DOI 10.1007/s00280-016-2979-9 ORIGINAL ARTICLE Histone deacetylase 2 regulates doxorubicin (Dox) sensitivity of colorectal cancer cells by targeting ABCB1 transcription Pingjiang Ye 1 Haibo Xing 2 Fang Lou 3 Kaifeng Wang 3 Qin Pan 3 Xiaoyun Zhou 3 Liu Gong 3 Da Li 3 Received: 24 December 2015 / Accepted: 26 January 2016 Springer-Verlag Berlin Heidelberg 2016 Abstract Purpose Histone deacetylases (HDACs) have been shown to regulate cell cycle, differentiation, and apoptosis of colorectal cancer (CRC) cells, while their roles in drug sensitivity remain unclear. The objectives of the present study were to investigate the effects of HDAC2 on drug resistance of CRC cells. Methods We measured the expression of class I HDACs (HDAC1, 2, 3, 8) in CRC and human normal colonic epithelial cells. Additionally, we inhibited HDAC2 via sirna or overexpressed it via pcdna/hdac2 transfection to evaluate its roles in doxorubicin (Dox) sensitivity. Results Our present study showed HDAC2 was significantly increased in CRC cell lines as compared to human normal colonic epithelial cells. Silencing of HDAC2 can obviously enhance the sensitivity of HCT-116 and SW480 cells to ddox. Further, knockdown of HDAC2 can significantly (p < 0.05) downregulate the expression of ABCB1, while not ABCG2, ABCC1, ABCA1, or ABCC2. Inhibition of HDAC2 decreased ABCB1 promoter activities and the phosphorylation of c-fos and c-jun, which can directly interact with the ABCB1 promoter and then promote its * Da Li lidazju@163.com 1 Department of Colorectal Surgery, Shaoxing People s Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing 312000, China 2 Department of Intensive Care, Xiasha Campus, Sir Run Run Shaw Hospital Affiliated to Zhejiang University, School of Medicine, Hangzhou 310019, China 3 Department of Medical Oncology, Sir Run Run Shaw Hospital Affiliated to Zhejiang University, School of Medicine, 3# Qingchun Eastern Road, Hangzhou 310018, China transcription. Overexpression of HDAC2 by pcdna/ HDAC2 transfection significantly increased the sensitivity of CRC cells to Dox and upregulated the levels of P-gp, p-c-fos, and p-c-jun. Conclusions Our data revealed that HDAC2 can regulate Dox sensitivity of CRC cells by targeting ABCB1 transcription. It suggested that HDAC2 might be an important target for CRC therapy. Further, the combination of HDAC2-specific inhibitor and anticancer drugs including Dox might be an efficiency approach to elevate the treatment outcome of CRC. Keywords Introduction HDAC2 CRC ABCB1 AP-1 Doxorubicin Human colorectal cancer (CRC), also called colon cancer or large bowel cancer, is a leading cause of cancer mortality in the worldwide [1]. It has been reported that there is around 1.2 million new CRC cases each year [2]. Although surgery remains the mainstay of treatment for CRC, there are still 50 % of patients died of tumor recurrence or metastasis in the second year of operation [3]. Chemotherapy is an another important therapy approach of CRC treatment, particularly for patients with advanced disease. However, a major obstacle in successful treatment in CRC is multidrug resistance (MDR), which occurs when cancer cells acquire simultaneous resistance to diverse chemotherapeutic agents. One of the important mechanisms conferring multidrug resistance is the overexpression of P-glycoprotein (P-gp), a member of ATP-binding cassette (ABC) superfamily of membrane transporter encoded by human ABCB1 gene [4]. The overexpression of P-gp can mediate the efflux of cytotoxic drugs and decrease their
intracellular concentration and therefore lead to chemotherapy failure. Suppression of P-gp function or inhibition of its expression represents logical approaches to overcome MDR in cancer chemotherapy. Therefore, the investigation of underlying mechanisms of drug resistance and P-gp expression is important to improve the therapeutic outcome of CRC patients. Histone deacetylases (HDACs), which remove acetyl groups from the N-acetyllysines on histone and nonhistone proteins, can repress gene expression and act as promising new cancer therapeutic targets for various cancers [5]. HDAC inhibitors such as butyrate, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and apicidin are considered as candidate drugs in therapy for leukemia