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Veliparib overcomes multidrug resistance in liver cancer cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Veliparib overcomes multidrug resistance in liver cancer cells Lin Chang a, *, 1, Yulan Hou a, 1, Lili Zhu a, Zhixue Wang a, Guotao Chen b, Chengfeng Shu a, Yali Liu a a b
Department of Laboratory Medicine, Bishan Hospital, Chongqing, China Department of Nephrology, Bishan Hospital, Chongqing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2019 Accepted 19 October 2019 Available online xxx
Overexpression of ATP-binding cassette (ABC) transporter is one of the most important factors taking responsibility for the progress of multidrug resistance (MDR) in multiple cancers. In this study, we investigated that veliparib, a PARP inhibitor which is in clinical development, could overcome ABCB1mediated MDR in liver cancer cells. Veliparib could significantly enhance the cytotoxic effects of a series of conventional chemotherapeutic drugs in ABCB1-overexpression liver cancer cells. Mechanism study showed that veliparib could significantly enhance the accumulation of doxorubicin in ABCB1overexpression liver cancer cells, without down-regulating the expression level of ABCB1. Finally, veliparib could significantly inhibit the ATPase activity of ABCB1 transporter. This study could provide information that combine veliparib with other chemotherapeutic drugs may benefit liver cancer patients. © 2019 Elsevier Inc. All rights reserved.
Keywords: Veliparib Liver cancer Multidrug resistance (MDR) ABC transporter
1. Introduction Liver cancer, the sixth most prevalent cancer all over the world, cause 0.6 million deaths per year [1]. A series of therapeutic strategies is available for liver cancer in clinic, for example, surgical resection or liver transplantation has been identified as valid approaches to liver cancer treatment. Moreover, local ablative therapy could recruit patients who loss surgical resection chance. Unfortunately, due to late diagnosis and/or advanced potential liver cirrhosis, liver cancer patients had to receive system chemotherapy [2]. On the other hand, intra-arterial delivery of chemotherapy agents, for example, transarterial chemoembolization (TACE), is also necessary when systemic chemotherapy is limited due to some severe side effects like systemic toxicity [3]. Therefore, chemotherapy is still one of a most crucial treatment modalities in the strategies of liver cancer therapy, especially for those patients with unresectable liver cancer [3e7]. However, the occurrence and development multidrug resistance (MDR) in cancer cells is a major obstacle in the treatment of liver cancer [8]. MDR is a phenomenon that lead cancer cells resistance to structurally and mechanically unrelated antineoplastic drugs [9].
* Corresponding author. Department of Laboratory Medicine, Bishan Hospital, Chongqing, 402760, China. E-mail address: [email protected] (L. Chang). 1 These authors contribute to this work equally.
Research carried out in decades has indicated that the mechanisms of MDR is mainly including apoptosis reduction, advanced DNA damage repair mechanisms, and/or the alteration of drug metabolism [10]. Nevertheless, one major mechanism contributing cancer cells resistance to anticancer drugs, especially chemotherapeutic drugs, is the overexpression the ABC (ATP-binding cassette) transporter on the membrane of cancer cells [11]. Expressing in both karyotes and eukaryotes, ABC transporters are large families that have 49 proteins and 48 of them have identified functions [12]. Structurally, the ABC transporters are constituted by transmembrane domains (TMDs) and nucleotidebinding domains (NBDs) [13]. The cavity in TMDs could combine a series of substrates by the energy from hydrolysis of ATP in NBDs. The ABC transporters are widely expressed in placenta, blood-brain barrier (BBB), intestines, livers and kidneys, and could transporter large number of endogenous substrates, including aliphatic acid, porphyrin, and sterol [14]. Meanwhile, some ABC transporter could also transport a series of anticancer drugs. ABCB1, for example, encoded by MDR1 gene, has high affinity to paclitaxel, doxorubicin, vincristine, and colchicine. These conventional chemotherapeutic drugs can be pumped out of the cancer cells by ABCB1, which will lead to down-regulation of intracellular concentration, as a result, MDR occurs [15]. It is documented that the overexpression of ABCB1 is contributed to chemoresistance in liver cancer [16]. Therefore, impeding the function of ABC transporter, especially the function of ABCB1 is a potential therapy strategy in liver cancer.
https://doi.org/10.1016/j.bbrc.2019.10.141 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Veliparib, a PARP1 and PARP2 inhibitor in clinical development has shown potent anticancer effects on a panel of cancer cells [17]. However, there is hardly any research of veliparib on MDR reversal. Here we reported for the first time that veliparib have the capacity to overcome ABCB1-mediated MDR in liver cancer cells.
combination of colchicine and veliparib for 72 h. Subsequently, cells were stained with V-FITC/PI for 30 min in the dark. A BD Accuri C6 (BD Biosciences, San Jose, CA, USA) was used to conduct the flow cytometry assays. An EVOS FL Auto Microscope System was used to catch the pictures.
2. Materials and methods
2.5. Western blot assays
2.1. Chemicals
Western blot assays were conducted by a conventional protocol. Firstly, cells were plated into 6-wells plate and then were treated with DMSO or veliparib for 0, 24, 48, and 72 h. Then, cells were extracted with RIPA lysis buffer. A BCA kit was used to determine the concentration of protein, and equal amount protein (30 mg) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking for 1 h with 5% nonfat milk, the membranes were incubated with primary antibodies in 4 C for overnight, then exposed to HRP-conjugated secondary antibodies for 1 h. The protein bands were visualized with ECL and analyzed with an Image J software.
