1. Introduction
As cancer is one of the leading causes of death worldwide, cancer treatment is always on the top of listed hot research topics [
1]. With advanced scientific researches and abundant medical resources in recent decades, diverse options have been developed to conquer cancer and related diseases. Nevertheless, the multi-drug resistance (MDR) of cancer treatment is still an ever-changing problem and more in-depth studies have been conducted to unveil the complicated characteristics of cancer treatment. Cancer MDR manifests cross resistance to several structurally and mechanically different chemo-agents and could be contributed to the following reasons [
2]. The change in tumor microenvironment [
3,
4,
5], decreased drug uptake [
6], adapted cell apoptotic pathways [
7,
8,
9], drug inactivation through metabolism [
10,
11], the influence of epigenetic regulation [
12,
13], mutation of drug target site [
14], and the increased drug efflux [
15] have been reported to play important roles in causing cancer MDR. Among the above mechanisms, the increased drug efflux by ATP-binding cassette (ABC) transporters has been regarded as the most influential cause. ABC transporter superfamily consists of several subfamilies, and P-glycoprotein (P-gp) is one of the most comprehensively studied proteins [
16]. P-gp is encoded by human
ABCB1 gene and can recognize various clinically used drugs, including antidepressants, HIV protease inhibitors, immunosuppressive agents, and chemotherapeutic drugs [
17,
18]. The diverse structures recognizing and effluxing the ability of P-gp, result in insufficient chemo-drug concentration inside cancer cells, therefore, causing cancer MDR.
There have been a series of P-gp inhibitor developments along the cancer MDR reversing agents discovering history, and the improvements have been based on previous failure experiences [
19]. First generation P-gp inhibitors are potent but toxic as the required dose is high; examples of this are quinidine and verapamil [
20]. Second generation inhibitors have exhibited better effects with lower IC
50, but the involvement of these inhibitors in CYP450 interaction has impeded their further application [
21,
22]. Third generation inhibitors, including tariquidar and zosuquidar, have demonstrated prominent MDR reversal effects. However, they have still faced failure in clinical studies [
23,
24]. Therefore, severe toxicity and interaction of the above chemical reagents have turned the research direction toward natural resources, aiming at discovering low toxic and potent structures from plants, fungi, or marine organisms.
Among various natural resources, phytochemicals such as flavonoids and phenolic acids get much attention due to their multiple pharmacological effects, including antioxidant and antitumor activity [
25,
26]. Several phytochemicals, such as cyanidin, catechin, quercetin, caffeic acid, and ellagic acid, have been related to the down-regulation of human LDL oxidation [
27]. Ellagic acid and ursolic acid have been reported to exhibit preventive and therapeutic effects against breast cancer cells [
28]. Caffeic acid (
Figure 1), a phenolic acid that widely exists in vegetables, fruits, and tea extracts, is well-known as a natural antioxidant [
29]. Besides, caffeic acid has also been identified to have anti-inflammatory, antibacterial, and antiviral effects [
30,
31]. With regards to cancer treatment, caffeic acid and its derivative, caffeic acid phenethyl ester (CAPE), exhibit some therapeutic effects toward lung cancer and breast cancer cells, as well as breast cancer pre-clinical models [
32,
33,
34]. CAPE has been well studied in previous researches, including its
MDR1 gene down-regulating effects in MCF-7 and MDA-MB-231 breast cancer cells [
34] and P-gp inhibitory effects in HeLa resistant cancer subline and human intestinal LS174T cell line [
35,
36]. Nevertheless, the P-gp inhibitory and MDR modulating information of the caffeic acid was insufficient and warrants further detailed investigation.
Therefore, in the present study, comprehensive researches of caffeic acid were conducted. The interaction of caffeic acid with human P-gp, as well as the inhibitory effects and mechanisms were assessed in P-gp over-expressing cell line ABCB1/Flp-InTM-293. The cancer MDR reversing ability of caffeic acid was then evaluated in both ABCB1/Flp-InTM-293 and KB/VIN MDR cancer cell lines. The present study demonstrated that caffeic acid is a promising candidate for P-gp inhibition and cancer MDR attenuation.
