Abstract
The aim of the research was to evaluate the influence of two P-glycoprotein (P-gp) inhibitors silymarin and quercetin on anticancer drug doxorubicin (DOX) and pegylated liposomal doxorubicin (PLD) delivery into breast cancer cells (2D cultures) and cancer cell spheroids (3D cultures) at different pH. The cytotoxicity of the compounds was assessed using MTT assay. Spheroids were generated using magnetic 3D Bioprinting method. The uptake of DOX and PLD into monolayer-cultured cells and spheroids was assessed by fluorescence microscopy. Both tested flavonoids did not increase DOX and PLD levels into monolayer-cultured 4T1 cells and 4T1 cell spheroids. However, both silymarin and quercetin enhanced DOX and PLD uptake into JC cell cultures. Silymarin and quercetin may modulate DOX and PLD transport into monolayer-cultured cells and three-dimensional cancer cell cultures depending on P-gp activity.
Keywords: Drug delivery, P-glycoprotein, multidrug resistance, silymarin, quercetin, cell spheroids
Introduction
P-glycoprotein (P-gp), or multidrug resistance protein 1 (MDR1), is an ATP-dependent multidrug transporter that actively effluxes its substrates including many chemotherapeutic drugs out of the cell [1]. Therefore, it plays a very important role in the development of multidrug resistance. It is known that P-gp is overexpressed in many types of tumors [2,3]. It was also observed that the expression of P-gp may increase after the exposure to chemotherapeutic agents [4]. Many anticancer drugs, such as anthracyclines (doxorubicin, daunorubicin) [5,6], podophyllotoxins (etoposide, teniposide) [7,8], taxanes (paclitaxel, doctetaxel) [9,10], vinca alcaloids (vinblastine, vincristine) [11,12] are the substrates of this efflux pump. Therefore, P-gp is one of the major obstacles to the efficacy of chemotherapy. It is hypothesized that the inhibition of this transporter may prevent P-gp mediated drug efflux in tumor cells and reverse multidrug resistance [2,13].
The idea to increase the chemosensitivity of tumor cells to anticancer agents has been extensively investigated for over three decades and many different P-gp inhibitors were developed. According to their specificity and toxicity P-gp inhibitors can be classified in four generations [14]. First generation P-gp inhibitors, e.g. verapamil, are pharmacologically active therapeutic agents that additionally inhibit P-gp. However, their clinical application was limited due to relatively low binding affinity and high toxicity at the required doses [15]. Second generation P-gp inhibitors, such as doxverapamil, were developed after structural modifications of first-generation inhibitors. These compounds exert lower toxicity compared to first generation inhibitors. However, they showed to be potent CYP 3A4 inhibitors, thus causing many pharmacokinetic interactions with other compounds [16]. Third generation P-gp inhibitors, such as tariquidar and zosuiqudar, do not interact with CYP 3A4 and exert high binding affinity to P-gp [17].
Unfortunately, these compounds did not show successful results in clinical trials and natural substances started to be investigated. Fourth generation P-gp inhibitors are plant-based compounds, that include flavonoids, coumarins, alkaloids, saponins, and terpenoids. Quercetin and silymarin are flavonoids that belong to the fourth generation P-gp inhibitors. These compounds are derived from fruits, vegetables, some of them are used as food supplements for a long time, therefore it is believed that they should be safer and better tolerated than first, second, or third generation inhibitors [18]. In our work, we selected two fourth-generation P-gp inhibitors - silymarin which is composed of several different flavonolignans and flavonoids (Figure 1) and quercetin (Figure 2), and evaluated their efficacy in increasing DOX and PLD levels in 4T1 and JC breast cancer cell cultures.
Figure 1.
Chemical structures of silymarin compounds.
Figure 2.
Chemical structure of quercetin.
Materials and methods
Materials
DOX hydrochloride and quercetin were bought from Abcam (Cambridge, UK). PLD was bought from FormuMax Scientific Inc. (Palo Alto, CA, USA). Silymarin was purchased from Sigma-Aldrich Co. (St Louis, MO, USA).
