Human ATP-Binding Cassette transporters ABCB1 and ABCG2 confer resistance to CUDC-101, a multi-acting inhibitor of histone deacetylase,
epidermal growth factor receptor and human epidermal growth factor receptor 2
Chung-Pu Wu a,b,c,*, Sung-Han Hsiao b, Ching-Ya Su b, Shi-Yu Luo b, Yan-Qing Li a, Yang-Hui Huang c, Chia-Hung Hsieh d,e and Chiun-Wei Huang f
Authors' Affiliations:
a Department of Physiology and Pharmacology, b Graduate Institute of Biomedical
Sciences, and c Molecular Medicine Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. d Graduate Institute of Basic Medical Science,
and e Department of Medical Research, China Medical University Hospital, Taichung, Taiwan. f Center for Advanced Molecular Imaging and Translation,
Chang Gung Memorial Hospital, Tao-Yuan, Taiwan.
* Corresponding author at: 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan. Phone: 886-3-2118800, ext. 3754. Fax: 886-3-2118700.
Keywords: Multidrug resistance; EGFR; HDAC; HER2; CUDC-101.
Running Title: ABCB1 and ABCG2 confer resistance to CUDC-101.
Abbreviations: MDR, multidrug resistance; ABC, ATP-binding cassette; HDAC,
histone deacetylase; EGFR, epidermal growth factor receptor, HER2, human epidermal growth factor receptor 2.
ABSTRACT
CUDC-101 is the first small-molecule inhibitor designed to simultaneously inhibit epidermal growth factor receptor (EGFR), human epidermal growth factor
receptor 2 (HER2) and histone deacetylase (HDAC) in cancer cells. Recently, in its first in human phase I study, CUDC-101 showed promising single agent activity
against advanced solid tumors and favorable pharmacodynamic profile. However, the risk of developing drug resistance to CUDC-101 can still present a significant
therapeutic challenge to clinicians in the future.One of the most common
mechanisms of developing multidrug resistance (MDR) in cancer is associated with the overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and
ABCG2. Together, they are able to reduce the efficacy and modify the
pharmacological properties of anti-cancer agents, including many small molecule tyrosine kinase inhibitors (TKIs). Here, we have investigated the impact of ABCB1 and ABCG2 on the efficacy of CUDC-101 in human cancer cells. We revealed that although CUDC-101 has potent antiproliferative and proapoptotic activities against
most cancer cell lines, the overexpression of ABCB1 or ABCG2 in cancer cells significantly reduced the activity of CUDC-101 against HDAC, EGFR and HER2, as well as its cytotoxicity and proapoptotic activity. Moreover, we showed that
CUDC-101 modulated the function of both transporters without affecting the protein expression of either ABCB1 or ABCG2. More importantly, our study provide support for the rationale of combining CUDC-101 with modulators of ABC drug transporters to improve drug efficacy and overcome multidrug resistance associated with the overexpression of ABCB1 and ABCG2.
1. Introduction
Conventional cancer chemotherapy involves treating cancer patients with cytotoxic agents. However, toxicity of these agents can cause unwanted side effects
and cancer treatment-related disease later. In contrast, targeted cancer therapy involves the use of selective agents that are designed to target and inhibit specific
molecular signaling pathway(s). This new approach appears to be more effective than conventional chemotherapy and less toxic to patients. Inhibition of key
mediators of cancer progression such as the epidermal growth factor receptors has been shown to be effective in the treatment of solid tumors and leukemia until the development of drug resistance. For instance, the initial use of imatinib to combat chronic myeloid leukemia (CML) was extremely successful until the discovery of
resistance to imatinib in patients, which subsequently prompted the development of second generation of tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib .
Nowadays, in addition to developing new TKIs, combination therapies using TKIs and other therapeutic agents have also been investigated.
