Research ArticleExperimental Studies

Differences in Transport Characteristics and Cytotoxicity of Epirubicin and Doxorubicin in HepG2 and A549 Cells

KATSUHITO NAGAI, SHUHEI FUKUNO, MAHO SHIOTA, MAYUKO TAMURA, SAYAKA YABUMOTO and HIROKI KONISHI
Anticancer Research December 2021, 41 (12) 6105-6112; DOI: https://doi.org/10.21873/anticanres.15430

Abstract

Background/Aim: Epirubicin (EPI), an epimer of doxorubicin (DOX), and DOX are anthracycline agents with broad-spectrum antitumor activity. The aim of the present study was to elucidate the transport characteristics of EPI and DOX in human hepatocellular carcinoma HepG2 cells and human non-small cell lung cancer A549 cells, and to examine the relationship of intracellular drug accumulation with their cytotoxic effects. Materials and Methods: Intracellular concentrations of EPI and DOX were measured using high-performance liquid chromatography (HPLC). Expression level of targeted genes was analyzed by real-time quantitative PCR. Cell viability was evaluated using the MTT assay. Results: Similar to DOX, EPI was taken up into HepG2 and A549 cells by organic cation transporter 6 and passive diffusion; however, the efficiency of saturable and non-saturable uptake of EPI was greater than that of DOX in both cell types. EPI served as a substrate of P-glycoprotein and multidrug associated protein (MRP) 1 and MRP2 similarly to DOX, but the efflux efficiency of each transporter was markedly different between EPI and DOX. The intracellular accumulation of EPI was significantly greater than that of DOX in all cells, and the accumulated level reflected the cytotoxic effects of these drugs. However, the intracellular drug amount did not correspond to the degree of cytotoxicity when compared between HepG2 and A549 cells, which can be explained by the higher expression of Bcl-xl in A549 cells. Conclusion: This study suggested that the transport characteristics are markedly different between EPI and DOX in HepG2 and A549 cells, and that intracellular accumulation is the predominant factor affecting the cytotoxicity of EPI and DOX in individual cells.

Key Words:

Hepatocellular carcinoma (HCC) and non–small-cell lung cancer (NSCLC) are the major causes of cancer-related deaths worldwide (1-3). Systemic chemotherapy using cytotoxic agents has become an important therapeutic approach for patients with HCC and NSCLC, especially for patients who have aggressive tumors at the time of diagnosis. Anthracycline antibiotics, such as epirubicin (EPI) and doxorubicin (DOX), have been widely used for many cancers, including HCC and NSCLC (4-6). Some of the demonstrated antitumor mechanisms of anthracyclines include intercalation into DNA, which inhibits the synthesis of macromolecules, and the suppression of DNA biosynthesis via the inhibition of topoisomerase II, followed by apoptosis (7). As the molecular targets of anthracyclines are expressed intracellularly, permeation across the plasma membrane is the rate-limiting step in the cytotoxicity of antitumor drugs.

The transport characteristics of DOX have been investigated from the viewpoint of kinetics and molecular biology, and they differ among cell types (8). P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) family, which belong to ABC transporters, are involved in DOX efflux, which is also related to acquired drug resistance (9, 10). The therapeutic effects of DOX were reported to be improved by the inhibition of P-gp or MRP (10). Furthermore, organic cation transporter 6 (OCT6) encoded by the SLC22A16 gene was reported to play a role in DOX uptake (11). As for EPI, its cytotoxicity in cancer cells was increased by reducing the expression levels of P-gp, MRP1 and MRP2 using RNA interference (12, 13). The chemosensitivity of diffuse large B-cell lymphoma cells to EPI was reported to increase through regulation of P-gp via the NF-κB pathway (14). Although these observations suggest the involvement of P-gp, MRP1 and MRP2 in the efflux of EPI, information on the kinetic behavior of EPI transport is insufficient to estimate the degree to which each transporter functions in the efflux and uptake of this agent.

The aim of the present study was to examine the transport characteristics of EPI in human hepatocellular carcinoma HepG2 cells and human non-small cell lung cancer A549 cells, and compare them with those of DOX. We also examined the relation between their cytotoxic effects and intracellular accumulation dominated by transport characteristics.

Materials and Methods

Chemicals. EPI hydrochloride, DOX hydrochloride and daunorbicin hydrochloride (DNR) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). HepG2 cells and A549 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). All other reagents were of commercial or analytical grade and required no further purification.