Veliparib was a product from Selleck (TX, USA), Dulbecco’s modified Eagle’s Medium (DMEM) and penicillin/streptomycin were purchased from Corning Incorporated (Corning, NY, USA), paclitaxel, doxorubicin, colchicine, fluorouracil, cisplatin and verapamil were purchased from MCE (Monmouth, NJ, USA). The monoclonal antibody of ABCB1 were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP conjugated secondary goat anti mouse antibody was purchased from Beyotime Biotechnology (Shanghai, China). Trypsin (0.25%) were purchased from Thermo Fisher Scientific Inc (Rockford, IL, USA). All other chemicals were purchased from Sigma Aldrich (MO, USA).
2.6. Accumulation assays 2.2. Cell lines and cell culture The human hepatoblastoma cells HepG2 were purchased from Cell Resource Center, Peking Union Medical College (Beijing, China), the doxorubicin selected HepG2/ADR cells were obtained from KeyGEN Biotch (Nanjing, China). We used 500 nM of doxorubicin to maintain the resistance fold of HepG2/ADR cells. Parental 293/ pcDNA3.1 and resistance 293/ABCB1 cells were established by transfecting 293 cells with empty vector pcDNA3.1 or pcDNA3.1 containing a full length ABCB1 gene. 293/pcDNA3.1 and 293/ABCB1 were kindly gifted by Zhejiang Normal University. G418 (2 mg/ml) was added into DMEM media to selected to maintain the resistance fold of 293/ABCB1 cells. Each cell line was maintained in DMEM media with 10% FBS and 1% penicillin/streptomycin at 37 C in a humidified atmosphere containing 5% CO2. Each above cell line showed an adherent growing monolayer. Each resistance cell line was cultured in drug free DMEM media for more than 10 days before the experiments. All cell lines used in this study were authenticated to be not contaminated by STR. 2.3. Cell viability analysis MTT assays were conducted to calculate the cell viability to evaluate the cytotoxic effects of veliparib in the cells we would use. MTT assays were also used to evaluate the reversal effect of veliparib. For the cytotoxic evaluation of veliparib, each kind of cell line was harvested, seeded into 96-well plate. Each well contained 3*103 cells. After adherent, veliparib was added for 48 h. For the evaluation of the reversal effect of veliparib, anti-carcinoma drugs were added into 96-well plate after incubating with veliparib for 2 h. MTT was added at 48 h time point for further 4 h further incubating. Subsequently, we used DMSO to solve the crystals. An BIORAD microplate was used to determine the OD value at 490 nm. We calculated the IC50 values as previously described [18]. In this study, verapamil that was reported as a potent ABCB1 inhibitor, was used as a positive reversal reagent [19]. Cisplatin was treated as a negative control of anticarcinoma drugs, for it is not a substrate of ABCB1 [20]. 2.4. Apoptosis analysis Apoptosis analysis was carried out by Annexin V-FITC/PI double staining. Briefly, cells were plated into 6-well plate and treated with DMSO (control group), veliparib only, colchicine only, or a
Fluorescence microscope was used to investigate the effect of veliparib on doxorubicin accumulation in HepG2 and HepG2/ADR cells. Briefly, HepG2 and HepG2/ADR cells were suspended at a concentration of 5*105 cells/mL in DMEM medium, treated with veliparib or verapamil for 2 h prior to doxorubicin exposing. After incubated with 4 mM of doxorubicin for 2 h, cells were washed with iced PBS twice, and fixed with 4% formaldehyde. A flow cytometry (C6, BD, USA) was used to measure the intracellular accumulation of doxorubicin in HepG2 and HepG2/ADR cells. We also used a fluorescence microscope to visualize the effect of veliparib on doxorubicin accumulation. 2.7. ATPase assays The effect of veliparib on ABCB1-associated ATPase assays were conducted by PREDEASY ATPase Kits (TEBU-BIO nv, Boechout, Belgium) with commercial protocols. Briefly, cell membranes were thawed and diluted before used. Sodium orthovanadate (Na3VO4) was used as an ATPase inhibitor. Various concentrations of veliparib were incubated with membranes for 5 min. The ATPase reactions were initiated by adding 5 mM Mg2þ ATP. Luminescence signals of Pi were initiated and measured after incubation at 37 C for 40 min with brief mixing. The changes of relative light units were determined by comparing Na3VO4-treated samples with veliparib treated groups. 2.8. Statistical analysis All data are expressed as the mean ± SD and analyzed using a one-way ANOVA. All experiments were repeated at least three times. Differences were considered significant when P < 0.05. 3. Results 3.1. Veliparib significantly enhanced the sensitivity of anticancer drugs in drug-selected resistance cell lines Firstly, to exclude the potential reversal effect caused by the cytotoxicity of veliparib, we conducted the MTT assays to choose relative non-toxic concentrations of veliparib for further experiments. As it is shown in Fig. 1A and B, over 80% of cells were survived after treated with less than 5 mM of veliparib, so we use 1 and 5 mM of veliparib for further reversal studies. To make sure that our
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Fig. 1. Effect of veliparib on cell viability and the situation of ABCB1 expression level in the cells we would use. (A) Chemical structure of veliparib. (B) Cell viability curves of HepG2 and HepG2/ADR cells treated by veliparib for 72 h. (C) Cell viability curves of 293/pcDNA3.1 and 293/ABCB1 cells treated by veliparib for 72 h. The expression level and relative intensity of ABCB1 in HepG2 and HepG2/ADR cells (D), and 293/pcDNA3.1 and 293/ABCB1 cells (E). Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
resistant cells were overexpressing of ABCB1, we conducted our Western blot to verify. As shown in Fig. 1C and D, HepG2/ADR, and 293/ABCB1 were overexpressed ABCB1. In our reversal study, veliparib significantly enhanced the anticancer effects of doxorubicin, paclitaxel, and colchicine in HepG2/ ADR cells dose dependently, the reversal effect of veliparib is at the comparable level of verapamil, a positive ABCB1 inhibitor (Fig. 2AeC). However, veliparib failed to significantly enhance the cytotoxic effect of cisplatin in HepG2/ADR cells (Fig. 2D). Additionally, veliparib did not significantly change the sensitivity of HepG2 cells to doxorubicin, paclitaxel, colchicine, or cisplatin (Fig. 2AeD). Based on above results, we made a hypothesis that veliparib could have the capacity to reverse ABCB1-mediated MDR in liver cancer cells.