3. Discussion
Caffeic acid, a dietary non-flavonoid phenolic compound, has been a popular candidate among several research fields. The present study has demonstrated its usability in cancer MDR. Caffeic acid can attenuate this severe resistant problem by inhibiting the efflux function of human P-gp. Through diverse modulating mechanisms, caffeic acid helps resistant cancer cells retain chemotherapeutic drugs inside their cells, promoting further apoptosis and cell death.
Through investigating the history of P-gp inhibitor development, the ideal characteristics of potential candidates have been revealed. The inhibitor itself is not a substrate of P-gp, but is one of the favorable properties [
19]. Our present research performed an experiment and the results indicated that caffeic acid was not P-gp’s substrate. In this way, more caffeic acid could stay inside the cells to help P-gp inhibition, resulting in a higher intracellular chemotherapeutic drugs concentration.
The inhibitory effects of caffeic acid on P-gp efflux function were demonstrated on three P-gp fluorescent substrates, calcein-AM, rhodamine123, and doxorubicin. The different binding modes of each substrate revealed the inhibitory mechanisms of caffeic acid on P-gp drug binding sites. A previous investigation found that doxorubicin was a R-site substrate while rhodamine123 exhibited both M and R sites binding affinity [
37,
38,
39]. Our efflux assay results indicated that caffeic acid showed uncompetitive inhibition on rhodamine123 transport and competitive inhibition on doxorubicin transport. Therefore, caffeic acid might compete the R drug binding site with doxorubicin, resulting in efflux inhibition. In terms of rhodamine123 inhibition, caffeic acid exhibited an allosteric modulation on M site, indirectly prohibiting the pump out behavior of P-gp.
In addition to drug binding sites, the interaction between caffeic acid and ATP binding sites of P-gp was also studied. According to the tested compound’s behavior toward P-gp ATPase regulation, substances could be categorized into three classes: dual regulators, stimulators, and inhibitors [
40,
41]. Dual regulators stimulate both basal and verapamil-stimulated ATPase activity at a lower dose, but inhibit the activity at a higher dose, such as paclitaxel and vinblastine. Stimulators like valinomycin and bisantrene increase ATPase activity dose-dependently while inhibitors decrease both basal and verapamil-stimulated ATPase activity, such as rapamycin and cyclosporine A. The results of ATPase assay in the present study indicated that caffeic acid exhibited stimulatory activity from 10 μg/mL to 100 μg/mL in a dose-dependent manner. Therefore, caffeic acid was an ATPase stimulator. Besides, the results of verapamil-stimulated ATPase activity further revealed the binding behavior of caffeic acid on ATPase binding sites. Caffeic acid increased verapamil-stimulated ATPase activity regardless of the dose, implying its binding site on ATPase was different from verapamil. This allosteric stimulation advanced the consumption of ATP, indirectly inhibiting P-gp efflux function.