and various solid tumors [6, 7]. There are 11 known human HDAC isoforms, including class I (HDAC1, 2, 3, 8), class IIa (HDAC4, 5, 7, 9), class IIb (HDAC6, 10), and class IV (HDAC11). Among all HDAC forms, HDAC2 is frequently dysregulated in cancers including CRC [6, 8, 9]. Previous studies indicate that overexpression of HDAC2 plays a pivotal role in the development of cancer due to its regulation of cell-cycle components at the transcription levels [10 12]. However, no attempt has been made so far to explain the roles and underlying mechanisms responsible for HDAC2 in chemoresistance. Doxorubicin (Dox) in combination with other anticancer agents has been proved a good therapeutic effect for various types of cancers such as bladder, testicular, ovarian, and lung cancers [13, 14]. As a DNA intercalating anthracycline antibiotic, Dox has also been proved a good therapeutic effect for the treatment of advanced colorectal cancer [15, 16]. Despite the extensive and long-standing clinical utilization of Dox, a major cause in the clinical chemotherapy failure in CRC patients is the development of cancer cell resistance to Dox [17]. The increased expression of MDR gene and other transporter genes and/or proteins such as multidrug resistance protein (MRP) are involved in Dox resistance [18, 19]. The inhibition of class I HDACs can reverse drug resistance in cancer cells [20], but the investigations about the roles of HDAC2 in chemoresistance are very limited. The present study found that HDAC2 is unregulated commonly in human CRC cells, and sirna-mediated silencing of HDAC2 can sensitize CRC cells to Dox via downregulation of ABCB1 expression. Materials and methods Cells and reagents Human colorectal cancer cell lines were obtained from the American Type Culture Collection within 6 months of this study. Routine morphological examination of the main cultures was performed. Dulbecco s modified Eagle s medium (DMEM) and RPMI 1640 were products of Gibco BRL (Gaithersburg, MD, USA). Dox and other chemicals were of reagent grade or better and purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise noted. Monoclonal antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA) excluding antibodies against MRP1, which were purchased from Bioworld Technology Inc. (Minneapolis, MN, USA). All compounds were solubilized in DMSO. Medium containing 0.5 % DMSO was used as the control. Cell culture and transfection Human CRC cell lines were cultured in DMEM or RPMI 1640 medium with 10 % FBS, 100 units/ml of penicillin, and 100 μg/ml of streptomycin in a humidified incubator with 5 % CO 2 at 37 C. For transfection, HCT-116 and SW480 cells were seeded into plates in order to reach 30 50 % confluence and transfected with pcdna3.1, pcdna3.1/hdac2, sirna negative control (sirna-nc: 5 -GGC TAC GTC CAG GAG CGC A-3 ), si-hdac2-1(5 - AAC AGA CGU UAA GGA AGA A -3 ), or si-hdac2-2 (5 -GGA UUA CAU CAU GCU AAG A-3 ) by use of Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer s instruction. Silencing was validated by qrt-pcr and Western blot analysis. Cell viability assay HCT-116 and SW480 cells were seeded in 96-well plates with 5 10 3 cells per well in 100 µl of cell culture medium and incubated at 37 C for 24 h. The cells were transfected with si-hdac2 or si-nc for 24 h and then further incubated with increasing concentrations of Dox for 48 h. Then, cells were incubated with 20 µl of 5 mg/ ml MTT at 37 C for 4 h. Medium was removed, and the precipitated formazan was dissolved in 100 µl of dimethyl sulfoxide (DMSO). After shaking the incubation for 10 min, the absorbance at 570 nm was detected using an uquant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). All experiments were performed in duplicates. The 50 % inhibitory concentration (IC 50 ) was calculated using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). RNA extraction and real time PCR After treatment, total cellular RNA was isolated by use of a Trizol Reagent RNA extraction kit according to the manufacturer s instructions (Molecular Research Center, Cincinnati, OH, USA). The first strand of cdna was