3.2. Veliparib significantly reversed ABCB1-mediated MDR in 293/ ABCB1 cells We conducted our reversal study subsequently in genetransfected cells, 293/ABCB1. As it is shown in Fig. 2EeH, veliparib
significantly enhanced the cytotoxic effects of doxorubicin, paclitaxel, and colchicine, but not cisplatin, in 293/ABCB1 cells. However, veliparib could not change the IC50 values of doxorubicin, paclitaxel, colchicine, or cisplatin in 293/pcDNA3.1 cells. 3.3. Veliparib significantly enhanced colchicine-induced apoptosis in ABCB1-overexpression liver cancer cells It is documented that colchicine is a known apoptosis inducer [21]. On the other hand, colchicine is also a substrate of ABCB1 transporter, so we used flow cytometry to detect the effect of veliparib on colchicine-induced apoptosis in HepG2/ADR cells. As it is shown in (Fig. 3A and B), veliparib (5 mM) could significantly enhance the percentage of colchicine-induced apoptosis cells in HepG2/ADR cells compared to the group of HepG2/ADR which is treated with only colchicine. 3.4. Veliparib did not affect the expression level of ABCB1 in liver cancer cells The reversal effect of veliparib may due to the down-regulation
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Fig. 2. Effects of veliparib on the IC50 values of different chemotherapeutic drugs in parental and ABCB1-overexpressing liver cancer cells, and in 293/pcDNA3.1 and 293/ ABCB1 cells. IC50 values of (A) doxorubicin, (B) paclitaxel, (C) colchicine, and (D) cisplatin in HepG2 and HepG2/ADR cells. IC50 values of (E) doxorubicin, (F) paclitaxel, (G) colchicine, and (G) cisplatin in 293/pcDNA3.1 and 293/ABCB1 cells. Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
of ABCB1 expression level in hepatocellular carcinoma cells. To verify the effect of veliparib on ABCB1 expression level, we conducted our Western blot assays. As shown in Fig. 3C, we did not detect any significant change of ABCB1 expression level after treated with veliparib in HepG2/ADR cells for up to 72 h.
3.5. Veliparib significantly enhanced the accumulation level of doxorubicin in ABCB1-overexpression cells Since veliparib could reverse ABCB1-mediated MDR in hepatocellular carcinoma cells, induce ABCB1-overexpression cells apoptosis, but not down-regulating the expression level of ABCB1
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Fig. 3. Mechanism study of veliparib on the reversal effects of ABCB1-mediated MDR. (A, B) Veliparib significantly increased the colchicine (1 mM)-induced apoptosis effects in HepG2/ADR cells. Scale bar: 200 mM. (C) The effects of veliparib on ABCB1 expression level. Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
transporter, we made a hypothesis that veliparib may have the capacity to directly influence the function of ABCB1 transporter. We then conducted our accumulation assays to verify our hypothesis. As shown in Fig. 4A-C, in ABCB1-overexpression HepG2/ADR cells, veliparib significantly enhanced the accumulation level of doxorubicin in a dose dependent manner compared with the accumulation level in HepG2/ADR cells treated with DMSO. 3.6. Veliparib significantly inhibited the ATPase activity of ABCB1 transporter We then used a commercial kit to evaluate the effect of veliparib on ATPase activity of ABCB1 transporter. As shown in Fig. 4D, veliparib significantly inhibited the ATPase activity of ABCB1 in a dose-dependent manner. 4. Discussion Though surgical resection has been considered the optimal treatment approach in liver cancer, only a small proportion of patients are suitable candidates for surgery, and the relapse rate is
high [22]. Therefore, chemotherapy, including chemoembolization before and adjuvant therapy after surgery, is still a major approach in the treatment of hepatoma, especially in late stage of liver cancer patients. However, MDR, including acquired and/or congenital resistance to chemotherapeutic drug remained a major obstacle in not only liver cancer, but also other types of cancers. In this study, we used in vitro ABCB1-overexpression liver cancer cells model to find that, veliparib, a clinical-developing PARP inhibitor have the capacity to reverse ABCB1-mediated MDR in liver cancer cells. To reduce the influence of veliparib induced cytotoxic effects, we first evaluated the proliferation inhibition effects of veliparib in the cells we would use in this study. Finally, we chose 1 and 5 mM of veliparib as reversal concentrations. In our succedent reversal study, veliparib showed a potent reversal effect in both drugselected liver cancer cells and ABCB1-gene-transfected cells. However, veliparib could not significantly decrease the IC50 values of anticancer drugs in parental cells. Moreover, for cisplatin, a nonABCB1-substrate drug, veliparib could not significantly alter its anticancer effect on both parental or resistance cells. These results indicated that veliparib could overcome ABCB1-mediated MDR in liver cancer cells. In our subsequently flow cytometry apoptosis
