The Multidrug Resistance-Reversing Activity of a Novel Antimicrobial Peptide

The overexpression of ATP-binding cassette (ABC) transporters is a common cause of multidrug resistance (MDR) in cancers. The intracellular drug concentration of cancer cells can be decreased relative to their normal cell counterparts due to increased expression of ABC transporters acting as efflux pumps of anticancer drugs. Over the past decades, antimicrobial peptides have been investigated as a new generation of anticancer drugs and some of them were reported to have interactions with ABC transporters. In this article, we investigated several novel antimicrobial peptides to see if they could sensitize ABCB1-overexpressing cells to the anticancer drugs paclitaxel and doxorubicin, which are transported by ABCB1. It was found that peptide XH-14C increased the intracellular accumulation of ABCB1 substrate paclitaxel, which demonstrated that XH-14C could reverse ABCB1-mediated MDR. Furthermore, XH-14C could stimulate the ATPase activity of ABCB1 and the molecular dynamic simulation revealed a stable binding pose of XH-14C-ABCB1 complex. There was no change on the expression level or the location of ABCB1 transporter with the treatment of XH-14C. Our results suggest that XH-14C in combination with conventional anticancer agents could be used as a novel strategy for cancer treatment.


Introduction
The incidence of cancer has increased as a result of growing population, aging, and other risk factors [1,2]. After decades of investigation and development, chemotherapies started to reduce the cancer incidence and cancer death rate. Despite that, the bright future of chemotherapies were gradually shadowed because of endogenous multidrug resistance (MDR) or MDR acquisition due to chemotherapies [3], leading to refractory cancers and tumor recurrences, which ultimately contribute to increasing cancer-related deaths. As MDR refers to the resistance of cancer cells to various different structural and functional chemotherapeutic drugs [4], it is a major factor of failure of cancer chemotherapy [5]. The mechanisms of MDR could be classified into several categories, with the major one being the enhancement of drug efflux by transporters on cancer cell membranes [6].
The ATP-binding cassette (ABC) transporter family is a protein superfamily with 49 different members categorized by gene sequence and structural similarities [3]. They are the main cell membrane transporters and are organized into seven subfamilies [7]. Many human ABC proteins, including are zwitterionic [30,36].In addition to binding to the cell membrane which results in cell death, antimicrobial peptides can also block the interaction between growth factors and their receptors, activating anti-angiogenic effects; inhibiting specific kinase/protease which promote the growth, invasion, and metastasis of tumor; and inhibiting special functional proteins to halt the progression of cancer [34,35]. These different mechanisms of cancer cell death may overcome the limitations of traditional chemotherapeutic agents.
We have designed three antimicrobial peptides XH-14A (FIKRIARLLRKIKR), XH-14B (FIKRIARLLRKIFR), and XH-14C (FIKRIARLLRKIWR) based on the template of BmKn2 (FIGAIARLLSKIF), an antibacterial peptide with 13 amino acids identified from ButhusmartensiiKasch [35]. These peptides displayed potency against both Gram-negative and Gram-positive bacteria. The replacements of one or more amino acid residues by other amino acid residues were involved in the design and modification of the antimicrobial peptides. As some antimicrobial peptides have anticancer activities and do not lead to drug resistance in cancer cells, the anticancer activity of these peptides was tested. The possible reversal effect of these novel peptides and whether the reversal activity is associated with the overexpression of ABCB1 transporter was the focus of this project. Our findings suggested that peptide XH-14C has potential ABCB1-overexpressing MDR reversing activity which would allow further design or modification of these peptides.
Cancers 2020, 12, x 3 of 15 preferential binding to cancer cells but not normal cells are zwitterionic [30,36].In addition to binding to the cell membrane which results in cell death, antimicrobial peptides can also block the interaction between growth factors and their receptors, activating anti-angiogenic effects; inhibiting specific kinase/protease which promote the growth, invasion, and metastasis of tumor; and inhibiting special functional proteins to halt the progression of cancer [34,35]. These different mechanisms of cancer cell death may overcome the limitations of traditional chemotherapeutic agents.
We have designed three antimicrobial peptides XH-14A (FIKRIARLLRKIKR), XH-14B (FIKRIARLLRKIFR), and XH-14C (FIKRIARLLRKIWR) based on the template of BmKn2 (FIGAIARLLSKIF), an antibacterial peptide with 13 amino acids identified from ButhusmartensiiKasch [35]. These peptides displayed potency against both Gram-negative and Gram-positive bacteria. The replacements of one or more amino acid residues by other amino acid residues were involved in the design and modification of the antimicrobial peptides. As some antimicrobial peptides have anticancer activities and do not lead to drug resistance in cancer cells, the anticancer activity of these peptides was tested. The possible reversal effect of these novel peptides and whether the reversal activity is associated with the overexpression of ABCB1 transporter was the focus of this project. Our findings suggested that peptide XH-14C has potential ABCB1-overexpressing MDR reversing activity which would allow further design or modification of these peptides.

