Introduction

Multidrug resistance (MDR) of cancer cells is a major factor limiting the effectiveness of cancer chemotherapy. A widely studied mechanism of MDR is associated with overexpression of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, which results in increased efflux of chemotherapeutic agents from cells. Among 48 human ABC transporters, a well-characterized P-glycoprotein (Pgp) is highly implicated with reduction of intracellular levels of anticancer drugs below their therapeutic threshold and a consequent decrease in their toxic effects [1–5].

A significant contribution of Pgp to the efflux of endogenous and exogenous molecules, which leads to drug resistance, provides an alternative rationale for the development of targeted therapies against this membrane transporter [6]. Direct inhibitors and modulators of Pgp were identified first in the 1980s, but three generations of Pgp inhibitors have been investigated extensively as a strategy for MDR reversal. However, concerns remain over safety and pharmacokinetic changes of therapeutic drugs in clinical trials [7]. Thus, strategies have been employed to overcome MDR using new chemotherapeutic agents designed as analogs of therapeutic compounds [8, 9], prodrugs [10, 11], Pgp-specific therapeutic antibodies [12], and nanoparticle-based drug delivery systems [13, 14]. These diverse strategies are emerging as promising candidates for the effective treatment of cancer, but the toxicology of long-term exposure to drugs or nanomaterials in humans needs to be studied and evaluated fully [15–17].

Alternatively, Pgp activity can be regulated by protein phosphorylation, particularly if induced by activated protein kinase C (PKC) isoforms; Pgp overexpression has been implicated to have a prominent role in MDR cells in humans [18–20]. Several reports of drug accumulation in human MDR cells have suggested increased activities/levels of PKC isoforms, mainly for PKCα, which phosphorylates the specific serine residues in the linker region of Pgp, thereby activating Pgp to increase ATPase activity and drug efflux [21–24]. It has also been shown that regulation of drug resistance with a PKC inhibitor which inhibits various PKC isoforms non-specifically leads to decreased phosphorylation of Pgp and drug efflux, suggesting the possibility of altered drug resistance in MDR cells [25]. Furthermore, PKCα activity has been proposed to be related to cancer stem cells, which have unique features such as self-renewal, differentiation, and chemoresistance to conventional therapeutic agents. In particular, recent studies have demonstrated that an intrinsic mechanism of resistance of cancer stem cells to chemotherapeutic drugs is acquired through high expression of ABC transporters [26–28]. Therefore, targeting ABC transporters such as Pgp through regulation of PKCα activity could help to reverse MDR, resulting in better anticancer strategies.

Here, we investigated if Pgp-mediated drug resistance was modulated by inhibition of PKCα activity in human MDR breast cancer cells, in which a PKC inhibitor was used in combination with an anticancer drug to overcome MDR. To assess levels of PKCα activity, a PKCα-specific peptide substrate (Alphatomega; FKKQGSFAKKK-NH2), which we have shown previously to have high specificity towards PKCα [29, 30], was used to compare phosphorylation levels in drug-sensitive MCF-7 cells with those in drug-resistant MCF-7/ADR cells. PKCα activity was modulated by the PKC inhibitor Ro-31-7549, which has highly specific inhibitory effects towards PKCα [30–32]. Inhibition of PKCα activity resulted in increased accumulation of anticancer drug and enhancement of its toxicity in resistant cells (Fig. 1). These observations provided evidence that Pgp phosphorylation induced by activated PKCα has a prominent role in the regulation of drug efflux.

Fig. 1
figure 1

Inhibition of intracellular PKCα activity for overcoming multidrug resistance in drug-resistant human breast cancer cells (schematic)

Full size image

Materials and methods

Cell culture

MCF-7 cells were cultured in modified Eagle medium containing 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 10 μg/mL insulin. MCF-7/ADR cells were kindly provided by Dr. Ick Chan Kwon (Center for Theragnosis, Korea Institute of Science and Technology, Seoul, Korea) and cultured in RPMI 1640 medium. All medium contained 10 % fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (all from Gibco Invitrogen, Grand Island, NY, USA). Cells were harvested in a humidified atmosphere of 5 % CO2 and 95 % air at 37 °C.