Cell culture. HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (Fujifilm Wako Pure Chemical Co., Osaka, Japan) containing 10% fetal bovine serum (Biowest USA, Riverside, MO, USA) at 37°C under a humidified atmosphere of 5% CO2 in air. A549 cells were maintained in RPMI1640 medium (Fujifilm Wako Pure Chemical Co.) containing 10% fetal bovine serum at 37°C under a humidified atmosphere of 5% CO2 in air.

Transport assay. After pre-incubation of HepG2 and A549 cells with Hanks’ balanced salt solution for 10 min with or without an inhibitor of OCT6, P-gp or MRP, the reaction was initiated by adding EPI and DOX at the indicated concentrations. After appropriate time intervals, the reaction was terminated by adding ice-cooled excess phosphate-buffered saline. The cells were lysed with ultrapure water.

Assay procedure. Concentrations of EPI and DOX were measured using the HPLC method described below. Fifty micro-liters of 2 μg/ml DNR, an internal standard, 75 μl of 4% zinc sulfate, and 75 μl of methanol were added to a 100-μl lysis sample. The mixture was then vortexed and centrifuged at 5,000 g for 10 min. One hundred micro-liters of supernatant was injected into the HPLC system. The mobile phase consisted of 15 mM acetate buffer (pH 2.5) and acetonitrile (v/v: 2:3), and the flow rate was set at 0.7 ml/min. The eluent was monitored using fluorescence detection with an excitation wavelength of 470 nm and an emission wavelength of 550 nm. The concentrations of EPI and DOX were calculated using the ratio of the corresponding peak area of these drugs to that of an internal standard. The protein concentration was measured based on the Bradford method using bovine serum albumin (Fujifilm Wako Pure Chemical Co.) as the standard (15), and used to calculate the intracellular amount of EPI and DOX.

Kinetic analysis of uptake rate of EPI and DOX. Sets of data points were fitted to the following equation to estimate the apparent kinetic parameters: V=Vmax × S/(Km + S) + kd × S, where V, Vmax, S, Km, and kd are the uptake velocity, maximum uptake velocity, substrate concentration, Michaelis constant, and nonsaturable transport rate constant, respectively. The individual parameters were calculated by the non-linear least-squares curve-fitting method (16).

Real-time quantitative PCR. Total RNA of HepG2 and A549 cells was extracted with ISOGEN (Fujifilm Wako Pure Chemical) and purified using a GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich) according to the manufacturer’s instructions. After reverse transcription using a commercial kit (GE Healthcare, Seattle, WA, USA), the expression level of targeted genes was analyzed by real-time quantitative PCR with MyiQ2 (Bio-Rad Lab Inc., Berkeley, CA, USA) using SYBR green as the fluorescent dye (Toyobo Co., Ltd., Osaka, Japan). cDNA was PCR-amplified at 95°C for 10 s, at 62°C (P-gp) or 57°C (others) for 10 s, and at 72°C for 30 s. In the initial experiments, the melting curve was analyzed to monitor PCR product purity. Relative mRNA expression of targeted genes was quantified using the comparative threshold cycle number for each sample. To adjust for variation in the amount of DNA, gene expression of the target sequence was normalized to that of GAPDH. The synthetic oligonucleotide primers (Hokkaido System Science Co., Ltd., Sapporo, Japan) were designed by Beacon Designer 8 (Bio-Rad Lab., Inc.) and are listed in Table I. mRNA levels were quantified based on standard curves and the results were expressed relative to values of HepG2 cells, which were an arbitrary value of 1.

View this table:
Table I.

Forward and reverse oligonucleotide primer sequences used for real-time PCR.

Cell viability. Cell viability was evaluated using the MTT assay based on our previous report with minor corrections (18). Cell viability was expressed relative to control values, which were given an arbitrary value of 100.

Statistical analysis. Data are expressed as mean±S.D. Differences between the means of two groups were compared using the Student’s unpaired t-test. Comparisons among groups were made by means of an analysis of variance (ANOVA) followed by the Dunnett’s test or the two-tailed multiple t-test with Bonferroni correction. Differences with a p-value of 0.05 or less were considered significant.

Results

Kinetics of transport behavior of EPI and DOX. The amount of EPI and DOX transported into HepG2 and A549 cells was measured (Figure 1). The intracellular amount of EPI and DOX in HepG2 and A549 cells increased linearly up to 30 min, followed by equilibrium. The amount of EPI in HepG2 and A549 cells was significantly greater than that of DOX. The amount of EPI in HepG2 cells was less than in A549 cells, whereas the amount of DOX was similar between the two cell types.