Cytotoxicity of ABC Substrates in Combination with The Peptide
To test the reversal effect of these three peptides in ABC transporter-mediated MDR, drug-selected MDR human cancer cell lines, KB-C2 and NCI-H460/MX20, and their parental cell lines, KB-3-1 and NCI-H460, were used to examine the ABCB1 substrates paclitaxel and doxorubicin and ABCG2 substrates mitoxantrone and topotecan. The drug resistance profile in Table 1 and Table   Figure 1. Dose-response curve of A) XH-14A, B) XH-14B and C) XH-14C, on drug-selected ABCB1-overexpressing cell lines (KB-C2) and its parental drug-sensitive cell line (KB-3-1) with gradient concentrations. Each point with error bar represents the mean ± SD of the cytotoxicity with different concentrations calculated from at least three independent triplicate experiments.

Cytotoxicity of ABC Substrates in Combination with the Peptide
To test the reversal effect of these three peptides in ABC transporter-mediated MDR, drug-selected MDR human cancer cell lines, KB-C2 and NCI-H460/MX20, and their parental cell lines, KB-3-1 and NCI-H460, were used to examine the ABCB1 substrates paclitaxel and doxorubicin and ABCG2 substrates mitoxantrone and topotecan. The drug resistance profile in Table 1 and Table S1 demonstrated that only peptide XH-14C significantly potentiated the cytotoxicity of paclitaxel and doxorubicin in ABCB1-overexpressing KB-C2, but none of the three peptides demonstrated reversal effects on ABCG2-overexpressing NCI-H460/MX20 cells. As the MDR in drug-selected cancer cells might be multifactorial, the transfected ABCB1-overexpressing cell line (HEK293/ABCB1) was used to verify the actual role of ABCB1 and similar reversal effects were observed (Table 2). In addition, no significant change in the drug resistant profile of cisplatin, which is not a substrate of ABCB1 transporter, in both drug-selected and transfected ABCB1-overexpression cells was observed with or without the peptide. These results indicated that peptide XH-14C could reverse MDR of cancer cells mediated by ABCB1-overexpression but not ABCG2-overexpression.

XH-14C on ATPase Activity of ABCB1 and ABCG2 Transporters
As the ABC transporters hydrolyze ATP to facilitate the efflux of anticancer drugs, the possible mechanism behind the reversal effect of XH-14C was investigated by analyzing the ATPase activity of ABCB1 and ABCG2 in the presence of XH-14C at various concentrations. Figure 2 demonstrated that XH-14C stimulated the ATPase activity of ABCB1 but had almost no effect on the ATPase activity of ABCG2 ( Figure S2). A maximal stimulation of 3.28-fold on ABCB1 ATPase activity and a maximal stimulation of 1.03-fold on ABCG-2 ATPase activity were observed. In addition, in a concentration-dependent manner, a concentration of 0.05 µM, which is much lower than the reversal effect concentration in the reversal assay, was calculated as the essential concentration for half simulation of ABCB1 ATPase activity. These results suggested that XH-14C may reverse ABCB1-mediated MDR by affecting the ATPase activity through interaction with ABCB1 protein. and finally represented as mean ± SD with unit of nM. 2 RF, resistant fold, which was calculated using the IC50 in the transfected ABCB1-overexpressing cell line HEK293/ABCB1 divided by the IC50 in the transfected empty-vector cell line HEK293/pcDNA3.1. # , represents p﹤0.001, compared to the value of HEK293/ABCB1 control group.