Cytotoxicity of doxorubicin (Dox) and PKC inhibitor (Ro-31-7549)

Cells (2 × 103) were seeded into 96-well plates and cultured for 24 h at 37 °C in a 5 % CO2. The medium in each well was replaced with 100 μL of fresh medium containing a different concentration of doxorubicin hydrochloride (0–100 nM; Wako Pure Chemicals, Osaka, Japan) and the PKC inhibitor Ro-31-7549 (0–50 μM; Merck Millipore, Billerica, MA, USA). After incubation for 24 or 72 h, the medium was replaced with 100 μL of fresh medium. Then, 10 μL of 4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt (WST-8; Dojindo Laboratories, Kumamoto, Japan) was added to each well. Cells were incubated for a further 2 h. Cell viability (%) was calculated by comparing the absorbance of treated cells to that of untreated control cells at 440 nm. This assay was conducted in triplicate.

Phosphorylation assay of the PKCα-responsive peptide substrate

MCF-7 and MCF-7/ADR cells were plated at 1 × 106 in 100-mm dishes or 2 × 105 in six-well plates at 37 °C in 2 mL medium containing 10 % FBS. At ∼70–80 % confluence, cells were stimulated with doxorubicin (0.01–10 nM) and Ro-31-7549 (0.05–100 μM) for 6 h, and then scraped in 1 mL buffer [10 mM HEPES, 10 % sucrose, Complete™ protease inhibitor cocktail (EDTA-free; Roche, Basel, Switzerland)]. Samples were centrifuged at 5000×g for 10 min at 4 °C and the supernatant was removed. Then, 0.1 mL of buffer was added to the precipitate and sonicated twice in ice for 15 s. Samples were centrifuged again at 5000×g for 15 min at 4 °C, and the resulting supernatant used for phosphorylation of the PKCα-specific peptide substrate (prepared by standard Fmoc-chemistry) [30]. Total protein concentration of cell lysates was measured using Protein Assay Dye reagent (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard. Phosphorylation of the peptide substrate responding to PKCα in MCF-7 and MCF-7/ADR cell lines was carried out in 100 μL buffer [10 mM HEPES, 10 mM MgCl2, 0.5 mM CaCl2, 2.0 μg/mL diacylglycerol, 2.5 μg/mL phosphatidylserine, and 200 μM adenosine triphosphate] containing 30 μM peptide substrate and cell lysates at protein concentrations of 100 or 200 μg/mL. Phosphorylation was allowed to proceed for 1 h at 37 °C. Resulting solutions were analyzed by matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS). Half-maximal inhibitory concentration (IC50) values were obtained using Prism (GraphPad, Avenida, CA, USA).

Confocal laser scanning microscopy

MCF-7 and MCF-7/ADR cells were plated at 1 × 105 in 35-mm glass-bottom dishes (Matsunami, Osaka, Japan) at 37 °C in 1 mL medium containing 10 % FBS for 24 h. Dishes were washed with phosphate-buffered saline (PBS) and incubated with or without 5 μM Ro-31-7549 for 30 min; cells incubated further with 100 μM doxorubicin for 2 h. Intracellular localization and accumulation of doxorubicin were observed by confocal laser scanning microscopy (CLSM, LSM 700; Carl Zeiss, Oberlochen, Germany) equipped with an EC Plan-Neofluar ×20/0.50 M27 objective lens.

Cellular accumulation of doxorubicin

MCF-7 and MCF-7/ADR cells were plated at 5 × 104 in 24-well plates at 37 °C in medium containing 10 % FBS for 24 h. The medium in each well was washed with PBS and replaced with Opti-MEM (Gibco), and then incubated with or without 50 μM Ro-31-7549 and doxorubicin, (0, 0.1, 1, or 10 nM) for 6 h. Estimation of cellular uptake was conducted by a Tali™ Image-based Cytometer (Invitrogen, Carlsbad, CA, USA).

Statistical analysis

Results are the mean ± S.D. from at least three independent experiments. Differences were determined with the Student’s t test. P < 0.05 was considered significant.

Results and discussion

Resistance to doxorubicin in human breast cancer cells

To investigate resistance to the therapeutic drug doxorubicin in drug-sensitive MCF-7 and resistant MCF-7/ADR cells (which are well-characterized human breast cancer cell lines for multidrug resistance [33, 34]), we first compared cell viability using the WST-8 assay in the presence of various concentrations of doxorubicin (Fig. 2a). Both cell types were exposed to increasing concentrations of doxorubicin (0.01–100 nM) for 72 h. As expected, MCF-7 cells had high sensitivity to doxorubicin (IC50 < 0.1 nM), whereas MCF-7/ADR cells showed ∼100-times higher resistance to doxorubicin than MCF-7 cells (IC50 > 10 nM). Then we examined the toxicity of the PKCα inhibitor Ro-31-7549 on both cell lines. Toxicity of Ro-31-7549 on these cell lines was weak and almost identical (Fig. 2b).