Inhibition of histone deacetylases (HDACs) with inhibitors such as SAHA (vorinostat) has been shown to cause cell cycle arrest and apoptosis, and has been approved for the treatment of cutaneous T-cell lymphoma (CTCL) . Studies showed
that inhibitors of HDAC and numerous TKIs can act synergistically to promote apoptosis and inhibit proliferation of cancer cells . In order to improve the response rate and bypass the development of acquired drug resistance in single
agent-targeting therapy, and to reduce the potential toxic effect in combination multi-targeting therapy, which have been frequently observed in TKI-based therapies, a novel small molecule CUDC-101 was rationally designed and synthesized . By
simultaneously targeting HDAC, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in cancer cells, CUDC-101
showed potent antiproliferative and proapoptotic activities against multiple drug sensitive and drug resistant cancer cell lines . Moreover, cancer cells that are
resistant to single-target TKIs (such as lapatinib and erlotinib) remained sensitive to CUDC-101, emphasizing the potential therapeutic advantages of multi-targeting agents over single-target agents . Very recently, by 1-hour intravenous (i.v.) infusion administration, CUDC-101 showed promising single agent activity against advanced
solid tumors and favorable pharmacodynamic profile in its first in human phase I study .
The overexpression of ATP-binding cassette (ABC) drug transporters is one of the most common mechanisms for the development of multidrug resistance (MDR),
and has been identified as a major obstacle in cancer chemotherapy . These drug transporters recognize wide varieties of therapeutic agents and actively efflux drug substrates out of cancer cells, causing MDR and cancer relapse . Human ABCB1
(also known as P-glycoprotein, Pgp) is a 170kDa cell membrane
phosphor-glycoprotein. It is the first member of the mammalian ABC protein family identified to transport drug substrates across cell membranes . By hydrolyzing ATP, the ATP-binding domains of ABCB1 provide energy to efflux drug substrates, including a large number of conventional anticancer agents, across cell membranes. In cancer cells, ABCB1 is known to confer resistance to conventional drugs such as etoposide,
taxanes, camptothecins, methotrexate, colchicines, Vinca alkaloids and anthracyclines . Alike ABCB1, human ABCG2 (also known as breast cancer
resistance protein, BCRP) is also a mammalian ABC protein . In cancer cells, the functional dimerized ABCG2 can hydrolyze ATP to actively transport and confer
resistance to many anticancer agents, including etoposide, methotrexate, topotecan, SN-38, flavopiridol and mitoxantrone . Moreover, both ABCB1 and ABCG2 are
expressed at barrier sites, such as the intestinal walls and endothelial cells at the blood-brain barrier (BBB) sites in normal brain tissue and primary brain tumors.
Collectively, they reduce the oral bioavailability and distribution of
addition to conventional chemotherapeutic agents, ABCB1 and ABCG2 have been shown to reduce the effectiveness of TKIs via active efflux , and have a negative impact on the penetration, distribution and efficacy of many TKIs . Knowing that CUDC-101 was designed based on the chemical structures of TKIs , it becomes important to determine the impact of ABCB1 and ABCG2 on the efficacy of CUDC-101 in cancer cells.
In the present study, we showed that the inhibitory activity of CUDC-101 on HDAC, EGFR and HER2 was greatly reduced by the function of ABCB1 and ABCG2. Moreover, we discovered that cells overexpressing ABCB1 or ABCG2
were significantly more resistant to CUDC-101 treatment, which can be fully reversed by inhibiting the function of ABCB1 and ABCG2 transporters. Together, this work provides evidence that the overexpression of ABCB1 and ABCG2 in cancer cells can contribute, at least in part, to the development of acquired resistance
to CUDC-101, and that the combination of CUDC-101 with modulators of ABC drug transporters may provide therapeutic benefits to patients.
2. Materials and methods
2.1. Chemicals
DMEM, fetal calf serum (FCS), trypsin-EDTA, penicillin, streptomycin and PBS were purchased from Gibco, Invitrogen (CA, USA). Cell Counting Kit-8 (CCK-8), MTT dye, Ko143, SAHA, calcein-AM, mitoxantrone and doxorubicin were purchased from Sigma (St. Louis, MO, USA), unless stated otherwise. Annexin V : FITC Apoptosis Detection Kit was purchased from BD Pharmingen (San Diego, CA, USA). CUDC-101 (99% purity by HPLC, Chiral HPLC) was purchased from ChemieTek (Indianapolis, IN, USA). Tariquidar was a generous gift
from Dr. Susan Bates (National Cancer Institute, NIH, Bethesda, MD, USA).