XH-14C on ATPase Activity of ABCB1 and ABCG2 Transporters
As the ABC transporters hydrolyze ATP to facilitate the efflux of anticancer drugs, the possible mechanism behind the reversal effect of XH-14C was investigated by analyzing the ATPase activity of ABCB1 and ABCG2 in the presence of XH-14C at various concentrations. Figure 2 demonstrated that XH-14C stimulated the ATPase activity of ABCB1 but had almost no effect on the ATPase activity of ABCG2 (FigureS2).A maximal stimulation of 3.28-fold on ABCB1 ATPase activity and a maximal stimulation of 1.03-fold on ABCG-2 ATPase activity were observed. In addition, in a concentration-dependent manner, a concentration of 0.05 µM, which is much lower than the reversal effect concentration in the reversal assay, was calculated as the essential concentration for half simulation of ABCB1 ATPase activity. These results suggested that XH-14C may reverse ABCB1-mediated MDR by affecting the ATPase activity through interaction with ABCB1 protein.

Interaction of XH-14C with ABCB1
To study the potential interaction between XH-14C and ABCB1 binding pocket, a docking analysis was performed. Figure 3 demonstrated that XH-14C binds tightly in the ABCB1 binding pocket and stabilized by hydrogen bonds and hydrophobic interactions. Specifically, the lysine of XH-14C was stabilized by a hydrogen bond with Gln721 of ABCB1. Also, the arginine of XH-14C was stabilized by a hydrogen bond with Gly985 of ABCB1. These hydrogen bonds provide essential forces to the binding affinity of XH-14C and ABCB1. Additionally, XH-14C was also buried in tight

Interaction of XH-14C with ABCB1
To study the potential interaction between XH-14C and ABCB1 binding pocket, a docking analysis was performed. Figure 3 demonstrated that XH-14C binds tightly in the ABCB1 binding pocket and stabilized by hydrogen bonds and hydrophobic interactions. Specifically, the lysine of XH-14C was stabilized by a hydrogen bond with Gln721 of ABCB1. Also, the arginine of XH-14C was stabilized by a hydrogen bond with Gly985 of ABCB1. These hydrogen bonds provide essential forces to the binding affinity of XH-14C and ABCB1. Additionally, XH-14C was also buried in tight hydrophobic cavities formed by residues including Phe724, Leu335, Tyr306, Gly342, Phe339 and Leu232.

Effects of XH-14C on ABCB1 Transporter's Function
The ABCB1 transporter functions as the drug pump on the cancer cell membrane to efflux the anticancer drugs out of cancer cells, therefore bringing down the intracellular level of anticancer drugs. The effects of XH-14C on accumulation and efflux of ABCB1 substrate paclitaxel were determined by [ 3 H]-paclitaxel measurement in KB-3-1 and ABCB1-overexpressing KB-C2 cells. The intracellular [ 3 H]-paclitaxel with anticancer drug alone in KB-C2 cells was more than 100-fold lower than that in KB-3-1 cells after 2 h incubation with XH-14C ( Figure 4A). When compared with [ 3 H]-paclitaxel alone, the combination with 3 µM XH-14C could notably improve the intracellular accumulation of

Effects of XH-14C on ABCB1 Transporter's Function
The ABCB1 transporter functions as the drug pump on the cancer cell membrane to efflux the anticancer drugs out of cancer cells, therefore bringing down the intracellular level of anticancer drugs. The effects of XH-14C on accumulation and efflux of ABCB1 substrate paclitaxel were determined by [ 3 H]-paclitaxel measurement in KB-3-1 and ABCB1-overexpressing KB-C2 cells. The observed in Figure 4B demonstrated that the efflux function of ABCB1 on KB-C2 cells was dramatically inhibited with the combination of 3 µM XH-14C as the concentration of [ 3 H]-paclitaxel in KB-C2 cells was similar to that in KB-3-1 cells after 2 h, while the drug intracellular concentration of KB-C2 cells decreased by about 50% without XH-14C. The cellular concentration of [ 3 H]-paclitaxel did not significantly change after 2 h treatment either with or without a reversal agent in KB-3-1 cells.