Cell cultures
4T1 triple-negative murine breast cancer cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). JC triple-negative murine breast cancer cell line was kindly provided by Prof. Chiara Riganti (University of Torino, Italy). Cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) GlutaMAX medium supplemented with 10% fetal bovine serum (FBS), 10,000 U/mL penicillin, and 10 mg/mL streptomycin. RPMI 1640, FBS, and antibiotic solution were purchased from Gibco (Carlsbad, CA, USA). Cells were cultured in a humidified atmosphere that contained 5% CO2 at 37°C.
Drug delivery in monolayer-cultured cells
The cells were seeded on collagen-coated coverslips in 24-well plates in a volume of 500 μL (50,000 cells/well) and placed in the incubator for 48 h. After the incubation, the medium was changed by a new medium of pH 6.0 or 7.4 and maintained in the incubator for 1 h. Later the medium was removed and the solutions of the same pH that contained 100 µM quercetin or silymarin or 0.2% DMSO were poured into the wells and incubated for 2 h. After that, the medium was replaced with the solutions of the same pH that contained 1 or 5 µM of DOX or PLD and placed in the incubator for 0.5, 1, 2 and 4 h. After that, the wells were rinsed with PBS, fixed with 4% paraformaldehyde (Thermo Scientific, Waltham, MA, USA) solution, and stained using 4’,6-diamidino-2-phenylindole (DAPI; Thermo Scientific). The photos were taken using fluorescence microscope Olympus FLUOVIEW FV1000 (Olympus Corp., Japan). Fluorescence intensity of DOX into cells and cell nuclei was calculated using ImageJ software (National Institutes of Health).
Drug delivery in cell spheroids
The spheroids were made using magnetic 3D Bioprinting method as described by Tseng et al. [19]. The spheroids were incubated with the medium of pH 6.0 or 7.4 for 1 h. After that, the medium was replaced with the solutions of the same pH that contained 100 µM quercetin or silymarin or 0.2% DMSO and incubated for 2 h. After that, the medium was replaced with 20 µM DOX or PLD solutions of the same pH. After 1, 2, 4 and 8 h of incubation the spheroids were rinsed with PBS and fixed with paraformaldehyde. The photos were taken using fluorescence microscope Olympus IX73. DOX and PLD delivery into spheroids was estimated using ImageJ software. Fluorescence intensity was assessed by tracing a radius from the center of the spheroid to the edge each degree around the spheroid. For analytical purposes, the spheroids were partitioned into three zones: edge (0-50 µM), middle (100-150 µM), and central zone (200-225 µM).
Statistical analysis
Statistical analysis was performed using Microsoft Office Excel 2016 (Microsoft Corporation, Redmond, WA, USA). Each experiment was repeated at least thrice independently. The results were shown as mean ± standard deviation. The analysis was done using Student’s t-test. A P-value less than 0.05 was considered as a significant difference.
Results
Effect of P-gp inhibitors on cell viability
Silymarin EC50 for both 4T1 and JC cell lines after 8 h were >250 μM. Quercetin EC50 for 4T1 and JC cells after 8 h were 40.3 μM and 161.2 μM, respectively. However, after 2 h of incubation quercetin EC50 for 4T1 was also >150 μM. Therefore, silymarin and quercetin concentrations of 100 μM were used in the following experiments. EC50 values of DOX and PLD after 8 h were >150 μM. It shows that 1, 5 and 20 µM concentrations of DOX and corresponding concentrations of PLD used in the research did not reduce cell viability during the experiments.
Effect of P-gp inhibitors on DOX and PLD delivery in cancer cells (2D cultures)
Neither silymarin, nor quercetin increased DOX delivery into 4T1 cells or their nuclei at pH 7.4 and 6.0. No significant difference in DOX fluorescence intensity between control group and cell affected with silymarin or quercetin was observed (Figure 3).
Figure 3.
The influence of P-glycoprotein inhibitors on doxorubicin (DOX) levels into 4T1 cells and their nuclei in medium of different pH. A. Fluorescence intensity of DOX in 4T1 cells at different time points at physiological pH; B. Fluorescence intensity of DOX in 4T1 cell nuclei at different time points at physiological pH; C. Fluorescence intensity of DOX in 4T1 cells at different time points at weakly acidic pH; D. Fluorescence intensity of DOX in 4T1 cell nuclei at different time points at weakly acidic pH; E. Photos of 4T1 cells after 4 h of incubation with DOX at physiological pH; F. Photos of 4T1 cells after 4 h of incubation with DOX at weakly acidic pH. Scale bar =50 μm. Abbreviations: QUE, quercetin; SIL, silymarin.