2.2. Cell lines and culture conditions
Human epidermal KB-3-1 cancer cells and ABCB1-overexpressing sublines KB-8-5-11, KB-C-1 and KB-V-1, as well as mouse NIH3T3 and NIH3T3-G185 fibroblast cells, human ovarian OVCAR-8 and NCI-ADR-RES cancer cells were cultured in DMEM, supplemented with 10 % FCS, 2 mM L-glutamine and 100 units
of penicillin/streptomycin/mL. KB-8-5-11 and KB-C-1 cells were maintained in media containing 10 ng/mL and 1 µg/mL of colchicine, respectively, whereas KB-V-1 cells were maintained in media containing 1 mg/mL vinblastine . NIH3T3-G185
cells were maintained in the presence of 60 ng/mL colchicine . pcDNA3.1-HEK293, MDR19-HEK293 (HEK293 cells transfected with human ABCB1) and R482-HEK293 (R482-HEK293 cells transfected with wild-type R482 ABCG2) cells were cultured in DMEM, supplemented with 10 % FCS, 2 mM L-glutamine, 100 units of penicillin/streptomycin/mL and 2 mg/mL G418 . Cell lines were generous gifts from Dr. Suresh V. Ambudkar (National Cancer Institute, NIH, Bethesda, MD, USA), all
maintained at 37 °C in 5 % CO2 humidified air. Cells were placed in drug-free medium 7 days prior to assay.
2.3. Immunoblotting
Antibodies anti-acetyl-Histone H3, anti-acetylated tubulin, anti-EGFR, anti- phospho-EGFR, anti-HER2, anti-phospho-HER2, C219, BXP-21 and anti-α-tubulin were used to detect class I HDAC, class II HDAC, total EGFR, phosphorylated
EGFR, total HER2, phosphorylated HER2, ABCB1 and ABCG2, respectively, and tubulin as positive control for Western blotting. The secondary antibodies used were
the Horseradish peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG. Signals were detected as described previously. .
2.4. Cytotoxicity assay
tested compounds according to the method described by Ishiyama et al . Briefly, cells were plated into 96-well plates at a density of 5,000 cells per well in 100 µL of culture medium at 37 °C for 24 h before adding drugs to make a final volume of 200 µL. Cells were incubated for an additional 72 h before development. When CCK-8 reagent was added into each well, the water-soluble tetrazolium WST-8 salt in
CCK-8 can be reduced by dehydrogenase activities in viable cells to give yellow color formazan dye. Therefore, the formation of formazan dye is directly proportional to
the number of viable cells. For the reversal of cytotoxicity assays, a nontoxic concentration of CUDC-101 or inhibitor of ABCB1 or ABCG2 was added into the cytotoxicity assay, and the extent of reversal was then calculated based on the relative resistance values.
2.5. Apoptosis assay
For the determination of apoptotic cells, 1 x 106 cells were treated with indicated regimens for 48 hours before harvested by a series of washing,
centrifugation, and resuspended in FACS buffer containing 1.25 µg/mL annexin V– FITC (PharMingen) and 0.1 mg/mL PI and incubated for 15 min at room
temperature. The labeled cells were then analyzed by FACScan (BD Biosciences) using the CellQuest software (Becton-Dickinson). Cells in the lower right dot-plot
quadrant (PS-positive and PI-negative) were counted as apoptotic and have intact plasma membranes, whereas cells in the upper right dot-plot quadrant (PS-positive and PI-positive) have leaky membranes and can be either necrotic or late apoptotic .
2.6. Fluorescent drug accumulation assay
ABCB1 and ABCG2-mediated efflux assays were carried out using a FACSort flow cytometer equipped with CellQuest software. Briefly, cells were harvested after
trypsinization by centrifugation at 500 x g and then resuspended in Iscove's modified Dulbecco's medium supplemented with 5 % FCS. Calcein-AM (0.25
µmol/L) or mitoxantrone (5 µmol/L) was added to 3 x 105 cells in 4 mL of Iscove's modified Dulbecco's medium in the presence or absence of tested drugs. The effect
of CUDC-101, tariquidar or Ko 143 on ABCB1-mediated calcein-AM efflux or ABCG2-mediated efflux of mitoxantrone, was measured and analyzed according to
the method described by Gribar et al .
2.7. Statistical analysis
GraphPad Prism software (La Jolla, CA, USA ) was used to plot the curves and statistical analysis. Data are presented as mean ± S.E.M, whereas IC50 values were calculated as mean ± SD from at least three independent experiments. Differences between any mean values were analyzed by two-sided Student’s t-test and results
were considered statistically significant at P < 0.05.