Whether the promising P-gp inhibitory effects of caffeic acid were helpful in reversing cancer MDR was than studied in our following experiments. In
ABCB1/Flp-In
TM-293 P-gp over-expressing cell line, caffeic acid significantly decreased the required doses of chemo-agents, including vincristine, paclitaxel, and doxorubicin. Under the treatment of 30 μg/mL caffeic acid, the IC
50 of paclitaxel largely decreased from 604.09 nM to 121.55 nM. This advanced cytotoxicity was related to the increased apoptotic effects revealed by cell cycle assay results. With caffeic acid as a combinatory agent, the percentage of subG1 apoptotic population induced by paclitaxel significantly increased in a dose-dependent manner. The above results were consistent with previous researches, which revealed that caffeic acid could sensitize ovarian carcinoma cells and lung cancer cells to cisplatin and paclitaxel, respectively [
33,
42]. Caffeic acid exhibited chemo-sensitizing effects in the combination group by cell cycle arresting in G2/M (caffeic acid 20 μg/mL with paclitaxel) and G1 (caffeic acid 25 μg/mL with paclitaxel). These effects were not only due to the modulation of P-gp, other cellular targets and multiple mechanistic possibilities may be involved and need further investigation. The MDR reversing ability of caffeic acid was also investigated in MDR cancer cell line KB/VIN. The results exhibited a trend on increasing the cytotoxicity of chemo-agents. With the treatment of 100 μg/mL caffeic acid, the cell viability decreased from nearly 100% to 67.91%, 61.18%, and 59.50% for doxorubicin, paclitaxel, and vincristine, respectively. However, compared to the promising results in
ABCB1/Flp-In
TM-293 cell line, the MDR modulating effects of caffeic acid in KB/VIN seemed to be less potent and did not show increased apoptosis in the cell cycle analyses, exhibiting cell type-dependent effects. This phenomenon could be explained by the regulation of caffeic acid on P-gp expression. As
Figure 5a,b shows, caffeic acid slightly decreased
ABCB1 gene expression in
ABCB1/Flp-In
TM-293 but increased the expression level in KB/VIN cell line. This up-regulating trend in KB/VIN diminished the functional inhibitory potency of caffeic acid, resulting in weaker MDR reversing effects. Previous research has revealed that the oxidative stress might have a role in the regulation of P-gp expression [
43]. As caffeic acid exhibited significant ROS-related anti-oxidant effects, the influence of caffeic acid on ROS production in KB/VIN cell line was performed. The results showed that caffeic acid significantly decreased ROS production in HeLaS3 cell line and slightly decreased ROS production in KB/VIN cell line. However, the doxorubicin-induced oxidative challenge was significantly reversed by caffeic acid in amounts of 10 μg/mL and 100 μg/mL in both HeLaS3 and KB/VIN cell lines. Therefore, the relationship between the reactive oxygen species levels and the up-regulation of
ABCB1 gene in KB/VIN might be related to the insufficient ROS regulation of caffeic acid. The above results indicated that the MDR reversal effects of caffeic acid might be cell line-dependent and warrant further detailed investigation.
The present study provided in-depth and comprehensive researches on the relationship between caffeic acid and human P-gp, and demonstrated the ability of caffeic acid on sensitizing MDR cancer cells toward chemotherapeutic drugs treatment. In order for caffeic acid to find a role in clinical application, some attempts could be applied to this phenolic prototype agent, including structural modification and pharmaceutical design.
4. Materials and Methods
4.1. Chemicals and Reagents
Acetic acid, β-Mercaptoethanol (β-ME), caffeic acid, dimethyl sulfoxide (DMSO), ethanol (Absolute; analytical grade), paclitaxel, rhodamine 123, sulforhodamine B (SRB), trichloroacetic acid (TCA), tris base, (±)-verapamil, and vincristine were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Calcein-AM was from AAT Bioquest (Sunnyvale, CA, USA), and doxorubicin was from US Biological (Woburn, MA, USA). Dulbecco’s Modified Eagle Medium, RPMI 1640 medium, fetal bovine serum (FBS), phosphate buffered saline (PBS; pH 7.2), Trypsin-EDTA, and hygromycin B were purchased from Invitrogen (Carlsbad, CA, USA). Zeocin was from InvivoGen (San Diego, CA, USA).
4.2. Cell Lines
Human cervical epithelioid carcinoma HeLaS3 was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan), and the multi-drug resistant human cervical cancer cell line KB/VIN was kindly provided by Dr. Kuo-Hsiung Lee (University of North Carolina, Chapel Hill, NC, USA) and maintained with vincristine regularly. The human P-gp stable expression cells (
ABCB1/Flp-In
TM-293) and parental cell line Flp-In
TM-293 were constructed as previously described [
44]. All cells were cultured in DMEM or RPMI-1640 containing 10% FBS at 37 °C in a humidified atmosphere of 5% CO
2.