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Fig. 4. Veliparib significantly increase the accumulation level of doxorubicin in HepG2/ADR cells. (A) Doxorubicin accumulation fluorescence in HepG2 and HepG2/ADR cells. Scale bar: 200 mM. (B) Flow cytometry assays to detect the doxorubicin accumulation level influenced by veliparib in HepG2/ADR cells. (C) Quantitative analysis of doxorubicin accumulation influenced by veliparib. (D) The inhibitory effect of veliparib on ABCB1 ATPase activity. Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
assays, veliparib could significantly enhance colchicine-induced apoptosis effects in HepG2/ADR cells. These findings proved the results in our reversal study. It is reported that down-regulation of ABCB1 could be a major factor that is responsible for the reversal of MDR [23]. However, in our Western blot results, no significant down-regulation of ABCB1 was found after treated HepG2/ADR cells with veliparib for 72 h, indicated that the reversal effects of veliparib was not related to the down-regulation of ABCB1 expression level. Nonetheless, further studies on whether veliparib could influence other proteins which are related with the functions of ABCB1 is still remained to be determined. Since veliparib have the capacity to reverse ABCB1-mediated MDR in liver cancer cells without the alteration of ABCB1 protein expression level, we made a hypothesis that veliparib could influence the efflux function of ABCB1 transporter in liver cancer cells. As shown in our accumulation assays, veliparib significantly increased the intracellular level of doxorubicin in HepG2/ADR cells in a dose-dependent manner, while no significant change of intracellular accumulation level of doxorubicin was detected in parental HepG2 cells after treatment of veliparib. These results indicated that veliparib could overcome ABCB1-medated MDR in liver cancer cells by directly inhibit the efflux function of ABCB1 transporter. It is well documented that the efflux function of ABC transporter relies on the hydrolysis of ATP [19]. To verify the effect of veliparib on ABCB1 transporter, we determined our ATPase assays. As predicted, veliparib inhibited the ATPase function of ABCB1 transporter in a dose-dependent. Veliparib is a potent PARP1 and PARP2 inhibitor, a phase I combination study of veliparib with carboplatin and gemcitabine
demonstrated promising preliminary antitumor activity in platinum-sensitive ovarian cancer patients with germline BRCA mutations [24]. Lin et al. have reported that inhibiting ABCB1 or ABCG2 could improve the treatment efficacy of veliparib when coadministrated with temozolomide [25] in glioblastoma, which indicated that veliparib may act as a substrate of ABCB1 and ABCG2. However, our ATPase assays suggested that veliparib inhibited the ATPase of ABCB1 transporter. Moreover, in our cytotoxic assays, there was no significant difference of cell viability between parental liver cancer cells and resistance liver cancer cells after treated with veliparib. Nevertheless, the results from Lin et al. showed a possibility that co-administration of veliparib with certain anticancer drug may benefit to those patients with potential MDR phenomenon. Another phase II clinical investigation evaluated the temozolomide and veliparib combination therapy for sorafenib-refractory advanced hepatocellular carcinoma. Unfortunately, though the combination of temozolomide and veliparib is well tolerated in patients with advanced liver cancer, the regimen failed to show survival benefit [26]. Moreover, veliparib significantly suppressed temozolomide resistant in gliomas cells, which enriched the evidence of veliparib that act as a reversal reagent in resistant cancer cells [27]. In conclusion, our study may provide a novel treatment strategy that combination veliparib with conventional chemotherapeutic drug may benefit liver cancer patients, especially those who are not sensitive to chemotherapeutic therapy due to the overexpression of ABC transporters. However, more MDR liver cancer cell lines would be used to further validate the reversal effect of veliparib in ABCB1overexpression MDR liver cancer cells. In addition, in vivo study will also be done in the near future to verify this kind of effect. At
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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least, our study provides clues at in vitro level that coadministration of veliparib with traditional chemotherapeutic drugs would be a potential treatment strategy for liver cancer treatment to exclude MDR. Availability of data and materials Not applicable.
[10] [11] [12]
[13] [14]
Authors contribution CL conceived the general idea. CL and HY performed experiments. CL, HY, ZL, and WZ analyzed the results. ZL, CG, SC, and LY wrote the first draft. CL and HY revised the manuscript. Ethics approval and consent to participate
[15]
[16]
[17]
Not applicable. [18]
Patient consent for publication Not applicable. Declaration of competing interest
[19]
The authors have declared no potential conflicts of interest. [20]
Acknowledgements This work was supported by Chongqing medical research program [grant numbers 2018QNXM042].