The experiments with PLD yielded similar results. Both P-gp inhibitors did not increase PLD delivery into 4T1 cells and their nuclei, as well (Figure 4).
Figure 4.
The influence of P-glycoprotein inhibitors on pegylated liposomal doxorubicin (PLD) levels in 4T1 cells and their nuclei in medium of different pH. A. Fluorescence intensity of PLD in 4T1 cells at different time points at physiological pH; B. Fluorescence intensity of PLD in 4T1 cell nucleus at different time points at physiological pH; C. Fluorescence intensity of PLD in 4T1 cells at different time points at weakly acidic pH; D. Fluorescence intensity of PLD in 4T1 cell nuclei at different time points at weakly acidic pH; E. Photos of 4T1 cells after 4 h of incubation with PLD at physiological pH; F. Photos of 4T1 cells after 4 h of incubation with PLD at weakly acidic pH. Scale bar =50 μm. Abbreviations: QUE, quercetin; SIL, silymarin.
However, different results were observed in experiments with JC cells. In this case both silymarin and quercetin increased DOX concentration in JC cells and their nuclei (Figure 5). At pH 7.4, quercetin increased DOX uptake into cell nuclei after 1, 2, and 4 h of incubation, meanwhile SIL enhanced DOX delivery after 2 and 4 h of incubation. After 4 h of incubation the fluorescence intensity of DOX in the nuclei of cells affected with silymarin or quercetin was approximately 1.3-fold higher compared to the control group. At pH 6.0 positive quercetin and silymarin effect on DOX delivery in JC cells was observed only at the end of the experiment (after 4 hours of incubation). At that point in time DOX fluorescence intensity in JC cell affected with P-gp inhibitors nuclei was 1.4 (quercetin group) and 1.5 (silymarin group) higher compared to control group.
Figure 5.
The influence of P-glycoprotein inhibitors on doxorubicin (DOX) levels in JC cells and their nuclei in medium of different pH. A. Fluorescence intensity DOX in JC cells at different time points at physiological pH; B. Fluorescence intensity of DOX in JC cell nuclei at different time points at physiological pH; C. Fluorescence intensity of DOX in JC cells at different time points at weakly acidic pH; D. Fluorescence intensity of DOX in JC cell nuclei at different time points at weakly acidic pH 6.0; E. Photos of JC cells after 4 h of incubation with DOX at physiological pH; F. Photos of JC cells after 4 h of incubation with DOX at weakly acidic pH. Scale bar =50 μm. The asterisks (*) indicate P<0.05. Abbreviations: QUE, quercetin; SIL, silymarin.
Both tested P-gp inhibitors increased PLD delivery into JC cells, as well (Figure 6). Quercetin improved PLD uptake after 2 and 4 h of incubation at weakly acidic and physiological pH. Silymarin increased PLD fluorescence intensity in JC cell nuclei only after 4 h of incubation.
Figure 6.
The influence of P-glycoprotein inhibitors on pegylated liposomal doxorubicin (PLD) delivery JC cells and their nuclei in medium of different pH. A. Fluorescence intensity of PLD in JC cells at different time points at physiological pH; B. Fluorescence intensity of PLD in JC cell nuclei at different time points at physiological pH; C. Fluorescence intensity of PLD in JC cells at different time points at weakly acidic pH; D. Fluorescence intensity of PLD in JC cell nuclei at different time points at weakly acidic pH; E. Photos of JC cells after 4 h of incubation with PLD at physiological pH; F. Photos of JC cells after incubating for 4 h with PLD in weakly acidic at pH. Scale bar =50 μm. The asterisks (*) indicate P<0.05. Abbreviations: QUE, quercetin; SIL, silymarin.
Effect of P-gp inhibitors on DOX and PLD delivery in tumor spheroids (3D cultures)
Silymarin and quercetin did not enhance DOX uptake into 4T1 spheroids at physiological and weakly acidic pH (Figure 7). No positive effect of these compounds was observed on PLD uptake in 4T1 spheroids, as well (Figure 8).
Figure 7.