synthesized with the Revert Aid First-Strand cdna Synthesis Kit (Thermo Scientific) according to the manufacturer s instructions. Expression values were measured in triplicate on a Roche LightCycler 480 and normalized to GAPDH expression. Results are computed as fold induction relative to controls. PCR cycles were as follows: 94 C for 4 min, followed by 40 cycles of 94 C for 1 min, 56 C for 1 min, and 72 C for 1 min. Primer pairs were as follows: GAPDH, forward 5 -GCA CCG TCA AGG CTG AGA AC-3 and reverse 5 -TGG TGA AGA CGC CAG TGG A-3 ; HDAC-1, forward 5 - TCC AAC ATG ACC AAC CAG AA -3 and reverse 5 - TTG TCA GGG TCC TCC TCA TC -3 ; HDAC-2, forward 5 - TGG AGG AGG CTA CAC AAT CC -3 and reverse 5 - TTT GAA CAC CAG GTG CAT GT -3 ; HDAC-3, forward 5 - CTC CCC TTT CCC TCA AAC TC -3 and reverse 5 - TTG CAT GGA AGG AGG AAC TG -3 ; HDAC-8, forward 5 - AGG TGA TGA GGA CCA TCC AG -3 and reverse 5 - ACC CTC CAG ACC AGT TGA TG -3 ; ABCB1, forward 5 - CCC ATC ATT GCA ATA GCA GG-3 and reverse 5 - TGT TCA AAC TTC TGC TCC TGA-3 ; ABCG2, forward 5 - AGA TGG GTT TCC AAG CGT TCA T-3 and reverse 5 - CCA GTC CCA GTA CGA CTG TGA CA-3 ; ABCC1, forward 5 - ATG TCA CGT GGA ATA CCA GC-3 and reverse 5 - GAA GAC TGA ACT CCC TTC CT-3 ; ABCA1, forward 5 - GTT CTC AGA TGC TCG GAG GCT TCT T-3 and reverse 5 - GAC AAT ACG AGA CAC AGC CTG GTA G-3 ; ABCC2, forward 5 - ACA GAG GCT GGT GGC AAC C-3 and reverse 5 - ACC ATT ACC TTG TCA CTG TCC ATG A-3. CT values were reported relative to GAPDH RNA. The cycle number when the fluorescence first reached a preset threshold (Ct) was used to quantify the initial concentration of individual templates for expression of mrna of genes of interest. All experiments were performed three times independently, and the average was used for comparison. Western blot analysis Cells were grown to 80 % confluence in 25 cm 2 culture flasks ( 1 10 7 cells per flask), washed, and centrifuged. The pellet was dispersed in a lysis buffer containing 50 mm Tris HCl (ph 7.4), 120 mm NaCl, 1 mm EDTA, 1 % TritonX-100, and 1 % (v/v) protease inhibitor cocktail (Sigma-Aldrich). The cell lysate was collected after centrifugation, and the protein concentration was determined using the Bio-Rad Detergent Compatible Protein Assay kit (Bio-Rad). Equal amount of proteins (20 μg per lane) from each sample was used for SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked overnight at 4 C with Tris-buffered saline containing 0.05 % (v/v) Tween-20 (TBS-T) and 5 % (m/v) nonfat dried milk, and incubated for 1 h at room temperature with primary antibodies. The membranes were washed with TBS-T and incubated for 1 h at room temperature with HRP-conjugated secondary antibody (Amersham Life Sciences, Cleveland, OH, USA). Protein bands were visualized using an enhanced chemoluminescence kit (Amersham Biosciences, Piscataway, NJ, USA). Monoclonal antibody GAPDH (Sigma-Aldrich) was used as a reference. ABCB1 promoter activity The analysis of ABCB1 promoter activity was conducted according to previous study [21]. Briefly, cells were transiently transfected with the ABCB1 promoter luciferase reporter construct ptl-mdr1 (Affymetrix) and pbabepuro plasmid using FuGENE6 reagent (Promega). After 12 h, cells were transfected with sinc, si-hdac2-1, or si- HDAC2-2 for 24 h. Luciferase activity (relative luciferase units, RLU) was normalized to protein amount as determined by BCA assay (ThermoFisher, Waltham, MA, USA). Experiments were performed three times with similar results. Statistical analysis Data are presented as mean ± SD and evaluated by Student s t test using SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Probability values of p < 0.05 were considered to be significant. Results HDAC2 is overexpressed in CRC cell lines We measured the expression of class I HDACs (HDAC1, 2, 3, 8) in four CRC cell lines HCT-116, SW480, SW620, and Lovo and compared with that in the human normal colonic epithelial cell line NCM460. Our results showed that among four members of class I HDACs, the mrna levels of HDAC2 were significantly (p < 0.05) increased in CRC cell lines as compared to human normal colonic epithelial cell line NCM460 (Fig. 1a). This was confirmed by the results of Western blot analysis, which showed that the protein levels of HDAC2 in CRC cell lines were obviously greater than that in NCM460 cells. Collectively, these data suggested that CRC cells have a much higher level of HDAC2 than NCM460 cells.