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Veliparib overcomes multidrug resistance in liver cancer cells Lin Chang a, *, 1, Yulan Hou a, 1, Lili Zhu a, Zhixue Wang a, Guotao Chen b, Chengfeng Shu a, Yali Liu a a b
Department of Laboratory Medicine, Bishan Hospital, Chongqing, China Department of Nephrology, Bishan Hospital, Chongqing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2019 Accepted 19 October 2019 Available online xxx
Overexpression of ATP-binding cassette (ABC) transporter is one of the most important factors taking responsibility for the progress of multidrug resistance (MDR) in multiple cancers. In this study, we investigated that veliparib, a PARP inhibitor which is in clinical development, could overcome ABCB1mediated MDR in liver cancer cells. Veliparib could significantly enhance the cytotoxic effects of a series of conventional chemotherapeutic drugs in ABCB1-overexpression liver cancer cells. Mechanism study showed that veliparib could significantly enhance the accumulation of doxorubicin in ABCB1overexpression liver cancer cells, without down-regulating the expression level of ABCB1. Finally, veliparib could significantly inhibit the ATPase activity of ABCB1 transporter. This study could provide information that combine veliparib with other chemotherapeutic drugs may benefit liver cancer patients. © 2019 Elsevier Inc. All rights reserved.
Keywords: Veliparib Liver cancer Multidrug resistance (MDR) ABC transporter
1. Introduction Liver cancer, the sixth most prevalent cancer all over the world, cause 0.6 million deaths per year [1]. A series of therapeutic strategies is available for liver cancer in clinic, for example, surgical resection or liver transplantation has been identified as valid approaches to liver cancer treatment. Moreover, local ablative therapy could recruit patients who loss surgical resection chance. Unfortunately, due to late diagnosis and/or advanced potential liver cirrhosis, liver cancer patients had to receive system chemotherapy [2]. On the other hand, intra-arterial delivery of chemotherapy agents, for example, transarterial chemoembolization (TACE), is also necessary when systemic chemotherapy is limited due to some severe side effects like systemic toxicity [3]. Therefore, chemotherapy is still one of a most crucial treatment modalities in the strategies of liver cancer therapy, especially for those patients with unresectable liver cancer [3e7]. However, the occurrence and development multidrug resistance (MDR) in cancer cells is a major obstacle in the treatment of liver cancer [8]. MDR is a phenomenon that lead cancer cells resistance to structurally and mechanically unrelated antineoplastic drugs [9].
* Corresponding author. Department of Laboratory Medicine, Bishan Hospital, Chongqing, 402760, China. E-mail address: [email protected] (L. Chang). 1 These authors contribute to this work equally.
Research carried out in decades has indicated that the mechanisms of MDR is mainly including apoptosis reduction, advanced DNA damage repair mechanisms, and/or the alteration of drug metabolism [10]. Nevertheless, one major mechanism contributing cancer cells resistance to anticancer drugs, especially chemotherapeutic drugs, is the overexpression the ABC (ATP-binding cassette) transporter on the membrane of cancer cells [11]. Expressing in both karyotes and eukaryotes, ABC transporters are large families that have 49 proteins and 48 of them have identified functions [12]. Structurally, the ABC transporters are constituted by transmembrane domains (TMDs) and nucleotidebinding domains (NBDs) [13]. The cavity in TMDs could combine a series of substrates by the energy from hydrolysis of ATP in NBDs. The ABC transporters are widely expressed in placenta, blood-brain barrier (BBB), intestines, livers and kidneys, and could transporter large number of endogenous substrates, including aliphatic acid, porphyrin, and sterol [14]. Meanwhile, some ABC transporter could also transport a series of anticancer drugs. ABCB1, for example, encoded by MDR1 gene, has high affinity to paclitaxel, doxorubicin, vincristine, and colchicine. These conventional chemotherapeutic drugs can be pumped out of the cancer cells by ABCB1, which will lead to down-regulation of intracellular concentration, as a result, MDR occurs [15]. It is documented that the overexpression of ABCB1 is contributed to chemoresistance in liver cancer [16]. Therefore, impeding the function of ABC transporter, especially the function of ABCB1 is a potential therapy strategy in liver cancer.
https://doi.org/10.1016/j.bbrc.2019.10.141 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Veliparib, a PARP1 and PARP2 inhibitor in clinical development has shown potent anticancer effects on a panel of cancer cells [17]. However, there is hardly any research of veliparib on MDR reversal. Here we reported for the first time that veliparib have the capacity to overcome ABCB1-mediated MDR in liver cancer cells.
combination of colchicine and veliparib for 72 h. Subsequently, cells were stained with V-FITC/PI for 30 min in the dark. A BD Accuri C6 (BD Biosciences, San Jose, CA, USA) was used to conduct the flow cytometry assays. An EVOS FL Auto Microscope System was used to catch the pictures.
2. Materials and methods
2.5. Western blot assays
2.1. Chemicals
Western blot assays were conducted by a conventional protocol. Firstly, cells were plated into 6-wells plate and then were treated with DMSO or veliparib for 0, 24, 48, and 72 h. Then, cells were extracted with RIPA lysis buffer. A BCA kit was used to determine the concentration of protein, and equal amount protein (30 mg) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking for 1 h with 5% nonfat milk, the membranes were incubated with primary antibodies in 4 C for overnight, then exposed to HRP-conjugated secondary antibodies for 1 h. The protein bands were visualized with ECL and analyzed with an Image J software.