Figure 1.
Figure 1.

Time courses of the intracellular amount of EPI and DOX in (A) HepG2 and (B) A549 cells. Cells were preincubated for 10 min at 37°C and then incubated with 5 μM EPI (○) or DOX (●) for the indicated times at 37°C. Each point represents the mean±SD from three independent experiments. *p<0.05.

Expression levels of OCT6, P-gp, MRP1, and MRP2. The expression levels of OCT6, P-gp, MRP1, and MRP2 in HepG2 and A549 cells are shown in Figure 2. OCT6 was expressed at similar levels in HepG2 and A549 cells. P-gp was expressed in HepG2 cells, but not in A549 cells. The expression levels of MRP1 and MRP2 in A549 were significantly higher than those in HepG2 cells.

Figure 2.
Figure 2.

Expression of OCT6, P-gp, MRP1, and MRP2 in HepG and A549 cells. Total RNA was extracted and purified from HepG2 and A549 cells, and the expression levels of OCT6, P-gp, MRP1, and MRP2 were measured by real-time PCR. The results represent the mean±SD from three independent experiments. *p<0.05.

Concentration dependence of EPI and DOX uptake. To examine the kinetics of EPI and DOX uptake by HepG2 and A549 cells, the uptake rate of THP and DOX was measured over the concentration range of 1-100 μM (Figure 3). The uptake rate in all groups became saturated as the drug concentration increased. The uptake rate of EPI by HepG2 and A549 cells was greater than of DOX. The kinetic parameters for EPI and DOX uptake by HepG2 and A549 cells are shown in Table II. The values of Vmax/Km, Kd, and Vmax/Km + Kd for EPI uptake were significantly higher than those for DOX in both cell types.

Figure 3.
Figure 3.

Concentration dependence of EPI and DOX uptake by HepG2 and A549 cells. Cells were preincubated for 10 min at 37°C and then incubated with the indicated concentrations of EPI (○) or DOX (●) for 15 min. Each point represents the mean±SD from three independent experiments.

View this table:
Table II.

Apparent kinetic constants for EPI and DOX uptake in HepG2 and A549 cells.

Effects of carnitine and temperature on EPI and DOX uptake. As shown in Figure 4, the uptake rate of EPI and DOX by HepG2 and A549 cells was significantly reduced by carnitine. The uptake rate of EPI and DOX at 0°C was significantly lower than that at 37°C in both cell types.

Figure 4.
Figure 4.

Effects of carnitine and temperature on EPI and DOX uptake by HepG2 and A549 cells. Cells were preincubated for 10 min at 37°C or 0°C with or without 1 mM carnitine, and then incubated with 5 μM EPI or DOX for 15 min under the same conditions as preincubation. Each point represents the mean±SD from three independent experiments. *p<0.05.

Effects of verapamil, PSC833 and MK-571 on the intracellular amount of EPI and DOX. The effects of verapamil, PSC833 and MK-571 on the intracellular amount of EPI and DOX in HepG2 and A549 cells are shown in Figure 5. In HepG2 cells, the intracellular amounts of EPI and DOX were increased to a similar extent by verapamil. The intracellular amount of EPI was significantly increased by treating HepG2 cells with PSC833, whereas the amount of DOX significantly decreased following treatment. The intracellular amount of EPI, but not DOX, was significantly increased by MK-571. In A459 cells, the degree of the increase in the intracellular amount of EPI by MK-571 was larger than that of DOX.

Figure 5.
Figure 5.

Effects of verapamil, PSC833 and MK-571 on the intracellular amount of EPI and DOX in HepG2 cells and A549 cells. Cells were preincubated for 10 min at 37°C with or without 500 μM verapamil, 5 μM PSC833 and 100 μM MK-571, and then incubated with 5 μM EPI or DOX for 60 min under the same conditions as preincubation. Each point represents the mean±SD from three independent experiments. *p<0.05.

Cytotoxicity of EPI and DOX. The cytotoxicity of EPI and DOX against Hep2 and A549 cells is shown in Table III. The cell viability of HepG2 and A549 cells decreased after exposure to EPI and DOX in a concentration-dependent manner. The survival rates following EPI exposure were significantly lower than those following DOX exposure in both cell types. The values of survival rate of HepG2 cells following exposure to DOX was significantly lower compared to of A549 cells, while no difference in the values following EPI exposure was observed between the two cell types.

