Gh.; carried out the experiment, wrote the manuscript, and designed the figure. H. A.; performed part of the experiments. A. B.; co-wrote the paper. F. M., Kh. J.; conceived the original idea and supervised the project. All authors contributed to the final manuscript.

Funding

This research work was funded as a part of the PhD thesis of Asefeh Dahmardeh Ghalehno by the Grant (Grant No. 960594) from the Scientific Board of Mashhad University of Medical Sciences.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

We thank staff members of the Cancer Institute of Imam Khomeini hospital of Tehran for their generous collaboration. The thank Norbert Nass for advice in generating drug-resistant tumor cells.

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No competing interests reported.

graphicalabstract.tiff
Graphical Abstract Long exposure of MCF-7 cells to tamoxifen can cause resistance to treatment with tamoxifen and an increase in the expression of the ABCG2 mRNA and protein levels. Tamoxifen resistance can lead to cross-resistance to mitoxantrone.

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published 13 Jun, 2023

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Tamoxifen resistance induction results in mitoxantrone resistance in MCF-7 breast cancer cells via upregulation of ABCG2 expression

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