Veliparib was a product from Selleck (TX, USA), Dulbecco’s modified Eagle’s Medium (DMEM) and penicillin/streptomycin were purchased from Corning Incorporated (Corning, NY, USA), paclitaxel, doxorubicin, colchicine, fluorouracil, cisplatin and verapamil were purchased from MCE (Monmouth, NJ, USA). The monoclonal antibody of ABCB1 were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP conjugated secondary goat anti mouse antibody was purchased from Beyotime Biotechnology (Shanghai, China). Trypsin (0.25%) were purchased from Thermo Fisher Scientific Inc (Rockford, IL, USA). All other chemicals were purchased from Sigma Aldrich (MO, USA).
2.6. Accumulation assays 2.2. Cell lines and cell culture The human hepatoblastoma cells HepG2 were purchased from Cell Resource Center, Peking Union Medical College (Beijing, China), the doxorubicin selected HepG2/ADR cells were obtained from KeyGEN Biotch (Nanjing, China). We used 500 nM of doxorubicin to maintain the resistance fold of HepG2/ADR cells. Parental 293/ pcDNA3.1 and resistance 293/ABCB1 cells were established by transfecting 293 cells with empty vector pcDNA3.1 or pcDNA3.1 containing a full length ABCB1 gene. 293/pcDNA3.1 and 293/ABCB1 were kindly gifted by Zhejiang Normal University. G418 (2 mg/ml) was added into DMEM media to selected to maintain the resistance fold of 293/ABCB1 cells. Each cell line was maintained in DMEM media with 10% FBS and 1% penicillin/streptomycin at 37 C in a humidified atmosphere containing 5% CO2. Each above cell line showed an adherent growing monolayer. Each resistance cell line was cultured in drug free DMEM media for more than 10 days before the experiments. All cell lines used in this study were authenticated to be not contaminated by STR. 2.3. Cell viability analysis MTT assays were conducted to calculate the cell viability to evaluate the cytotoxic effects of veliparib in the cells we would use. MTT assays were also used to evaluate the reversal effect of veliparib. For the cytotoxic evaluation of veliparib, each kind of cell line was harvested, seeded into 96-well plate. Each well contained 3*103 cells. After adherent, veliparib was added for 48 h. For the evaluation of the reversal effect of veliparib, anti-carcinoma drugs were added into 96-well plate after incubating with veliparib for 2 h. MTT was added at 48 h time point for further 4 h further incubating. Subsequently, we used DMSO to solve the crystals. An BIORAD microplate was used to determine the OD value at 490 nm. We calculated the IC50 values as previously described [18]. In this study, verapamil that was reported as a potent ABCB1 inhibitor, was used as a positive reversal reagent [19]. Cisplatin was treated as a negative control of anticarcinoma drugs, for it is not a substrate of ABCB1 [20]. 2.4. Apoptosis analysis Apoptosis analysis was carried out by Annexin V-FITC/PI double staining. Briefly, cells were plated into 6-well plate and treated with DMSO (control group), veliparib only, colchicine only, or a
Fluorescence microscope was used to investigate the effect of veliparib on doxorubicin accumulation in HepG2 and HepG2/ADR cells. Briefly, HepG2 and HepG2/ADR cells were suspended at a concentration of 5*105 cells/mL in DMEM medium, treated with veliparib or verapamil for 2 h prior to doxorubicin exposing. After incubated with 4 mM of doxorubicin for 2 h, cells were washed with iced PBS twice, and fixed with 4% formaldehyde. A flow cytometry (C6, BD, USA) was used to measure the intracellular accumulation of doxorubicin in HepG2 and HepG2/ADR cells. We also used a fluorescence microscope to visualize the effect of veliparib on doxorubicin accumulation. 2.7. ATPase assays The effect of veliparib on ABCB1-associated ATPase assays were conducted by PREDEASY ATPase Kits (TEBU-BIO nv, Boechout, Belgium) with commercial protocols. Briefly, cell membranes were thawed and diluted before used. Sodium orthovanadate (Na3VO4) was used as an ATPase inhibitor. Various concentrations of veliparib were incubated with membranes for 5 min. The ATPase reactions were initiated by adding 5 mM Mg2þ ATP. Luminescence signals of Pi were initiated and measured after incubation at 37 C for 40 min with brief mixing. The changes of relative light units were determined by comparing Na3VO4-treated samples with veliparib treated groups. 2.8. Statistical analysis All data are expressed as the mean ± SD and analyzed using a one-way ANOVA. All experiments were repeated at least three times. Differences were considered significant when P < 0.05. 3. Results 3.1. Veliparib significantly enhanced the sensitivity of anticancer drugs in drug-selected resistance cell lines Firstly, to exclude the potential reversal effect caused by the cytotoxicity of veliparib, we conducted the MTT assays to choose relative non-toxic concentrations of veliparib for further experiments. As it is shown in Fig. 1A and B, over 80% of cells were survived after treated with less than 5 mM of veliparib, so we use 1 and 5 mM of veliparib for further reversal studies. To make sure that our
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Fig. 1. Effect of veliparib on cell viability and the situation of ABCB1 expression level in the cells we would use. (A) Chemical structure of veliparib. (B) Cell viability curves of HepG2 and HepG2/ADR cells treated by veliparib for 72 h. (C) Cell viability curves of 293/pcDNA3.1 and 293/ABCB1 cells treated by veliparib for 72 h. The expression level and relative intensity of ABCB1 in HepG2 and HepG2/ADR cells (D), and 293/pcDNA3.1 and 293/ABCB1 cells (E). Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
resistant cells were overexpressing of ABCB1, we conducted our Western blot to verify. As shown in Fig. 1C and D, HepG2/ADR, and 293/ABCB1 were overexpressed ABCB1. In our reversal study, veliparib significantly enhanced the anticancer effects of doxorubicin, paclitaxel, and colchicine in HepG2/ ADR cells dose dependently, the reversal effect of veliparib is at the comparable level of verapamil, a positive ABCB1 inhibitor (Fig. 2AeC). However, veliparib failed to significantly enhance the cytotoxic effect of cisplatin in HepG2/ADR cells (Fig. 2D). Additionally, veliparib did not significantly change the sensitivity of HepG2 cells to doxorubicin, paclitaxel, colchicine, or cisplatin (Fig. 2AeD). Based on above results, we made a hypothesis that veliparib could have the capacity to reverse ABCB1-mediated MDR in liver cancer cells.