3. Results
3.1. CUDC-101 inhibits EGFR, HER2, and HDAC in drug sensitive cancer cells, but not in ABCB1- or ABCG2-overexpressing MDR cancer cells
Given that CUDC-101 is a multi-targeting inhibitor originally designed to block EGFR, HER2 signaling pathways and the activity of HDAC , we first
evaluated the effect of CUDC-101 on EGFR and HER2 phosphorylation, as well as acetylation of histone H3 and H4 in drug sensitive and drug resistant cancer cell
lines. Human epidermal KB-3-1 and colon S1 cancer cells, and their ABCB1-overexpressing KB-V-1 and ABCG2-ABCB1-overexpressing S1-M1-80 MDR sublines were used in this experiment. As expected, we found that the phosphorylation and total
protein levels of receptor tyrosine kinases (RTKs) EGFR and HER2 were inhibited by CUDC-101 in parental KB-3-1 and S1 cancer cells (Fig. 1A and B, left panels)
after exposing cells to 1 μM of CUDC-101 for 24 hours. However, we were surprised to discover that CUDC-101 had limited effect on EGFR and HER2
phosphorylation in MDR KB-V-1 cells or S1-M1-80 cells (Fig. 1A and B, right
panels). Gefitinib, an inhibitor of EGFR and a known drug substrate of ABCB1 and
or S1-M1-80 cells. On the other hand, we found that in the presence of ABCB1 reference inhibitor tariquidar (1 μM) or ABCG2 reference inhibitor Ko143 (1 μM),
CUDC-101 and gefitinib were able to inhibit EGFR and HER2 phosphorylation in drug resistant KB-V-1 and S1-M1-80 cells to the same extent as in drug sensitive KB-3-1 and S1 cancer cells (Fig. 1A and B). Moreover, treatment with EGF, TKI
alone or in combination with reference inhibitor of ABCB1 or ABCG2 had no significant effect on ABCB1 or ABCG2 protein expression the protein in these cell
lines. Next, we determined the effect of CUDC-101 on histone acetylation in drug sensitive and drug resistant human cancer cell lines. The human KB-3-1, KB-V-1, S1 and S1-M1-80 cancer cells were maintained in the presence or absence of CUDC-101 (1 μM), SAHA (20 μM) and tariquidar (1 μM) or Ko 143 (1 μM) for 24 hours, harvested and processed for immunoblotting as indicated in Materials and
methods. We found that CUDC-101 significantly increased the acetylation of
histone H3 and H4 in KB-3-1 and S1 cells, but not in KB-V-1 or S1-M1-80 cells (Fig. 1C and D). In contrast, SAHA, a reference inhibitor of HDAC, was not affected by the function of ABCB1 or ABCG2. Moreover, in the presence of
tariquidar or Ko 143, the levels of histone acetylation in drug resistant sublines were restored to the same extent as drug sensitive parental cells. Collectively, our results
significantly reduced by the function of ABCB1 and ABCG2 in human cancer cells.
3.2. ABCB1- and ABCG2-overexpressing cells are resistant to CUDC-101
In order to examine the effect of ABCB1 or ABCG2 on the antiproliferative effect of CUDC-101, we determined the cytotoxicity of CUDC-101 in multiple cancer cell lines. As shown in Fig. 2A and 2B, it was apparent that the ABCB1-overexpressing human epidermal KB-V-1 and ovarian NCI-ADR-RES cancer cells
(closed circles) were resistant to CUDC-101 as compared with the ABCB1-negative parental cells (open circles). Similarly, the ABCG2-overexpressing S1-M1-80 (closed circles, Fig. 2C), human breast MCF-FLV1000 (closed circles, Fig. 2D) and
MCF7-AdVp3000 (closed squares, Fig. 2D) cancer cells were also resistant to CUDC-101 as compared with the parental cells (open circles). As shown in Table 1,
the resistance factor (RF) value was calculated by dividing the IC50 value of MDR subline by the IC50 value of the respective parental line, representing the degree of cellular resistance to CUDC-101 caused by ABCB1 or ABCG2 .