The influence of P-glycoprotein inhibitors on doxorubicin (DOX) levels in 4T1 cell spheroids at physiological and weakly acidic pH. A. Fluorescence intensity of DOX in edge and middle zones of 4T1 cell spheroids incubated with quercetin at physiological and weakly acidic pH; B. Fluorescence intensity of DOX in edge and middle zones of spheroids incubated with silymarin at physiological and weakly acidic pH; C. Photos of 4T1 cell spheroids after the incubation with DOX at physiological pH; D. Photos of 4T1 cell spheroids after the incubation with DOX at weakly acidic pH. Scale bar =200 μm. Abbreviations: QUE, quercetin; SIL, silymarin.
Figure 8.
The influence of P-glycoprotein inhibitors on pegylated liposomal doxorubicin (PLD) levels in 4T1 cell spheroids at physiological and weakly acidic pH. A. Fluorescence intensity of PLD in edge and middle zoned of 4T1 cell spheroids incubated with quercetin at physiological and weakly acidic pH; B. Fluorescence intensity of PLD in spheroids incubated with silymarin at physiological and weakly acidic pH; C. Photos of spheroids after the incubation with PLD at physiological pH; D. Photos of spheroids after the incubation with PLD at weakly acidic pH. Scale bar =200 μm. Abbreviations: QUE, quercetin; SIL, silymarin.
However, during experiments with JC spheroids it was observed that after 1 and 8 h of incubation quercetin increased DOX uptake into the edge zone of spheroids at pH 7.4 (Figure 9). At pH 6.0 quercetin increased DOX uptake into the edge zone of JC spheroids after 1, 2, 4 and 8 h of incubation. Data obtained with silymarin was very similar, however, in weakly acidic medium silymarin increased DOX fluorescence intensity in the edge zone of spheroids only after 4 and 8 h of incubation.
Figure 9.
The influence of P-glycoprotein inhibitors on doxorubicin (DOX) levels into JC cell spheroids at physiological and weakly acidic pH. A. Fluorescence intensity of DOX in edge and middle zones of spheroids incubated with quercetin at physiological and weakly acidic pH; B. Fluorescence intensity of DOX in edge and middle zones of spheroids incubated with silymarin at physiological and weekly acidic pH; C. Photos of JC cell spheroids after the incubation with DOX at physiological pH 7.4; D. Photos of JC cell spheroids after the incubation with DOX at weakly acidic pH. Scale bar =200 μm. The asterisks (*) indicate P<0.05. Abbreviations: QUE, quercetin; SIL, silymarin.
Results from experiments with PLD showed that quercetin increased PLD fluorescence intensity in the edge zone of JC spheroids only at the end of the experiment - after 8 h of incubation (Figure 10). Silymarin was more effective in this case. It enhanced PLD uptake into the edge zone of spheroids after 1, 2, 4 and 8 h of incubation at both physiological and weakly acidic conditions. At pH 7.4 this P-gp inhibitor slightly increased PLD uptake also into middle zone of JC spheroids after 4 and 8 h of incubation.
Figure 10.
The influence of P-glycoprotein inhibitors on pegylated liposomal doxorubicin (PLD) levels in JC cell spheroids at physiological and weakly acidic pH. A. Fluorescence intensity of PLD in edge and middle zones of spheroids incubated with quercetin at physiological and weakly acidic pH; B. Fluorescence intensity of PLD in edge and middle zones of spheroids incubated with silymarin at physiological and weakly acidic pH; C. Photos of JC cell spheroids after the incubation with PLD at physiological pH; D. Photos of JC cell spheroids after the incubation with PLD at weakly acidic pH. Scale bar =200 μm. The asterisks (*) indicate P<0.05. Abbreviations: QUE, quercetin; SIL, silymarin.
Discussion
Our results showed that both tested P-gp inhibitors are not effective DOX and PLD transport modulators in 4T1 cell cultures. However, both quercetin and silymarin enhanced DOX and PLD uptake in monolayer-cultured JC cells and their spheroids. This difference can be explained by the different P-gp activity in 4T1 and JC cell lines. It is known that JC cells overexpress P-gp and do not express multidrug resistance protein 1 which makes this cell line a good option to study P-gp inhibitors [20,21]. Meanwhile in 4T1 cells P-gp expression is lower. Liu et al. found that the EC50 value of liposomal DOX for the JC cell line is about four times higher than the EC50 value for the 4T1 cell line [22]. This means that JC cells are about four times more resistant to DOX than 4T1.