3.2. Veliparib significantly reversed ABCB1-mediated MDR in 293/ ABCB1 cells We conducted our reversal study subsequently in genetransfected cells, 293/ABCB1. As it is shown in Fig. 2EeH, veliparib
significantly enhanced the cytotoxic effects of doxorubicin, paclitaxel, and colchicine, but not cisplatin, in 293/ABCB1 cells. However, veliparib could not change the IC50 values of doxorubicin, paclitaxel, colchicine, or cisplatin in 293/pcDNA3.1 cells. 3.3. Veliparib significantly enhanced colchicine-induced apoptosis in ABCB1-overexpression liver cancer cells It is documented that colchicine is a known apoptosis inducer [21]. On the other hand, colchicine is also a substrate of ABCB1 transporter, so we used flow cytometry to detect the effect of veliparib on colchicine-induced apoptosis in HepG2/ADR cells. As it is shown in (Fig. 3A and B), veliparib (5 mM) could significantly enhance the percentage of colchicine-induced apoptosis cells in HepG2/ADR cells compared to the group of HepG2/ADR which is treated with only colchicine. 3.4. Veliparib did not affect the expression level of ABCB1 in liver cancer cells The reversal effect of veliparib may due to the down-regulation
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Fig. 2. Effects of veliparib on the IC50 values of different chemotherapeutic drugs in parental and ABCB1-overexpressing liver cancer cells, and in 293/pcDNA3.1 and 293/ ABCB1 cells. IC50 values of (A) doxorubicin, (B) paclitaxel, (C) colchicine, and (D) cisplatin in HepG2 and HepG2/ADR cells. IC50 values of (E) doxorubicin, (F) paclitaxel, (G) colchicine, and (G) cisplatin in 293/pcDNA3.1 and 293/ABCB1 cells. Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
of ABCB1 expression level in hepatocellular carcinoma cells. To verify the effect of veliparib on ABCB1 expression level, we conducted our Western blot assays. As shown in Fig. 3C, we did not detect any significant change of ABCB1 expression level after treated with veliparib in HepG2/ADR cells for up to 72 h.
3.5. Veliparib significantly enhanced the accumulation level of doxorubicin in ABCB1-overexpression cells Since veliparib could reverse ABCB1-mediated MDR in hepatocellular carcinoma cells, induce ABCB1-overexpression cells apoptosis, but not down-regulating the expression level of ABCB1
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Fig. 3. Mechanism study of veliparib on the reversal effects of ABCB1-mediated MDR. (A, B) Veliparib significantly increased the colchicine (1 mM)-induced apoptosis effects in HepG2/ADR cells. Scale bar: 200 mM. (C) The effects of veliparib on ABCB1 expression level. Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
transporter, we made a hypothesis that veliparib may have the capacity to directly influence the function of ABCB1 transporter. We then conducted our accumulation assays to verify our hypothesis. As shown in Fig. 4A-C, in ABCB1-overexpression HepG2/ADR cells, veliparib significantly enhanced the accumulation level of doxorubicin in a dose dependent manner compared with the accumulation level in HepG2/ADR cells treated with DMSO. 3.6. Veliparib significantly inhibited the ATPase activity of ABCB1 transporter We then used a commercial kit to evaluate the effect of veliparib on ATPase activity of ABCB1 transporter. As shown in Fig. 4D, veliparib significantly inhibited the ATPase activity of ABCB1 in a dose-dependent manner. 4. Discussion Though surgical resection has been considered the optimal treatment approach in liver cancer, only a small proportion of patients are suitable candidates for surgery, and the relapse rate is
high [22]. Therefore, chemotherapy, including chemoembolization before and adjuvant therapy after surgery, is still a major approach in the treatment of hepatoma, especially in late stage of liver cancer patients. However, MDR, including acquired and/or congenital resistance to chemotherapeutic drug remained a major obstacle in not only liver cancer, but also other types of cancers. In this study, we used in vitro ABCB1-overexpression liver cancer cells model to find that, veliparib, a clinical-developing PARP inhibitor have the capacity to reverse ABCB1-mediated MDR in liver cancer cells. To reduce the influence of veliparib induced cytotoxic effects, we first evaluated the proliferation inhibition effects of veliparib in the cells we would use in this study. Finally, we chose 1 and 5 mM of veliparib as reversal concentrations. In our succedent reversal study, veliparib showed a potent reversal effect in both drugselected liver cancer cells and ABCB1-gene-transfected cells. However, veliparib could not significantly decrease the IC50 values of anticancer drugs in parental cells. Moreover, for cisplatin, a nonABCB1-substrate drug, veliparib could not significantly alter its anticancer effect on both parental or resistance cells. These results indicated that veliparib could overcome ABCB1-mediated MDR in liver cancer cells. In our subsequently flow cytometry apoptosis
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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Fig. 4. Veliparib significantly increase the accumulation level of doxorubicin in HepG2/ADR cells. (A) Doxorubicin accumulation fluorescence in HepG2 and HepG2/ADR cells. Scale bar: 200 mM. (B) Flow cytometry assays to detect the doxorubicin accumulation level influenced by veliparib in HepG2/ADR cells. (C) Quantitative analysis of doxorubicin accumulation influenced by veliparib. (D) The inhibitory effect of veliparib on ABCB1 ATPase activity. Data are expressed as mean ± SD, and representative of at least three independent experiments in triplicate are shown. * indicates p < 0.05 versus control group.