Fig. 1 The expression of class I HDACs in CRC cell lines. The mrna (a) and protein (b) levels of class I HDACs (HDAC1, 2, 3, 8) were detected in four CRC cell lines HCT-116, SW480, SW620, and Lovo, and human normal colonic epithelial cell line NCM460 by qrt-pcr and Western blot analysis, respectively. Data were presented as mean ± SD of three independent experiments Fig. 2 Silencing of HDAC2 enhances the sensitivity of CRC cells to Dox. a HCT-116 or SW480 cells were transfected with si-nc, si- HDAC2-1, or si-hdac2-2 for 24 h; then, the expression of HDAC2 was measured by Western blot analysis; after transfected with si-nc, si-hdac2-1, or si-hdac2-2 for 24 h, HCT-116 b or SW480 c cells were exposed to increasing concentrations of Dox for another 48 h; then, cell viability was measured by MTT assay. Data were presented as mean ± SD of six independent experiments Silencing of HDAC2 enhances the sensitivity of CRC cells to Dox Then, we investigated the roles of HDAC2 in mediating Dox sensitivity of CRC cells by MTT assay and IC 50 calculation. The knockdown efficiency of HDAC2 by sih- DAC2-1 and sihdac2-2 in HCT-116 and SW480 cells was confirmed by Western blot analysis (Fig. 2a). Compared to si-nc group cells, HDAC2-ablated HCT-116 (Fig. 2b) and SW480 (Fig. 2c) cells were more sensitive to Doxinduced growth inhibition. As shown in Fig. 2b, the IC 50 of HCT-116 in si-nc-treated cells (198 nm) was significantly greater than those of sihdac2-1 (67 nm) and sihdac2-2 (81 nm) cells (p < 0.05 for each). The IC 50 of SW480 in si-nc-treated cells (252 nm) was significantly greater than those of sihdac2-1 (79 nm) and sihdac2-2 (57 nm) cells (p < 0.05 for each). Together, these data revealed that silencing of HDAC2 enhances the sensitivity of CRC cells to Dox. Silencing of HDAC2 downregulates the expression of ABCB1 in CRC cells Previous studies suggested that candidate transporters that efflux doxorubicin include ABCB1, ABCG2, ABCC1, ABCA1, and ABCC2 [22, 23]. We therefore investigated the mrna expression of ABCB1, ABCG2, ABCC1, ABCA1, and ABCC2 in HDAC2-silenced CRC cells. The results showed that silencing of HDAC2 can significantly (p < 0.05) downregulate the expression of ABCB1, while not others, in both
Fig. 3 Silencing of HDAC2 downregulates the expression of ABCB1 in CRC cells. HCT-116 (a) or SW480 (b) cells were transfected with si-nc, si-hdac2-1, or si-hdac2-2 for 24 h; then, the mrna expression of ABCB1, ABCG2, ABCC1, ABCA1, and ABCC2 was measured by qrt-pcr; c HCT-116 and SW480 cells were transfected with si-nc, si-hdac2-1, or si-hdac2-2 for 48 h; the protein levels of P-gp, BCRP, and MRP1 were measured by Western blot analysis. Data were presented as mean ± SD of three independent experiments. ** p < 0.01 compared with control HCT-116 (Fig. 3a) and SW480 (Fig. 3b) cells. This was further confirmed by Western blot analysis that si-hdac2 can downregulate the protein expression of P-gp (ABCB1 protein) in both HCT-116 and SW480 cells, while not obviously influence the expression of BCRP (ABCG2 protein) or MRP1 (ABCC1 protein). The results suggested that silencing of HDAC2 may enhance the sensitivity of CRC cells to Dox via downregulation of ABCB1 expression. AP 1 is involved in the modulation effects of HDAC2 on ABCB1 expression Transcription factors including AP-1 and NF-κB have been reported to regulate ABCB1 promoter activity [21, 24, 25]. To investigate the mechanism of HDAC2-mediated ABCB1 regulation, a construct containing 1 kb of the ABCB1 promoter driving a luciferase reporter (ptl- ABCB1) was transiently co-transfected with either an empty vector (pbabe-puro) into HCT-116 cells. Cells were then transfected with si-nc, si-hdac2-1, or si-hdac2-2 for 24 h, and lysates were measured for luciferase activity. ABCB1 promoter activity was notably decreased in HDAC2 knockdown cells (Fig. 4a). Based on data demonstrating the role of AP-1 and NF-κB in ABCB1 regulation, a panel of AP-1 and NF-κB proteins were assessed by Western blot analysis in HCT-116 and SW480 cells following 24-h si- HDAC2 transfection. As shown