View this table:
Table III.

Cytotoxicity of EPI and DOX against HepG2 and A549 cells.

Expression levels of ERCC1 and Bcl-xl. The expression levels of ERCC1 and Bcl-xl in HepG2 and A549 cells are shown in Figure 6. There was no significant difference in the expression level of ERCC1 between the two cell types, but the expression level of Bcl-xl in A549 cells was significantly higher than that in HepG2 cells.

Figure 6.
Figure 6.

Expression of ERCC1 and Bcl-xl in HepG and A549 cells. Total RNA was extracted and purified from HepG2 and A549 cells, and the expression level of ERCC1 and Bcl-xl was measured by real-time PCR. The results represent the mean±SD from three independent experiments. *p<0.05.

Discussion

Chemotherapy using anthracycline antibiotics plays an important role in cancer treatment together with surgery and radiation therapy. Although there are many factors responsible for the susceptibility and resistance of cancer cells to anthracyclines (19), an understanding of the transport characteristics of EPI and DOX across the plasma membrane is of particular importance for assessing the level of drug exposure. In the present study, we examined the transport characteristics of EPI and DOX in HepG2 and A549 cells, and investigated their relationship with cytotoxicity. To the best of our knowledge, this is the first study to provide experimental information on the detailed transport characteristics of EPI.

The transport characteristics were markedly different between EPI and DOX in both HepG2 and A549 cells. First, the uptake characteristics of EPI and DOX by HepG2 and A549 cells were examined. The uptake of EPI and DOX by OCT6-expressing HepG2 and A549 cells was concentration-dependent and exhibited saturation kinetics, which is consistent with the observation that these uptake rates depended on temperature and the presence of carnitine, a OCT6 inhibitor. However, the uptake clearance (Vmax/Km) and non-saturable transport rate constants (Kd) of EPI were higher than those of DOX in both HepG2 and A549 cells. It is of interest that the ability of non-saturable uptake indicative of passive diffusion into HepG2 and A549 cells was different between EPI and DOX. This observation was consistent with the uptake rate of EPI at 0°C being greater than that of DOX in both cell types. One reason for these phenomena may be the difference in chemical properties between EPI and DOX despite mutual structural similarity, which was supported by the intracellular accumulation of DOX in HepG2 cells being suppressed by PSC833, an inhibitor of P-gp, and intracellular accumulation of EPI being conversely increased by PSC833. There was no information available on the inhibitory effects of PSC833 on transporting functions associated with uptake. It was previously reported that the uptake activity mediated by OCT1 is inhibited by pretreatment with cyclosporine, which possesses a structure similar to PSC833 (20). However, our previous study adopting kinetic analysis demonstrated that DOX is not a substrate of OCT1 (21). Therefore, it is unclear why the intracellular accumulation of DOX in HepG2 cells was suppressed by PSC833 treatment and further studies are needed in this regard. Taken together, our study suggested that the ability of saturable and non-saturable uptake of EPI was greater than that of DOX in both HepG2 and A549 cells, although EPI was taken up into both cell types by OCT6 and passive diffusion, similar to DOX.

Next, the efflux characteristics of EPI and DOX from HepG2 and A549 cells were examined. P-gp was expressed in HepG2 cells, but not in A549 cells, and higher expression of MRP1 and MRP2 was observed in A549 cells than in HepG2 cells. The intracellular accumulation of EPI in HepG2 cells was increased by verapamil (P-gp inhibitor) and MK-571 (MRP1 and MRP2 inhibitor). In a previous study, the mRNA expression of P-gp, MRP1 and MRP2 in Hela cells increased after EPI exposure (22). Knockdown of galectin-3, treatment with PEGylated liposomal hepcidin 2-3 and silencing of the RNA-binding protein HuR increased the intracellular accumulation of EPI in cancer cells secondary to suppression of the mRNA expression of P-gp, MRP1 and MRP2 (23-25). The induction of P-gp was observed in EPI-resistant (EPI-R) cell lines developed from parental MDA-MB-231 cells, but chemosensitivity of EPI-R cells was restored by short hairpin RNA interference against P-gp (12). In addition, EPI-mediated apoptosis increased after the introduction of PEGylated liposomal antisense oligonucleotides against P-gp, MRP1 and MRP2 (13). These studies strongly support EPI being a substrate of these transporters, similar to DOX. As the uptake ability of EPI was greater than that of DOX in both cell types, the impact of the increase in intracellular accumulation of EPI following treatment with the inhibitors of efflux transport should be marked. Indeed, the degree of increase in intracellular accumulation of EPI in HepG2 and A549 cells following treatment with MK-571 was larger than that of DOX. However, the degree of increase in intracellular accumulation in HepG2 cells after verapamil treatment was similar between EPI and DOX. This may be attributed to the greater efficiency of ERI efflux mediated by the MRP family than that of DOX. On the other hand, P-gp likely plays a major role in the efflux of DOX instead of MRP, providing an explanation for the lack of alteration in intracellular accumulation of DOX in the presence of MK-571. This suggests that although there is functional similarity in transporters excreting EPI and DOX from HepG2 and A549 cells, the efflux ability of each transporter differs markedly between these drugs.