Fig. 2
figure 2

Cell viability with doxorubicin (a) and Ro-31-7549 (b) in MCF-7 (sensitive) and MCF-7/ADR (resistant) cells for 72 h

Full size image

Effect of a PKCα inhibitor on drug-sensitive and drug-resistant cell lines

PKCα activity was estimated from phosphorylation levels of a PKCα-specific peptide substrate (FKKQGSFAKKK-NH2) with MCF-7 and MCF-7/ADR cell lysates at a protein concentration of 200 μg/mL. We have established this method to be a simple and rapid quantification method with MALDI-TOF-MS analyses [29]. Basal activity of PKCα in MCF-7/ADR was higher than MCF-7 (Ro-31-7549 = 0 μM, Fig. 3), a finding that was in accordance with previous reports [35, 36]. Addition of Ro-31-7549 led to a gradual decrease in the phosphorylation level of MCF-7 and MCF-7/ADR cells. IC50 values of Ro-31-7549 were 0.76 μM for MCF-7 and 15 μM for MCF-7/ADR cells, respectively. The higher IC50 value in MCF-7/ADR cells reflected (again) the higher activity of PKCα in this cell line.

Fig. 3
figure 3

Inhibition of PKCα activity by addition of an inhibitor of protein kinase (Ro-31-7549). After 6 h incubation with Ro-31-7549, cells were scraped and sonicated at 4 °C. Samples were then centrifuged and the supernatant used as cell lysates. Phosphorylation of the PKCα-specific peptide substrate with cell lysates at a protein concentration of 200 μg/mL was undertaken at 37 °C for 1 h and analyzed by MALDI-TOF-MS (n = 3)

Full size image

Effects of doxorubicin on PKCα activity and its inhibition by Ro-31-7549

To investigate the effect of doxorubicin on PKCα activity, a phosphorylation assay was carried out in MCF-7 and MCF-7/ADR cells after exposure to different concentrations of doxorubicin for a short period (6 h), in which the toxicity in both cell lines is negligible. MCF-7 cells showed no change in phosphorylation irrespective of doxorubicin concentration (Fig. 4a). In contrast, MCF-7/ADR cells showed a gradual increase in phosphorylation with increasing doxorubicin concentration. This increase in PKCα activity in response to doxorubicin treatment may correlate with an increase in Pgp-mediated drug efflux in MCF-7/ADR cells.

Fig. 4
figure 4

Effects of doxorubicin and Ro-31-7549 on phosphorylation of the PKCα-specific peptide substrate. After 6 h incubation with doxorubicin alone (a) and with both doxorubicin and Ro-31-7549 (b), cells were scrapped and sonicated at 4 °C. Samples were then centrifuged and the supernatant used as cell lysates. Phosphorylation of the PKCα-specific peptide substrate with cell lysates at a protein concentration of 100 μg/mL was carried out at 37 °C for 1 h and analyzed by MALDI-TOF-MS (n = 3). Data are the mean ± S.D., n.s., not significant, *P < 0.05, ***P < 0.005

Full size image

Then, we examined the effect of a combination of Ro-31-7549 with doxorubicin. Ro-31-7549 inhibited phosphorylation reactions in MCF-7 (23.6 to 3.6 %) and MCF-7/ADR (41.8 to 0.5 %) cells, irrespective of the presence of doxorubicin (Fig. 4b).

Reversal of resistance to doxorubicin by Ro-31-7549 in drug-resistant cells

To ascertain if inhibition of PKCα activity leads to suppression of Pgp-mediated drug efflux in MDR cells, cellular accumulation of doxorubicin was monitored by CLSM in MCF-7 and MCF-7/ADR cells. In the absence of Ro-31-7549, significant accumulation of doxorubicin was observed in drug-sensitive MCF-7 cells, whereas their accumulation was suppressed substantially in drug-resistant MCF-7/ADR cells (Fig. 5). This lower accumulation of doxorubicin in MCF-7/ADR cells showed marked efflux of doxorubicin by highly expressed Pgp in resistant cells [34]. However, in the presence of Ro-31-7549, cellular uptake of doxorubicin was enhanced extensively in MCF-7/ADR cells, suggesting significant suppression of Pgp-mediated doxorubicin efflux due to inhibition of PKCα activity. Results demonstrated that doxorubicin efflux was inhibited by Ro-31-7549. This is the first report focusing on MDR reversal by a PKCα-specific inhibitor.