Our results showed that CUDC-101 was significantly less effective against all of the ABCB1 or ABCG2-overexpressing human cancer sublines tested, with RF values ranging from approximately 3 to 85 (Table 1). Next, to further confirm the role of ABCB1 and ABCG2 in mediating CUDC-101 resistance, we determined the
ABCG2. We discovered that ABCB1-transfected human embryonic kidney MDR19-HEK293 cells (closed circles, Fig. 2E) and ABCG2-transfected R482-HEK293 (closed squares, Fig. 2E) were both highly resistant to CUDC-101, with
calculated RF values of approximately 15 and 36, respectively. Similarly, we showed that the human ABCB1-transfected mouse NIH3T3-G185 fibroblast cells, were also resistant to CUDC-101 than the parental NIH3T3 cells (Table 1).
3.3. Inhibition of ABCB1 and ABCG2 function can resensitize ABCB1- and ABCG2-overexpressing cancer cells to CUDC-101
Knowing that ABCB1 and ABCG2 confer resistance to CUDC-101 in human cancer cells, we examined whether we can resensitize ABCB1- and
ABCG2-overexpressing cells to CUDC-101 by blocking the function of ABCB1 and ABCG2. First, we studied the effect of tariquidar and Ko 143 on CUDC-101
induced apoptosis in KB-3-1, KB-V-1, S1 and S1-M1-80 cancer cell lines. The basal level of apoptosis in both KB-3-1 and KB-V-1 cells was approximately 11 %. After
exposing cells to CUDC-101 (1 μM) for 48 hrs, we found the percentage of apoptotic KB-3-1 cells increased greatly, while less apoptotic KB-V-1 cells were
observed (Fig. 3A). Treatment with CUDC-101 resulted in approximately 55 % of early and late apoptosis/necrosis in KB-3-1 cells in contrast to 26 % in KB-V-1
cells. However, in the presence of tariquidar (1 μM), CUDC-101-induced apoptosis in resistant KB-V-1 cells increased to approximately 62 %, which is comparable to 61 % in KB-3-1 cells (Fig. 3B). Similarly, we found that treatment with CUDC-101 resulted in an increase of approximately 42 % of early and late apoptosis/necrosis in S1 cells in contrast to no effect in S1-M1-80 cells (Fig. 3C), whereas in the presence
of ABCG2 inhibitor Ko 143 (1 μM), CUDC-101-induced apoptosis increased significantly in resistant S1-M1-80 cells to approximately 43 %, which is
comparable to the level calculated in parental S1 cells (Fig. 3D). We next asked whether we can restore the chemosensitivity of ABCB1- or ABCG2-overexpressing cells to CUDC-101 by blocking the function of ABCB1 and ABCG2. Our results
show that 1 μM of tariquidar significantly reversed ABCB1-mediated resistance to CUDC-101 in KB-V-1 cancer cells and MDR19-HEK293 cells, whereas 1 μM of Ko 143 significantly reversed ABCG2-mediated resistance to CUDC-101 in
S1-M1-80, MCF7-FLV1000, MCF7-AdVp3000 cancer cells and R482-HEK293 cells (Table 2).