assays, veliparib could significantly enhance colchicine-induced apoptosis effects in HepG2/ADR cells. These findings proved the results in our reversal study. It is reported that down-regulation of ABCB1 could be a major factor that is responsible for the reversal of MDR [23]. However, in our Western blot results, no significant down-regulation of ABCB1 was found after treated HepG2/ADR cells with veliparib for 72 h, indicated that the reversal effects of veliparib was not related to the down-regulation of ABCB1 expression level. Nonetheless, further studies on whether veliparib could influence other proteins which are related with the functions of ABCB1 is still remained to be determined. Since veliparib have the capacity to reverse ABCB1-mediated MDR in liver cancer cells without the alteration of ABCB1 protein expression level, we made a hypothesis that veliparib could influence the efflux function of ABCB1 transporter in liver cancer cells. As shown in our accumulation assays, veliparib significantly increased the intracellular level of doxorubicin in HepG2/ADR cells in a dose-dependent manner, while no significant change of intracellular accumulation level of doxorubicin was detected in parental HepG2 cells after treatment of veliparib. These results indicated that veliparib could overcome ABCB1-medated MDR in liver cancer cells by directly inhibit the efflux function of ABCB1 transporter. It is well documented that the efflux function of ABC transporter relies on the hydrolysis of ATP [19]. To verify the effect of veliparib on ABCB1 transporter, we determined our ATPase assays. As predicted, veliparib inhibited the ATPase function of ABCB1 transporter in a dose-dependent. Veliparib is a potent PARP1 and PARP2 inhibitor, a phase I combination study of veliparib with carboplatin and gemcitabine
demonstrated promising preliminary antitumor activity in platinum-sensitive ovarian cancer patients with germline BRCA mutations [24]. Lin et al. have reported that inhibiting ABCB1 or ABCG2 could improve the treatment efficacy of veliparib when coadministrated with temozolomide [25] in glioblastoma, which indicated that veliparib may act as a substrate of ABCB1 and ABCG2. However, our ATPase assays suggested that veliparib inhibited the ATPase of ABCB1 transporter. Moreover, in our cytotoxic assays, there was no significant difference of cell viability between parental liver cancer cells and resistance liver cancer cells after treated with veliparib. Nevertheless, the results from Lin et al. showed a possibility that co-administration of veliparib with certain anticancer drug may benefit to those patients with potential MDR phenomenon. Another phase II clinical investigation evaluated the temozolomide and veliparib combination therapy for sorafenib-refractory advanced hepatocellular carcinoma. Unfortunately, though the combination of temozolomide and veliparib is well tolerated in patients with advanced liver cancer, the regimen failed to show survival benefit [26]. Moreover, veliparib significantly suppressed temozolomide resistant in gliomas cells, which enriched the evidence of veliparib that act as a reversal reagent in resistant cancer cells [27]. In conclusion, our study may provide a novel treatment strategy that combination veliparib with conventional chemotherapeutic drug may benefit liver cancer patients, especially those who are not sensitive to chemotherapeutic therapy due to the overexpression of ABC transporters. However, more MDR liver cancer cell lines would be used to further validate the reversal effect of veliparib in ABCB1overexpression MDR liver cancer cells. In addition, in vivo study will also be done in the near future to verify this kind of effect. At
Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141
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least, our study provides clues at in vitro level that coadministration of veliparib with traditional chemotherapeutic drugs would be a potential treatment strategy for liver cancer treatment to exclude MDR. Availability of data and materials Not applicable.
[10] [11] [12]
[13] [14]
Authors contribution CL conceived the general idea. CL and HY performed experiments. CL, HY, ZL, and WZ analyzed the results. ZL, CG, SC, and LY wrote the first draft. CL and HY revised the manuscript. Ethics approval and consent to participate
[15]
[16]
[17]
Not applicable. [18]
Patient consent for publication Not applicable. Declaration of competing interest
[19]
The authors have declared no potential conflicts of interest. [20]
Acknowledgements This work was supported by Chongqing medical research program [grant numbers 2018QNXM042].
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Please cite this article as: L. Chang et al., Veliparib overcomes multidrug resistance in liver cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.141