The intracellular accumulation of EPI in both cell types was significantly greater than that of DOX. When comparing between cells, the intracellular accumulation of EPI in HepG2 cells was less than that in A549 cells, whereas the degree of DOX accumulation was similar between the two cell types. In the assessment of cytotoxicity, EPI exhibited greater cytotoxicity than DOX when the effects on each cell were examined individually. These results may be explained by the difference in intracellular accumulation between EPI and DOX in the two cell types. On the other hand, HepG2 cells were more sensitive to both EPI and DOX than A549 cells considering the disparity in their intracellular concentrations. Therefore, the difference in cytotoxicity of EPI and DOX between the two cell types cannot be fully explained only by the intracellular accumulation of these drugs. In HepG2 cells with acquired resistance to DOX, increased susceptibility to DOX was reported following reduction in the expression level of ERCC1, a DNA repair factor (26). The previous study also demonstrated a higher level of the antiapoptotic protein Bcl-xl in HuH6 cells than in HepT1 cells, with the former being more resistant to DOX than the latter (27). In the present study, higher expression of Bcl-xl, but not ERCC1, was observed in A549 cells than in HepG2 cells, suggesting that the difference in cytotoxicity between the cell types may be, at least in part, due to the higher expression level of Bcl-xl in A549 cells. Based on this study, in the same cell type, the intracellular drug accumulation may be a more important factor for cytotoxicity.

Information on the transport characteristics of anthracyclines across the plasma membrane is valuable for the implementation of appropriate cancer chemotherapy. First, carriers associated with the efflux of anthracyclines are responsible for the development of drug resistance by cancer cells. In particular, the expression of MRP1 was reported to be an important factor for the resistance to anthracyclines by different types of cancer (28). Therefore, the overexpression of MRP1 in cancer cells may be a more serious problem for the use of EPI than for DOX in terms of resistance development. The importance of the downregulation of OCT6 in the resistance of tumor cells to anthracyclines has not been elucidated yet, but the low uptake ability of DOX was reported to be involved in de novo resistance of M5076 ovarian sarcoma cells (18). Next, the ingenious utilization of transport characteristics may be a promising strategy for selectively delivering anthracyclines to tumor cells. Indeed, pirarubicin, an anthracycline derivative, was reported to be incorporated into human leukemia HL60 cells and fresh human mononuclear cells by differed transport mechanisms (29). In this regard, further studies on the detailed transport mechanisms of EPI will be required using different cancer and normal cells.

In conclusion, the present study demonstrated that the transport characteristics markedly differ between EPI and DOX in HepG2 and A549 cells. The intracellular accumulation of EPI and DOX dominated by the transport characteristics was a primary factor for determining cytotoxicity in the same cell type. Our study provides useful information for cancer treatment using anticancer drugs.

Footnotes

  • Authors’ Contributions

    Conceptualization: KN, HK. Methodology: KN, SF. Formal analysis: KN, MT. Investigation: MS, SY. Writing-Original Draft Preparation: KN. Writing/Review and Editing: HK.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Received August 7, 2021.
  • Revision received October 15, 2021.
  • Accepted October 18, 2021.

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Differences in Transport Characteristics and Cytotoxicity of Epirubicin and Doxorubicin in HepG2 and A549 Cells
KATSUHITO NAGAI, SHUHEI FUKUNO, MAHO SHIOTA, MAYUKO TAMURA, SAYAKA YABUMOTO, HIROKI KONISHI
Anticancer Research Dec 2021, 41 (12) 6105-6112; DOI: 10.21873/anticanres.15430
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