3.4. CUDC-101 antagonizes ABCB1- and ABCG2-mediated transport
In order to further evaluate the drug-drug interactions of CUDC-101 with drug substrates of ABCB1 and ABCG2, we examined the short-term effect of CUDC-101
on ABCB1 and ABCG2-mediated drug efflux and drug resistance in MDR19-HEK293 and R482-MDR19-HEK293 cells, as well as ABCB1 and ABCG2 protein
expression in KB and S1 cancer cell lines. First, we examined the effect of CUDC-101 on ABCB1-mediated efflux of calcein-AM and ABCG2-mediated efflux of mitoxantrone, known fluorescent substrates of ABCB1 and ABCG2 . These
experiments were performed in the absence or presence of increasing concentrations of CUDC-101, 3 μM of tariquidar or 5 μM of Ko 143, as indicated and described previously . Without having significant effects on parental pcDNA-HEK293 cells (Fig. 4A and C), CUDC-101 inhibited ABCB1-mediated calcein-AM transport from MDR19-HEK293 cells (Fig. 4B) and ABCG2-mediated mitoxantrone transport from
R482-HEK293 cells (Fig. 4D) in a concentration-dependent manner. Next, we evaluated the effect of CUDC-101 on ABCB1-mediated resistance to doxorubicin and ABCG2-mediated resistance to mitoxantrone in MDR19-HEK293 and R482-HEK293 cells. By competing with the transport of another drug substrate, some drug
substrates of ABCB1 or ABCG2 are capable of reversing drug resistance in cells overexpressing ABCB1 or ABCG2 . A maximum non-toxic concentration of 100
nM CUDC-101 was chosen based on the cytotoxicity curves (Fig. 2), and the reversal effect of CUDC-101 in HEK293 cells transfected with either ABCB1 or ABCG2 is summarized in Table 3. The relative resistance (RR) value was calculated
by dividing the IC50 value of HEK293 transfected with ABCB1 or ABCG2 by the IC50 value of the parental HEK293 cells in the presence or absence of a particular inhibitor. Tariquidar and Ko 143 were used as positive controls to reverse drug resistance conferred by ABCB1 or ABCG2, respectively. Our results show that at the concentrations tested, CUDC-101 was able to partially restore sensitivity of
ABCG2-overexpressing R482-HEK293 cells to mitoxantrone in a concentration-dependent manner, but had no significant effect on ABCB1-mediated resistance to doxorubicin (Table 3). Lastly, we examined the potential effect of CUDC-101 on the
protein expression of ABCB1 and ABCG2 in human cancer cells by treating KB-3-1, KB-V-KB-3-1, S1 and S1-M1-80 cancer cells with increasing concentrations of CUDC-101 for 72 hrs. Our results indicate that short-term exposure of these cancer cells to CUDC-101 had no significant effect on the protein expression level of either
ABCB1 (Fig. 5A and B) or ABCG2 (Fig. 5C and D).
4. Discussion
CUDC-101 is a novel, potent multi-targeted inhibitor designed to inhibit EGFR, HER2 and HDAC in human cancer cells. CUDC-101 also attenuates
compensatory pathways that are often utilized by cancer cells to escape conventional inhibition of EGFR/HER2 pathway. Overall, CUDC-101 showed potent
antiproliferative and proapoptotic activities against tumor cells, and demonstrated favorable pharmacodynamic profile in its first in human Phase I study .
Unfortunately, in addition to EGFR mutant and ALK rearranged tumors , the overexpression of ATP drug transporters ABCB1 and ABCG2 can often lead to acquired resistance to many TKIs and limit the bioavailability and CNS penetration
of these agents . Considering the fact that CUDC-101 was designed partially based on the structures of TKIs (such as erlotinib and lapatinib), we decided to examine
the potential impact of ABCB1 and ABCG2 on the effectiveness of CUDC-101 in human cancer cells.
As mentioned earlier, a key characteristic of CUDC-101 is the inhibition of cancer cell growth through inhibition of EGFR and HER2, as well as class I and class II HDACs. Therefore, we compared the activity of CUDC-101 in
drug-sensitive and drug-resistant cancer cell lines overexpressing ABCB1 or ABCG2. First, we observed that both CUDC-101 and gefitinib struggled to inhibit the
phosphorylation of EGFR and HER2 in ABCB1-overexpressing human KB-V-1 and ABCG2-overexpressing human S1-M1-80 cancer cells. Similarly, at tested
concentration, CUDC-101 was unable to inhibit the activity of HDACs in KB-V-1 or S1-M1-80 cells, suggesting the involvement of ABCB1 and ABCG2 in reducing
the activity of CUDC-101 in cancer cells. Our suspicion was further supported by the fact that the inhibitors of ABCB1 and ABCG2 were able to restore the activity of CUDC-101 in KB-V-1 and S1-M1-80 cells (Fig. 1).
