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Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression
Biochemical Pharmacology xxx (2016) xxx–xxx
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Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression Weicheng Wang a,1, Wenjie Guo b,1, Lele Li a,1, Zan Fu a, Wen Liu b, Jian Gao b, Yongqian Shu a, Qiang Xu b,⇑, Yang Sun b,⇑, Yanhong Gu a,⇑ a b
Department of Oncology, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China
a r t i c l e
i n f o
Article history: Received 27 July 2016 Accepted 22 September 2016 Available online xxxx Keywords: Andrographolide BAX 5-FU-resistant Human colorectal cancer
a b s t r a c t 5-FU is the first line therapy for colorectal cancer, however, treatment effect is often hampered by the development of drug resistance or toxicity at high doses. Andrographolide is a natural diterpenoid from Andrographis paniculata which has anti-bacterial, anti-antiviral and anti-inflammation activities. In the current study, we test the hypothesis that Andrographolide reverses 5-FU resistance in colorectal cancer and examine the underlying mechanism. In vitro and vivo studies indicated that Andrographolide treatment significantly re-sensitizes HCT116/5-FUR cells (HCT116 cells which are 5-FU resistant) to cytotoxicity of 5-FU. Mechanism analysis showed that Andrographolide/5-FU co-treatment elevated apoptosis level of HCT116/5-FUR cells with highly increased level of BAX. By using biotin-Andrographolide pull down and cellular thermal shift assay, we found out that Andrographolide can directly target to BAX. Andrographolide-BAX interaction prevented BAX degradation, enhancing mitochondria-mediated apoptosis thus reversed 5-FU resistance while BAX silence diminished this effect. Further, by analyzing patient samples who received 5-FU involved chemotherapy, we found that expression level of BAX is correlated with PFS. Our results here provide a novel combination treatment strategy, especially for patients with 5FU-resistant tumors expressing low level of BAX. Meanwhile, we also proposed that BAX expression may be a predicted and prognosis marker of 5-FU involved chemotherapy. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Colorectal cancer (CRC) is one of the most commonly diagnosed solid tumors worldwide. Colorectal cancer is the second leading cause of cancer deaths in the United States and the third most common malignant neoplasm worldwide [1]. Current therapies for the treatment of CRC mainly comprise 5-Fluorouracil (5-FU)based chemotherapies that are used individually or in combination with oxaliplatin or anti-angiogenic agents, and/or anti-epidermal growth factor agents [2,3]. As a false substrate to thymidylate synthase enzyme that incorporates into DNA and RNA, 5-FU treatment causes the growth restrain and apoptosis of cancer cells [4]. Although CRC incidence rates have declined somewhat, current
⇑ Corresponding authors at: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, China (Q. Xu and Y. Sun). Department of Oncology, The First Affiliated Hospital with Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China (Y. Gu). E-mail addresses: [email protected] (Q. Xu), [email protected] (Y. Sun), [email protected] (Y. Gu). 1 Contributed equally to this work.
therapies are associated with significant side effects, high expense and recurrence rates can be as high as 50–60%, primarily due to the development of acquired resistance to 5-FU-based chemotherapeutics, as more than 15% of patients are resistant to 5-FU-based chemotherapies [5,6]. Thus, resistance to 5-FU has been a major obstacle in advanced colorectal cancer chemotherapy and confines its use in clinical practice. Thus novel and safe treatment strategies which can help overcome chemoresistance and enhance CRC response to 5-FU-based chemotherapies are desperately needed. Andrographolide, a natural diterpenoid from Andrographis paniculata, has been used as a herbal medicine in China for thousands of years. Andrographolide has been reported to exert antibacterial, antiviral, anti-inflammation and neuroprotective activities [7–9]. Our previous studies have proved that Andrographolide can alleviate colitis and suppress colitis-associated colon cancer [10,11]. Recent studies showed that Andrographolide alone can induce apoptosis of different cancer cells via regulating pro-apoptotic Bcl-2 family members [12]. Another group has reported that Andrographolide can inhibit tumor growth in mice although at a relatively high dose (about 200 mg/kg) [13]. What’s more, Andrographolide can potentiate the cytotoxic effect of various
http://dx.doi.org/10.1016/j.bcp.2016.09.024 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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chemotherapy drugs in different cell lines via enhancing apoptosis pathways or via suppression of pro-survival autophagy [14,15]. However, in colorectal cancer, no investigations about the effects and mechanism of Andrographolide on CRC 5-FU resistance have been reported. In the present study, we investigated the effect of Andrographolide in combination with 5-FU on various growth regulatory parameters and extensive characterization of underlying mechanisms in a 5-FU-resistant CRC cell line in vivo and vitro. Our data firstly reveal that the bind and up-regulation of BAX expression by Andrographolide is one of the principle mechanisms for resensitizing 5-FU-resistant CRC cells to 5-FU. At the same time, we found that BAX expression level maybe a prognosis marker for 5FU-based treatment. These findings highlight the potential possibility of using this natural, safe and relatively inexpensive compound as potential adjunctive treatments in improving the overall treatment response of patients with CRC in future.