Effect of XH-14C on the expression of ABCB1
Western blotting was used to analyze and quantify the protein expression level of ABCB1. To normalize and calculate the expression level, GAPDH protein (37 kDa) was used as a loading control and the relative expression level of ABCB1 was calculated as the measurement of ABCB1 expression divided by the measurement of GAPDH expression. As shown in Figure 5

Effect of XH-14C on the expression of ABCB1
Western blotting was used to analyze and quantify the protein expression level of ABCB1. To normalize and calculate the expression level, GAPDH protein (37 kDa) was used as a loading control and the relative expression level of ABCB1 was calculated as the measurement of ABCB1 expression divided by the measurement of GAPDH expression. As shown in Figure 5

Effects of XH-14C on the Subcellular Localization of ABCB1
Immunofluorescent staining was used to quantify the expression of ABCB1 and the subcellular localization of ABCB1 on cell membranes. Figure 6 indicates that ABCB1 transporter was

Effects of XH-14C on the Subcellular Localization of ABCB1
Immunofluorescent staining was used to quantify the expression of ABCB1 and the subcellular localization of ABCB1 on cell membranes. Figure 6 indicates that ABCB1 transporter was overexpressed on the cell membrane of KB-C2 cells but not on the membrane of KB-3-1 cells. With the same concentration of 3 µM XH-14C and different treatment time periods (24,48, and 72 h), no remarkable alternation of expression level or subcellular distribution of ABCB1were observed in KB-C2 cells.
expression after incubation with different concentrations of XH-14C for 72 h in KB-C2 cells. KB-3-1 cells without any treatment used as the negative control of ABCB1 protein expression. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as loading control.(B) The relative intensity of the expression of ABCB1 compared with the expression of GAPDH. Expression level quantification by gray scale values calculated by ImageJ software(NIH, Bethesda, MD, USA).

Effects of XH-14C on the Subcellular Localization of ABCB1
Immunofluorescent staining was used to quantify the expression of ABCB1 and the subcellular localization of ABCB1 on cell membranes. Figure 6 indicates that ABCB1 transporter was overexpressed on the cell membrane of KB-C2 cells but not on the membrane of KB-3-1 cells. With the same concentration of 3 µM XH-14C and different treatment time periods (24, 48, and 72 h), no remarkable alternation of expression level or subcellular distribution of ABCB1were observed in KB-C2 cells.