To further confirm our findings, we examined the cytotoxicity of CUDC-101 in multiple cancer cell lines and MDR cancer cell lines overexpressing human ABCB1 or ABCG2. CUDC-101 was reported to be an effective drug against a broad range of human cancer cell lines with IC50 values ranging from 0.03 to 0.8 μM, but with an exception of human MNNG/HOS sarcoma cells (IC50 value of 1.81 μM) . In our
study, we confirmed that CUDC-101 is effective against human epidermal, ovary, breast and colon cancer cells, with IC50 values ranging from 0.13 to 0.82 μM, which
is comparable to previously reported values . In contrast, we found that the ABCB1 or ABCG2-overexpressing cancer sublines were significantly more resistant to
CUDC-101, with RF values ranging from 2.96 to 85.23 (Table 1). Interestingly, knowing that ABCB1 is highly expressed in MNNG/HOS cells, causing resistance to doxorubicin , we suspected that the higher IC50 value for CUDC-101 reported previously in MNNG/HOS cells was caused by the activity of ABCB1. It is worth noting that the human KB-8-5-11, KB-C-1 and KB-V-1 cell lines, three
ABCB1 protein expression levels, were all resistant to CUDC-101 with calculated RF values of approximately 3, 6 and 23, respectively (Table 1). This data suggest
that the degree of cellular resistance to CUDC-101 correlates with the amount of ABCB1 protein expression, at least amongst the KB sublines. Furthermore, we
found clear differences in CUDC-101 induced apoptosis in drug sensitive cancer cells and cancer cells overexpressing ABCB1 or ABCG2 (Fig. 3). The fact that we can restore the apoptotic response and cytotoxicity of ABCB1 and
ABCG2-overexpressing cells, including HEK293 cells transfected with human ABCB1 or ABCG2, to CUDC-101 by inhibiting the function of ABCB1 and ABCG2 (Table 2
and Fig. 4), indicated that ABCB1 and ABCG2 can confer significant resistance to CUDC-101 and potentially cause a therapeutic problem in the future.
Next, many ABC transporter-interacting compounds, including some clinical active drugs, are known to act as competitive inhibitors of these transporters , capable of reversing drug resistance mediated by ABCB1 or ABCG2, or reducing
the protein expression of ABCB1 and ABCG2 in cancer cells . Therefore, we examined the drug-drug interactions of CUDC-101with drug substrates of ABCB1 and ABCG2, and explored the possibility of using CUDC-101 to reverse drug resistance mediated by ABCB1 and ABCG2, or to down-regulate the protein
expression of ABCB1 and ABCG2 in cancer cells. Unfortunately, our data showed that although CUDC-101 can inhibit the function of ABCB1 and ABCG2, at non-toxic concentrations, the ability of CUDC-101 to reverse doxorubicin and
mitoxantrone resistance, or down-regulate the expression of these transporters in human cancer cells is somewhat limited. However, even though CUDC-101 did not
modulate the expression of ABCB1 or ABCG2 in cancer cells treated with 101 for a short period of 72 hours, the effect of prolong clinical usage of CUDC-101in patients remains to be seen.
In conclusion, although the clinical relevance of cell-based studies remains controversial , our study indicates that the overexpression of ABCB1 and ABCG2 in
human cancer cells reduces the activity of CUDC-101 to inhibit EGFR, HER2 and HDAC, which lead to acquired resistance to CUDC-101 (summarized in Fig. 6). Moreover, the activity and cellular sensitivity of CUDC-101 can be significantly restored by inhibiting the function of ABCB1 and ABCG2 in cancer cells.
Considering that ABCB1 and ABCG2 also play a significant role in the distribution and oral bioavailability of many therapeutic drugs , the overall effect of ABCB1 and ABCG2 on the pharmacology of CUDC-101 remains to be determined. We believe that further investigation is warranted for an effective therapeutic strategy to
overcome acquired resistance to CUDC-101.
Conflict of Interest None.
Acknowledgments
The authors thank Dr. Susan Bates and Dr. Suresh V. Ambudkar (National Cancer Institute, NIH) for generously providing tariquidar and cell lines. This work was supported by funds from the Chang Gung Medical Research Program
(CMRPD1D0151) and Ministry of Science and Technology of Taiwan (MOST- 103-2320-B-182 -011).
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Figure Legends
Fig. 1. CUDC-101 blocks EGFR and HER2 signaling, and HDAC activity in drug sensitive human cancer cells. (A) KB-3-1, KB-V-1 cells, (B) S1 and S1-M1-80 cells
were treated with DMSO (control), 1 µM of CUDC-101, 1 µM of gefitinib,1 µM of tariquidar or Ko143 for 24 h before processed for immunoblotting as detailed in
Materials and methods. Human EGF (50 ng/mL) was added (indicated as +EGF) to
the culture medium for 15 min to stimulate EGFR phosphorylation. (C) KB-3-1 and KB-V-1 cells, (D) S1 and S1-M1-80 cells were treated with DMSO (control), 1 µM of CUDC-101, 20 µM of SAHA, 1 µM of tariquidar or Ko143 as indicated for 24 h before Western analyses were performed as described in Materials and methods .