4.3. Cytotoxicity Determination Assay (SRB Assay)
The method has been described in our previous research [
45]. Briefly, after 72 h of treatment of a series of concentrations of chemotherapeutic drugs with or without caffeic acid, 50% trichloroacetic acid (TCA) was added to fix cells for 30 min, and then the cells were washed with water and air-dried. After that, cells were stained with 0.04% sulforhodamine B (SRB) for 30 min, and then the unbound dye was removed by washing cells with 1% acetic acid and air-dried. The bound stain was solubilized in 10 mM Tris Base and the absorbance was measured using a BioTek Synergy HT Multi-Mode Microplate Reader (Winooski, VT, USA) at 515 nm.
4.4. MDR1 Shift Assay
The method has been described in our previous research [
46]. The conformation change of P-gp after the addition of caffeic acid was examined by using a MDR1 Shift Assay kit (EMD Millipore Corp., Billerica, MA, USA) according to the manufacturer’s protocol. UIC2 shift was shown in the presence of a P-gp substrate such as vinblastine. A total of 5 × 10
5–1 × 10
6 cells were prepared per reaction and resuspended with warm UIC2 binding buffer. Cells were incubated at 37 °C for 10 min and then treated with DMSO or vinblastine or test compounds. Cells were incubated at 37 °C for 30 min and then treated with IgG2a (negative control antibody) or UIC2 working solution (P-gp conformational sensitive antibody). Cells were incubated at 37 °C for 15 min and then washed with iced UIC2 binding buffer twice. A secondary antibody, goat anti-mouse IgG ALEXA 488, was added at 4 °C for 15 min, and then iced UIC2 binding buffer was added. The fluorescence was measured by FACS analysis (BD FACSCanto
TM II System, South City-I, Haryana, India).
4.5. Intracellular Calcein Accumulation Assay
The method has been described in our previous research [
46]. For the screening of an inhibitory effect on human P-gp efflux function, intracellular calcein accumulation assay was performed. Briefly, 1 × 10
5 cells/well were seeded in 96-well black plates for 24 h. Before the assay, cells were washed and pre-incubated with warm Hanks’ balanced salt solution (HBSS) for 30 min and subsequently with caffeic acid for 30 min. After pre-incubation and three times washing with PBS, the calcein-AM was added (substrate of P-gp), and the calcein fluorescence generated within the cells was detected by BioTek Synergy HT Multi-Mode Microplate Reader using an excitation wavelength of 485 nm and emission wavelength of 528 nm at 37 °C temperature every 3 min for 30 min. Each experiment was performed at least three times, each in triplicate on different days.
4.6. Rhodamine123 and Doxorubicin Efflux Assay
The method has been described in our previous research [
46]. 1 × 10
5 cells/well were placed on 96-well plates and incubated overnight. Before the efflux assay, cells were washed and pre-incubated with warm HBSS for 30 min, and subsequently with caffeic acid for 30 min. Then, the cells were treated with rhodamine123 for 30 min or doxorubicin for 3 h at 37 °C. After being washed with warm PBS, cells were allowed to efflux fluorescent rhodamin123 and doxorubicin for 10 min and 2 h, respectively. Supernatant samples (100 μL) were transferred to 96-well black plates. The fluorescence of rhodamine123 and doxorubicin was measured using a BioTek Synergy HT Multi-Mode Microplate Reader (excitation/emission: 485/528 nm for rhodamine123, 485/590 nm for doxorubicin). Each experiment was performed at least three times, each in triplicate on different days. Kinetic parameters were estimated by nonlinear regression using Scientist v2.01 (MicroMath Scientific Software, Salt Lake City, UT, USA) according to the following equation:
where V denoted the efflux rate; V
max, the maximal efflux rate; K
m, the Michaelis-Menten constant; and C is the substrate concentration.