Discussion
The efflux of anticancer drugs constitutes the majority of MDR, which is a known molecular mechanism of chemotherapeutic failure. Therefore, development of novel reversal agents that could inhibit the efflux functions of ABC transporters is an urgent need to advance chemotherapy. ABCB1 is a typical transporter of the ABC protein superfamily. So far, there are a number of studies focused on the investigation of reversal agents for ABCB1 [37,38]. However, the potent reversal agents investigated in vitro did not show similar effectsin vivo; in addition, they had the problems of bioavailability, neurotoxicity, and synergetic effects.
Antimicrobial peptides are novel anticancer drugs with less possibility of causing drug resistance. Some primary differences between the cell membranes of human tumor and normal cells are the reasons for the selective higher cytotoxicity of antimicrobial peptides in cancer cells than normal cells, especially for the peptides with a positive charge [36]. The interaction between the antimicrobial peptides and the cell membranes may elevate the permeability of cancer cell membrane or even disrupt the plasma membrane. This greater diffusion promotes entry of anticancer drugs into the drug-resistant cancer cells [39]. Some studies have indicated that the antimicrobial peptides also have antimigratory effect, which could significantly prevent the migration of cancer cells [40]. The present study investigated the anticancer effect and the possible MDR reversal effects of some antimicrobial peptides.
First, the cytotoxicity assay on three antimicrobial peptides demonstrated that these peptides could kill drug-resistant cancer cells at a similar concentration compared with their IC 50 values against the parental cells. This result may be beneficial for the future use of cancer therapy to overcome MDR caused by overexpression of ABCB1 and ABCG2. This result indicated that the peptides could abort drug resistant effects mediated by ABCB1 and ABCG2, which suggests that they may have the potential to reverse ABCB1-and ABCG2-mediated MDR. Furthermore, the tests on combination with anticancer drugs that are ABCB1 or ABCG2 substrates, showed that peptide XH-14C potentiates the cytotoxicity of paclitaxel and doxorubicin in ABCB1-overexpressing cancer cells. To restrict the reversal factors to only the overexpression of ABCB1, ABCB1 gene-transfected cell line HEK293/ABCB1 was used for further confirmation. In contrast, XH-14C had no effect on the cytotoxicity of cisplatin, a non-substrate drug of ABCB1 transporter, in all tested cell lines, indicating that XH-14C only affects ABCB1 transporter. The cytotoxicity of XH-14C and the drug resistance profile of ABCB1 substrates collectively demonstrated that XH-14C could act as a potential inhibitor of ABCB1.
Interestingly, the results of ATPase assay showed that XH-14C stimulated the ATPase activity of ABCB1. As we focus on the reversal agent of ABC transporters, to further examine the possible reversal effect of XH-14C on ABCB1 overexpressing cells, the interplay between XH-14C with ABCB1 was simulated by molecular docking analysis. The simulation results demonstrated that XH-14C could bind tightly to the drug-binding pocket of ABCB1 with several hydrogen bonds, which implied that XH-14C may compete with ABCB1 substrates as it could bind to ABCB1 much more tightly than the ABCB1 substrates. Such binding reduced the amount of the anticancer drugs being pumped out by ABCB1 transporter. In previous studies, a large proportion of tyrosine kinase inhibitors (TKIs), a kind of inhibitor involved in ABC transportermediated MDR reversal, could stimulate ATP hydrolysis [41]. Some are competitive inhibitors, as they could bind to the membrane transporter much more tightly than the substrate, preventing the substrate from being transported out by these transporters. With this assumption, the cytotoxicity assay of XH-14C should display some differences between the IC 50 values of ABCB1-overexpressing cells to their parental cells as they might be substrates of ABCB1, which is conflicted with previous results that the cytotoxicity values of XH-14C were similar in ABCB1 overexpression cells compared to their corresponding parental cells. Therefore, XH-14C may not be an ABCB1 substrate but can stimulate ATPase activity by another mechanism. One of the third generation ABCB1 reversing TKI, tariquidar, is a non-competitive inhibitor able to stimulate ABCB1 ATPase activity. One possible assumption of this condition is that this kind of reversal agents could trap ABCB1 transporter in allosteric conformation which could stimulate the activity of ATPase while they are unable to be subjected to further conformational changes essential for drug outflow [42]. Although we did not have enough evidence to confirm this assumption, we speculate that XH-14C may actively stimulate the ATPase activity by capturing the ABCB1 transporter to a conformation that could not bind to ABCB1 substrates. The results of the docking analysis ( Figure S3) showed that XH-14C and paclitaxel were binding to the same drug-binding pocket but different specific positions and interactions with different residues of ABCB1. Although the mechanisms of anticancer and reversal effect of these peptides are still not elucidated, according to the results here, these peptides show no substrate properties of ABC transporters. We mentioned in the introduction before that one possible anti-cancer mechanism of antimicrobial peptides is after binding to the cell membrane, by electrostatic attraction, antimicrobial peptides can disrupt membrane integrity, leading to the leakage and depolarization of metabolites. Another interpretation is a non-membrane mechanism that antimicrobial peptides target cell apoptosis after being transported inside the cell via membrane perturbation or formation of pores [32,33].Based on previous studies that investigated the anticancer effect of AMPs, these peptide samples might act on the mitochondria and stimulate the release of cytochrome C [40], which should also be assessed by evaluating cell apoptosis and intracellular glutathione change induced by the peptides using flow cytometry in the future.
As the ABCB1 transporter functions as an anticancer drug pump to enhance the efflux of anticancer drugs, the reversal agent should basically inhibit the function of the ABCB1 transporter. Drug accumulation and efflux assays using [ 3 H]-paclitaxel, a substrate of ABCB1, showed that the intracellular level of [ 3 H]-paclitaxel in ABCB1-overexpressing KB-C2 cells was dramatically enhanced in the presence of XH-14C compared with the control group. Although XH-14C did not completely elevate the intracellular concentration of [ 3 H]-paclitaxel to a level of that in the parental cell line KB-3-1, its effects in augmenting drug accumulation were comparable to the positive reversal agent of ABCB1, verapamil.In this limited 4 h treatment with XH-14C, the results were consistent with the drug resistance profile of paclitaxel that the IC 50 value in KB-C2 decreased as a result of the increased intracellular concentration of paclitaxel. Not only the accumulation assay, but also the time course of efflux assay of [ 3 H]-paclitaxel verified that 3 µMof XH-14C could notably inhibit the efflux function of ABCB1 in KB-C2 cells, is more effective than verapamil, and almost similar to that in the parental KB-3-1 cells. One possible mechanism behind the block of the function of ABCB1 could be that XH-14C may inhibit the overexpression of ABCB1 in the MDR cells [3] so that the drug-resistant cells were re-sensitized to anticancer drugs. While the results of Western blotting showed that XH-14C did not downregulate the overexpressionof ABCB1 in KB-C2 cells with XH-14C treatment. Another possible reversal mechanism is that altering the localization of the ABCB1 transporter [3]. The results of immunofluorescence assay suggested that XH-14C did not affect the translocation of ABCB1 up to 72 h. There was no effect on either protein expression level or transporter localization even at higher concentration and longer time period. All the results above gave the conclusion that 3 µM XH-14C increased the accumulation of chemotherapeutic drugs by directly inhibiting ABCB1 transporter's efflux function.