in Fig. 4b, the phosphorylation of c-fos and c-jun was obviously downregulated upon silencing of HDAC2, while the phosphorylation of p65 and IκBα was not significantly changed. Similar results were also observed in SW480 cells. Collectively, these data suggested that AP-1 is involved in the modulation effects of HDAC2 on ABCB1 expression. Overexpression of HDAC2 decreases Dox sensitivity via upregulation of ABCB1 To verify the role of HDAC2 in modulation of Dox sensitivity of CRC cells, we overexpressed HDAC2 in HCT- 116 and SW480 cells by transfection pcdna3.1/hdac2 plasmid. Western blot analysis showed that HDAC2 was overexpressed in pcdna3.1/hdac2 plasmid-transfected HCT-116 and SW480 cells (Fig. 5a). Compared to the vector control group (pcdna3.1), cells with overexpression of HDAC2 were significantly (p < 0.05) less sensitive to Dox-induced growth inhibition of HCT-116 (Fig. 5b) and SW480 (Fig. 5c) cells. Further, overexpression of HDAC2 can increase the mrna of ABCB1 (Fig. 5d) and protein expression of P-gp, p-c-fos, and p-c-jun in both HCT- 116 and SW480 cells (Fig. 5e). These data suggested that overexpression of HDAC2 can decrease Dox sensitivity via upregulation of ABCB1. Discussion Selective inhibition of HDAC2 can suppress the progression of hepatocarcinoma and other cancers [10]. Although HDAC2 has been globally detected in colon adenoma and carcinoma [9], its role in the progression and drug sensitivity of CRC cells remains underexplored. In this report, we demonstrated that among class I HDACs (HDAC1, 2, 3, 8), HDAC2 are significantly increased in CRC cell lines as compared to human normal colonic epithelial cells. Silencing of HDAC2 can obviously enhance the sensitivity of CRC cells to Dox. Silencing of HDAC2 can significantly (p < 0.05) downregulate the expression of ABCB1, while
Fig. 4 AP-1 is involved in the modulation effects of HDAC2 on ABCB1 expression. a HCT-116 and SW480 cells were transiently cotransfected with 1 μg ABCB1 reporter construct (ptl-mdr1) and 1 μg empty vector (pbabepuro) for 12 h; then, cells were transfected with si-nc, si-hdac2-1, or si-hdac2-2 for 24 h. Luciferase activity was assessed in total cell lysates. Results are shown normalized to the amount of lysate. b HCT-116 and SW480 cells were transfected with si-nc, si-hdac2-1, or si-hdac2-2 for 24 h, and then, the total and phosphorylation of c-fos, c-jun, p65, and IκBα were measured by Western blot analysis. Data were presented as mean ± SD of three independent experiments. ** p < 0.01 compared with control not ABCG2, ABCC1, ABCA1, or ABCC2, which are candidate transporters that efflux Dox. Further, ABCB1 promoter activity was notably decreased in HDAC2 knockdown cells. It might be due to that the activities of AP-1 are inhibited by knockdown of HDAC2. Overexpression of HDAC2 by plasmid transfection can decrease Dox sensitivity of CRC cells via upregulation of ABCB1 and activation of AP-1. Taken together, these data revealed that HDAC2 can regulate Dox sensitivity of CRC cells by targeting ABC transporters ABCB1. To date, there are only few reports about the specific roles of HDACs and cancer progression. Among four classes of HDACs, classes I and II have been considered as the most important ones since they are components of large transcriptional corepressor complexes [26, 27]. Our present study showed that among class I HDACs (HDAC1, 2, 3, 8), HDAC2 were significantly increased in CRC cell lines. This was confirmed by the results that there was a close positive association between HDAC2 and colorectal cancer progression [9]. Increased HDAC2 expression is found in the majority of human colon cancer explants, as well as in intestinal mucosa and polyps of adenomatosis polyposis coli (APC)- deficient mice [6]. HDAC2-deficient mice have reduced body size and decreased intestinal tumor rates compared with the normal ones [28]. These data suggested a key role of HDAC2 in intestinal carcinogenesis, and the specific inhibition of HDAC2 might be important for CRC treatment. Our study revealed that HDAC2 regulates the