Different results obtained from the experiments performed in cells with different P-gp activity support the hypothesis that DOX and PLD transport modulation demonstrated by tested compounds quercetin and silymarin acts through a P-gp inhibition mechanism. In 4T1 cell line that shows lower P-gp activity both quercetin and silymarin did not enhance DOX and PLD uptake into cells and their nuclei or cell spheroids. Meanwhile in JC cell line which overexpresses P-gp both flavonoids enhanced DOX and PLD uptake in monolayer-cultured cells, their nuclei and cell spheroids. It is important to note that the pH of the medium affected the uptake of DOX into cells, as well. Under acidic conditions, the uptake of DOX and PLD into cells is lower than at physiological pH. This can be explained by the ion retention mechanism, according to which in acidic environment weakly basic DOX ionizes and the positive charge limits its ability to penetrate the cell membrane.
Our findings are in line with the results published by other researchers. Desrini et al. found that cell incubation for 25 days with 750 nM quercetin increased the susceptibility of the derived DOX-resistant MCF-7 breast cancer cell line to DOX by two to three times [23]. To our knowledge, the efficacy of quercetin in the wild-type MCF-7 cell line has not been studied, so it can be hypothesized that in this case the efficacy of quercetin in improving DOX cell uptake would be significantly lower or DOX efficacy would not be improved at all, as shown by our experiments with 4T1 cell line. This is confirmed by the results published by Borska et al., which showed that quercetin increased the cytotoxicity of daunorubicin to daunorubicin-resistant pancreatic cancer EPP85-181RDB cells, but not to the daunorubicin-sensitive pancreatic cancer cell line EPP85-181P. In another study done by Li et al., it was also shown that quercetin increased the cytotoxicity of DOX in DOX-resistant breast cancer MCF-7 and MDA-MB-231 cells that overexpress P-gp [24]. Furthermore, Wang et al. found that quercetin increased DOX cytotoxicity against liver cancer SMMC7721 and QGY7701 cells [25].
There are limited data on the efficacy of silymarin as a P-gp inhibitor in modulating DOX levels in the cell. Zhang et al. showed that 100 μM silymarin increases DOX cytotoxicity to DOX-resistant MCF-7/ADR and MDA435/LCC6 breast cancer cells [26]. Silibinin, one of the most active compounds in silymarin, has also been shown to significantly increase the cytotoxicity of DOX against the DOX-resistant breast cancer cells MDA-MB-435/DOX [27]. On the other hand, a high concentration (200 µM) of silibinin was used in this study and the authors themselves showed that such a concentration of silibinin is toxic to cancer cells. Therefore, these results do not necessarily imply that silibinin increased DOX concentration in the cells; cell viability may have decreased because of the P-gp inhibitor silibinin.
There are very little published data on studies with P-gp inhibitors in cancer cell spheroids. Hassan et al. found that quercetin at a concentration of 50 μM increases DOX cytotoxicity against pancreatic cancer ASPC-1 and liver cancer HepG2 monolayer cells characterized by high P-gp activity and their spheroids by up to 60% [28]. Barros et al. found that resveratrol, which also exert a P-gp inhibitory effect, increases DOX cytotoxicity in PANC-1 pancreatic cancer cell spheroids [29]. Although the data published by these researchers and our results obtained with JC cells support the hypothesis that silymarin and quercetin may be effective chemosensitizers, they did not demonstrate this activity in 4T1 cells. In view of this, it can be stated that the efficacy and potential applicability of the tested flavonoids is conditional and depends on the activity of P-gp in the cell line.
Conclusions
Quercetin and silymarin do not improve DOX and PLD uptake into 4T1 breast cancer cells with low P-gp expression and their spheroids but increase DOX and PLD levels in JC breast cancer cells that overexpress P-gp and their spheroids. In monolayer-cultured cells, both inhibitors show similar results on enhancing DOX and PLD levels. However, silymarin is slightly more effective in increasing DOX and PLD uptake in JC cell spheroids.
Acknowledgements
The research was supported by the Science Foundation of Lithuanian University of Health Sciences project “Application of P-glycoprotein inhibitors on improving doxorubicin and its pegylated formulation delivery in 2D and 3D cell cultures”, 2020.
Disclosure of conflict of interest
None.
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