4.7. P-gp ATPase Activity Assay
The method has been described in our previous research [
46]. For the evaluation of P-gp ATPase activity of caffeic acid, Pgp-Glo
TM Assay System from Promega (Madison, WI, USA) was used. In a 96-well untreated white plate, 25 μg of recombinant human P-gp membranes were incubated with Pgp-Glo
TM Assay Buffer (untreated control), 200 μM verapamil (positive control for drug induced P-gp ATPase activity), 100 μM sodium orthovanadate (selective inhibitor for P-gp ATPase activity), or a series of concentrations of caffeic acid. The reaction was initiated by adding 5 mM MgATP and incubated for 40 min at 37 °C, followed by stopping the reaction with 50 μL ATPase Detection Reagent for 20 min at room temperature. Luminescence was measured using a BioTek Synergy HT Multi-Mode Microplate Reader, and data were presented as Change in Luminescence (ΔRLU).
4.8. Real-Time Quantitative RT-PCR
The method has been described in our previous research [
46].
ABCB1 mRNA expression levels were quantified by real-time RT-PCR. Total RNA was extracted from HeLaS3, KB/VIN, Flp-In
TM-293, and
ABCB1/Flp-In
TM-293 cells using Qiagen RNeasy kit (Valencia, CA, USA). Taqman Assay-On-Demand
TM reagents of primers and probes for
ABCB1 (Hs00184500_m1) and
GAPDH (Hs02758991_g1) genes were provided by Applied Biosystem (Foster City, CA, USA). The relative
ABCB1 mRNA expression levels were normalized to the amount of
GAPDH in the same cDNA and evaluated by StepOnePlus
TM Real-Time PCR System (Applied Biosystems
®).
4.9. Intracellular Total ROS Activity Assay
The influence of caffeic acid on intracellular reactive oxygen species (ROS) was evaluated with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit (Catalog number: 22900) purchased from AAT Bioquest (Sunnyvale, CA, USA). Briefly, 4 × 104 cells/well were seeded in 96-well black plates for 24 h. Then the cells were stained with AmpliteTM ROS Green working solution for 1 h; after that, caffeic acid with or without chemotherapeutic drugs were added to induce ROS production at room temperature for at least 15 min. The fluorescence was measured using a BioTek Synergy HT Multi-Mode Microplate Reader at 490/525 nm (same as FITC filter).
4.10. Cell Cycle Analysis
The method has been described in our previous research [
45]. Cells were plated to 6-well plates with serum-free medium for starvation. Twenty-four hours later, cells were treated with chemotherapeutic drugs with or without caffeic acid for 72 h. After that, cells were harvested and washed in cold phosphate-buffered saline (PBS), followed by fixing in ice-cold 70% ethanol for at least 24 h. Then, cells were incubated with 50 μg/mL PI at 4 °C for 24 h in the dark. Cells were then analyzed by FACS analysis (BD FACSCanto
TM II System with excitation laser 488 nm, measuring at emission 575 nm for PI).
4.11. Molecular Docking
Molecular docking helps us in predicting the intermolecular framework formed between a protein and a small molecule and suggests the binding modes responsible for inhibition of the protein. In this study, the existing structure of P-gp (PDB entry 6QEX) was used as a template for docking caffeic acid (PubChem CID: 689043) putative ligands using Discovery Studio 4.5. After removing all crystallized H
2O molecules from the former construction, hydrogen was added into the CDOCKER module. CDOCKER is a powerful CHARMm-based docking method that has been used to generate highly accurate docked poses. In this refinement application, the ligands were conceded to tilt around the rigid receptor [
47].
4.12. Statistical Analysis
Statistical differences were evaluated by ANOVA followed by post hoc analysis (Tukey’s test) or Student’s t-test. The statistical significance was set at p value < 0.05.