Representative Western blots of three independent experiments are shown. Values are presented as mean ± SEM calculated from more than three independent
experiments .*P < 0.05; **P < 0.01 ; ***P < 0.001, versus the same treatment in parental cells.
Fig. 2. The overexpression of human ABCB1 or ABCG2 reduces the cytotoxicity of CUDC-101 in human cancer cells. The cytotoxicity of CUDC-101 in (A) human epidermal cancer KB-3-1 cell line (○) and ABCB1-overexpressing KB-V-1 subline (●); (B) human ovarian cancer OVCAR-8 cell line (○) and ABCB1-overexpressing
NCI-ADR-RES subline (●); (C) human colon cancer S1 cell line (○) and ABCG2-overexpressing S1-M1-80 subline (●); (D) human breast cancer MCF7 cell line (○), ABCG2-overexpressing MCF7-FLV1000 (●) and MCF7-AdVp3000 sublines (■); as well as (E) human pcDNA-HEK293 cells (○) and ABCB1-transfected
MDR19-HEK293 (●) and ABCG2-transfected R482-MDR19-HEK293 (■) cells, was determined over a period of 72 h as described previously . Points, mean from at least three
independent experiments; bars, SEM.
Fig. 3. CUDC-101 induces apoptosis in human cancer cell lines. (A and B) KB-3-1, KB-V-1, (C and D) S1 and S1-M1-80 cells were treated with either DMSO, 1 µM of 101, 1 µM of tariquidar, 1 µM of Ko 143 or in combination of 1 µM CUDC-101 and 1 µM of tariquidar or 1 µM CUDC-CUDC-101 and 1 µM of Ko 143 as indicated
for 48 h before cells were isolated and analyzed. Apoptotic cells were quantified by flow cytometry after staining with annexin V-FITC and PI. Live cells (annexin V
-PI-) appear in the lower left quadrant; early apoptotic cells (annexinV+PI-) appear in the lower right quadrant; necrotic or late apoptotic cells (annexin V+PI+) appear in
the upper right quadrant. Histograms shown are representative of three independent experiments, whereas quantified values are means from at least three independent experiments; bars, SEM. *P < 0.05; **P < 0.01 ; ***P < 0.001, versus no treatment
control value.
Fig. 4. CUDC-101 inhibits ABCB1-mediated efflux of calcein-AM and ABCG2-mediated efflux of mitoxantrone. The accumulation of fluorescent calcein in (A) parental pcDNA-HEK293 and (B) ABCB1-overexpressing MDR19-HEK293 cells; or fluorescent mitoxantrone in (C) parental pcDNA-HEK293 and (D) ABCG2-overexpressing R482-HEK293 cells, was measured in the absence or presence of increasing concentrations of CUDC-101 or 3 μM of tariquidar, a reference inhibitor
of ABCB1 (A and B), or 5 μM of Ko 143, a reference inhibitor of ABCG2 (C and D), and analyzed immediately by flow cytometry as described in Materials and
methods. Representative histograms of three independent experiments are shown.
Fig. 5. Short-term exposure of human MDR cancer cells to CUDC-101 has no significant effect on the protein expression of ABCB1 or ABCG2. Immunoblot detection and quantification of ABCB1 in total cell lysate protein (10 μg) in KB-3-1 and KB-V-1 cells (A and B) or ABCG2 in S1 and S1-M1-80 cells (C and D) treated
with increasing concentrations of CUDC-101, respectively for 72 h, as described previously . α-Tubulin was used as an internal control for equal loading. Values are presented as mean ± SD calculated from three independent experiments.
Fig. 6. Schematic diagram showing the potential impact of ABCB1 and ABCG2 transporters on the effectiveness of CUDC-101 in cancer cells overexpressing ABCB1 or ABCG2. Ideally, CUDC-101 inhibits histone deacetylase (HDAC),
epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in cancer cells. The action of ABCB1 or ABCG2 can confer significant resistance to 101, resulting in reduced effectiveness of CUDC-101.