Fig. 5
figure 5

Intracellular distribution of doxorubicin in MCF-7 (a) and MCF-7/ADR (b) cells. Cellular accumulation and distribution of doxorubicin (100 nM) was observed by confocal laser scanning microscopy by using doxorubicin’s fluorescence. After treatment with doxorubicin for 2 h with/without pre-incubation of 5 μM Ro-31-7549 for 30 min, images were obtained. Scale bar = 50 μm

Full size image

Cellular accumulation of doxorubicin was evaluated quantitatively by an image-based cytometer (Fig. 6a, b) and the obtained results summarized in Fig. 6c, d. MCF-7 cells showed increasing cellular accumulation of doxorubicin in a dose-dependent manner in the presence and absence of Ro-31-7549 (Fig. 6a). Median fluorescence intensity is summarized in Fig. 6c. Effect of Ro-31-7549 on enhancement of doxorubicin accumulation was small. In MCF-7/ADR cells, cellular accumulation of doxorubicin was much improved in the presence of Ro-31-7549 (Fig. 6b). Median fluorescence intensity of Ro-31-7549-treated cells was much higher than that of non-treated cells and increased gradually, suggesting dose-dependent accumulation of doxorubicin (Fig. 6d). Such MDR reversal in MCF-7/ADR suggested that Ro-31-7549 suppressed the Pgp-mediated drug efflux via PKCα inhibition.

Fig. 6
figure 6

Effects of Ro-31-7549 (50 μM) on accumulation of doxorubicin in MCF-7 (a) and MCF-7/ADR (b) cells. Median of cellular doxorubicin fluorescence is normalized to cellular doxorubicin fluorescence in doxorubicin (0.1 nM) alone as control (c, d). Data are the mean ± S.D., n.s., not significant, **P < 0.001, ***P < 0.005, significant difference in the presence and absence of Ro-31-7549

Full size image

Finally, enhancement of the anticancer activity of doxorubicin by PKCα inhibition was investigated after treatment of cells with a combination of the two drugs for 24 h. In MCF-7 cells (Fig. 7a), the presence of Ro-31-7549 weakly enhanced the toxicity of doxorubicin, and this enhancement disappeared at the high concentration of doxorubicin (10 nM). In MCF-7/ADR cells (Fig. 7b), Ro-31-7549 enhanced the toxicity of doxorubicin significantly at all the doxorubicin concentrations examined. Interestingly, doxorubicin toxicity against resistant cells reached almost the same level with doxorubicin-sensitive MCF-7 cells at 10 nM of doxorubicin (∼45 % in both cases). Taken together, these results demonstrated that the anticancer activity of doxorubicin can be improved synergistically by inhibition of PKCα activity, which leads to enhanced cellular accumulation of doxorubicin via suppression of Pgp-mediated drug efflux in drug-resistant MCF-7/ADR cells.

Fig. 7
figure 7

Effects of co-administration with doxorubicin and Ro-31-7549 on viability in MCF-7 (a) and MCF-7/ADR (b) cells. After 24 h incubation with different concentrations of doxorubicin and 5 μM Ro-31-7549, viability was evaluated in sensitive and resistant cell lines (n = 3). Data are the mean ± S.D., **P < 0.001, ***P < 0.005, significant difference in the presence and absence of Ro-31-7549

Full size image

Conclusions

Inhibition of PKCα activity enhanced the accumulation of doxorubicin in MCF-7/ADR cells. Use of the PKCα-specific inhibitor Ro-31-7549 could lead to a decrease of phosphorylation reactions and consequently induce increased drug accumulation through suppression of Pgp-mediated drug efflux in drug-resistant MCF-7/ADR cells. This observation may suggest that Pgp overexpression in MDR cancer cells is linked to PKCα activities, induces phosphorylation reactions in the linker region of Pgp, and provides evidence of reversal of intrinsic anticancer drug resistance. Moreover, reversal of intracellular accumulation of a drug could induce increase drug efficacy in drug-resistant human breast cancer cells. Hence, regulation of PKCα activity and reversal of drug efflux via Ro-31-7549 combined with doxorubicin is a possible alternative to current approaches with many other therapeutic anticancer drugs for combating MDR.