Chemicals and Reagents
All peptides were synthesized by solid phase methods using Fmoc N-terminal protected amino acids as previously described [43]. Before the in vitro study, all peptides were purified to >95% by RP-HPLC and the quality was analyzed and evaluated by mass spectrometry (MS) analysis. The phosphate buffered saline (PBS), paclitaxel, cisplatin, and topotecan were purchased from Sigma Chemical Co (St. Louis, MO, USA). Mitoxantrone was a product from Enzo life Sciences (Farmingdale, NY, USA).

Cell Lines and Cell Culture
The drug-selected ABCG2-overexpressing non-small cell lung cancer (NSCLC) cell line NCI-H460/MX20 and its parental NCI-H460 cell line, and the ABCB1 gene-transfected HEK293 cells, HEK293/ABCB1, and its parental empty vector transfected HEK293 cells, HEK293/pcDNA3.1 were provided by Drs. Susan E. Bates and Robert Robey (NIH, MD). The drug-resistant ABCB1 overexpressing human epidermal carcinoma cell line KB-C2 and its parental KB-3-1 cell line were generously provided by Dr. Shin-Ichi Akiyama (Kagoshima University, Kagoshima, Japan). All cell lines were cultured in complete Dulbecco's modified Eagle's medium(DMEM) (Hyclone, GE Healthcare Life Sciences, Pittsburgh, PA, USA) with the addition of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Hyclone, GE Healthcare Life Sciences, Pittsburgh, PA, USA). The transfected cell lines were selected in complete medium with addition of 2 mg/mL of Geneticin (Enzo Life Sciences, Farmingdale, NY, USA) [44] for selection, KB-C2 cells were continually treated with complete medium and 2 mg/mL of colchicine [45], and NCI-H460/MX20 cells were cultured with 20 ng/mL mitoxantrone added to the complete medium. All drug-treated cell lines were recovered for more than 2 weeks without treatment before their use. The culture condition was in a humidified incubator at 37 • C with 5% CO 2 .

MTT Colorimetric Assay
Cytotoxicity of the peptides on different cell lines and the cytotoxicity of ABCB1 and ABCG2 substrates were measured and calculated from the results of MTT assay [46] as previously described. The final absorbance of each well was measured by Microplate Spectrophotometer (Fisher Sci., Fair Lawn, NJ, USA) at 570 nm. Verapamil (Sigma Chemical Co., St. Louis, MO, USA) or Ko143 (Enzo life Sciences, Farmingdale, NY, USA) was used as the positive reversal agent, respectively.