sensitivity of CRC cells to Dox. Silencing of HDAC2 enhanced the sensitivity of CRC cells to Dox, while overexpression of HDAC2 exhibited opposite effects. This was consistent with recent studies that HDAC2 can mediate therapeutic resistance of pancreatic cancer cells [29]. Inhibition of HDACs can increase the cytotoxicity to anticancer drugs of cancer cells [30]. Further, synergy effects of Dox and HDAC inhibitor have been reported in acute myeloid leukemia [31]. Valproic acid, and several other HDAC inhibitors, can also enhance sensitivity to Dox of cancer cells [32, 33]. Therefore, the combination of HDAC2-specific inhibitor and anticancer drugs including Dox might be an efficiency approach to elevate the treatment outcome of CRC and other cancers. The inhibition of ABCB1 has been reported to mediate the Dox transport and chemoresistance in various cancer cells [34, 35]. Our present study reveals that silencing of HDAC2 can significantly (p < 0.05) downregulate the expression of ABCB1 via suppressing its promoter activity, while it has limited effects on the expression of ABCG2, ABCC1, ABCA1, or ABCC2. The expression of ABCB1 is mediated at least at two distinct levels, mrna turnover and translational regulation [36]. We showed that ABCB1 expression is also regulated at promoter level in response to silencing of HDAC2 in CRC cells. Various transcription factors such as AP-1 and NF-κB can induce the expression
Fig. 5 Overexpression of HDAC2 can decrease Dox sensitivity of CRC via upregulation of ABCB1. a HCT-116 and SW480 cells were transiently transfected with pcdna3.1 or pcdna/hdac2 for 24 h, and then, the expression of HDAC2 was measured by Western blot analysis; after transfected with pcdna3.1 or pcdna/hdac2 for 24 h, HCT-116 b or SW480 c cells were exposed to increasing concentrations of Dox for another 48 h, and then, cell viability was measured by MTT assay; d HCT-116 and SW480 cells were transiently transfected with pcdna3.1 or pcdna/hdac2 for 24 h; the mrna levels of ABCB1 were measured by qrt-pcr; e HCT-116 and SW480 cells were transiently transfected with pcdna3.1 or pcdna/ HDAC2 for 48 h; the levels of P-gp, p-c-fos, c-fos, p-c-jun, and c-jun were measured by Western blot analysis. Data are presented as mean ± SD of three independent experiments. ** p < 0.01 compared with control of ABCB1 gene [37]. The present study revealed that induction of ABCB1 expression by HDAC2 does not appear to involve NF-κB due to the fact that total and phosphorylation of p65 and IκBα are not significantly changed in HDAC2-silenced CRC cells. Inversely, silencing of HDAC2 decreased the phosphorylation of c-fos and c-jun, which can directly interact with the ABCB1 promoter and then promote its transcription [24, 38]. The activation of AP-1 and overexpression of c-jun and c-fos genes have been observed in various drug-resistant cells [39, 40], while targeted inhibition of c-fos can sensitize drug-resistant cancer cells to chemotherapeutic agents via P-gp inhibition [41]. Increased levels of c-jun and ABCB1 in other drugresistant cancer cell lines have also been observed [42, 43]. Together, our data suggested that HDAC2 can activate AP-1 and then promote increasing the transcription of ABCB1 gene in CRC cells. It has been reported that various other signals and transcription factors such as ERK1/2, p38-mapk, JNK, PTEN, and SP1 also regulate the transcription and expression of ABCB1 [44]. Whether these molecules also participated in HDAC2-elicited ABCB1 expression needs further study. In conclusion, our present study revealed that the HDAC2 is overexpressed in CRC cells. Its specific knockdown can increase the sensitivity of CRC cells to Dox via upregulation of ABCB1 though activation of AP-1, while overexpression of HDAC2 can decrease Dox sensitivity. It suggested that HDAC2 might be an important target for CRC therapy and combination of HDAC2- specific inhibitor and anticancer drugs including Dox might be an efficiency approach to elevate the treatment outcome of CRC.
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