ATPase Assay
The vanadate-sensitive membrane vesicles ABCB1 and ABCG2 ATPase activity were determined as previously described [45,47], and the ABCB1-and ABCG2-overexpressing cell membranes were purchased from BD Biosciences (San Jose, CA, USA). The ATPase activities were calculated based on the measurement of inorganic phosphate (IP)detected using a spectrophotometer at 800 nm.

Molecular Docking Simulation
The initial conformations of peptide XH-14C were generated using online server PEP-FOLD3 [48]. PEP-FOLD3 predicts peptide structures via de novo approaches from amino acid sequencing based on structural alphabet SA letters [48]. The generated structures were then subjected to a 10 ns molecular dynamics equilibration using Desmond (DE Shaw Research Group, NY, USA) [49]. The simulation protocol was similar to that previously described, with modification [45]. In brief, the simulation system was first built in the Maestro Module System Builder. The NpT runs under 300 K and 1.015 bar pressure for 10 ns. The conformations of XH-14C were picked from time frames when the system reached dynamic equilibration. ABCB1 model (4M2T) was obtained from the Protein Data Bank (PDB). Peptide-protein docking was performed using rigid-body docking programs ZDOCK, which uses the Fast Fourier Transform based docking algorithms [50]. The best scoring pose was selected for further visualization and analysis. Ligand-protein interactions were analyzed using LigPlot+ [51].

Drug Accumulation and Efflux Assay
[ 3 H]-paclitaxel (31 Ci/mmol, Moravek Biochemicals, Brea, CA, USA) was used to measure the drug accumulation and efflux as previously described [46]. The only difference is that after the cells were seeded evenly in 24-well plate, the cells were incubated in the presence or absence of XH-14C and a parallel positive reversal agent for 72 h. Liquid scintillation cocktail used to measure the radioactivity was purchased from MP Biomedicals, Inc (St. Ana, CA, USA) and the radioactivity of different groups were read with the Packard TRI-CARB1 190'A liquid scintillation analyzer. A parallel well of cells for each group was set up for cell number counting at the end of the assay for the purpose of data normalization.

Western Blotting Analysis
To investigate whether XH-14C can alter the expression of ABCB1, Western blotting was performed as previously described [52] with the primary monoclonal anti-ABCB1 (MDR1) antibody produced in mouse (Sigma Chemical Co, St. Louis, MO, USA), GAPDH Mouse Anti-Human Clone: GA1R (Thermo Fisher Scientific Inc., Rockford, IL, USA), and the anti-mouse HRP-linked secondary antibody (Cell Signaling Technology, Danvers, MA, USA), all diluted to 1:1000. The developed protein expression level was measured and analyzed by ImageJ software (NIH, Bethesda, MD, USA).

Statistical Analysis
All the values were calculated and presented as mean ± SD. All experiments were performed at least three times independently. Statistical differences between multiple groups were calculated Cancers 2020, 12,1963 12 of 15 by one-way ANOVA followed by Dunnett's test and were considered significant when p value was below 0.05.

Conclusions
This present study reports for the first time that a novel antimicrobial peptide could re-sensitize ABCB1 overexpression-mediated MDR cancer cells by directly inhibiting the efflux function of ABCB1 transporter. Past potent reversal agents investigated in vitrohad the problems of bioavailability, neurotoxicity, and synergetic effects, the much larger molecular peptides and their specific binding to cancer cells may avoid these possible disadvantages. This study identified a promising therapeutic strategy and may provide a potential clinical direction on overcoming ABCB1-mediated MDR in cancers. In the future, the clinical therapeutic effect of XH-14C needs to be explored in in vivo studies with xenograft animal models and clinical trials. Also, pharmacokinetic studies are necessary to verify the bioavailability of this peptide. Further studies are warranted to confirm the structure specialty of these peptides and whether they could be contributed to helping patients suffering chemotherapy-induced MDR and improving clinical outcomes in patients receiving chemotherapy.