2. Methods 2.1. Chemicals and reagents Primary antibodies against BAX, BCL2 and Actin, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against Biotin, PARP, Caspase-3, GAPDH antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipofectamine 3000 and JC-1 was bought from Life Technology (Carlsbad, CA, USA). TUNEL assay kits were bought from Vazyme Biotech Co., Ltd (Nanjing, China). Aneexin V/PI staining kit were bought from Beyotime Company (Nantong, China). Softlink Soft Release Avidin Resin was purchased from PROMEGA (Madison, WI, USA). GTVisinTM anti-mouse/anti-rabbit immunohistochemical analysis KIT was purchased from Gene Company (Shanghai, China). 5-FU was bought from KingYork Group Co. Ltd. (Tianjin, China). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Cell culture The HCT116 human colon cancer cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The 5-FU-resistant HCT116 (HCT116/5-FUR) cell line was induced in our lab. Cells were respectively cultured in DMEM culture medium (Gibco) supplemented with 10% FBS (fetal bovine serum, Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin, under a humidified 5% CO2 atmosphere at 37 °C in incubator.
2.3. Induction of 5-FU-resistant HCT116 cell line 5-FU-resistant HCT116 variants (HCT116/5-FU R1, HCT116/5FU R2 and HCT116/5-FU R) of each cell line were derived from each original parental (PT) cell line (HCT116) by utilizing serial passage in the presence of increasing 5-FU concentrations (continuous exposure). Initially, cells were treated with 5-FU (10 lM) for 72 h. The media and dead cells were removed and cells were allowed to recover for a further 72 h and then treated with higher concentration of 5-FU. This development period was carried out for approximately 6 months and finally we got the HCT116/5-FU R subline while HCT116/5-FU R1, HCT116/5-FU R2 were subline with partly resistant to 5-FU during this process. HCT116/5-FU R subline was then maintained continuously in the presence of 1 mM 5-FU for a further 3 months to make it stable.
2.4. Animals Nude mice (B6 background), 6–8 weeks of age, were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). They were maintained with free access to pellet food and water in plastic cages at 21 ± 2 °C and kept on a 12 h light/dark cycle. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, the United States) and the related ethical regulations of our university. All experimental protocols were approved by Ethic Committee of Nanjing University. All efforts were made to reduce the number of animals used and to minimize animals’ suffering. 2.5. Human tissue samples Human tissue samples were got from Department of Oncology, The First Affiliated Hospital with Nanjing Medical University. The study was approved by the Ethic Committee of Nanjing University and Nanjing Medical University and the methods and experimental protocols were carried out in accordance with the approved guidelines in Nanjing University. Written informed consent was obtained from all donors involved. 2.6. Xenograft model HCT116/5-FUR cells (2 106) were injected subcutaneously into the right flank of nude mice. Once the tumor reached 200 mm3, treatments were initiated as follows: group 1, PBS (vehicle); group 2, Andrographolide at 25 mg/kg of body weight once daily, ip; group 3, 5-FU at 25 mg/kg of body weight every 3 days, ip; group 4, combination of Andrographolide and 5-FU. Drugs were administered on days 0–24. The body weight of mice was measured daily. The body weight and tumor volume were measured daily after treatment begin and tumor volumes were calculated using the formula V = ab2/2. After 24 days, mice were sacrificed and solid tumors were separated. 2.7. Cell viability assay The cells were plated at a density of approximately 4 103 viable cells per well in 96-well plates. Various concentrations of compound were used to treat cells in triplicates. After incubation for the indicated time, MTT assay was performed to measure cell viability by a 96-well plate reader (Elx 800, BIOTECH). 2.8. Cell apoptosis assay Cells were incubated with Annexin V/PI at room temperature for 15 min in dark and then analyzed by FACS Calibur flow cytometry (Becton Dickinson). Annexin V+/PI and Annexin V+/PI+ cells were considered as apoptotic cells in the early and late phase, respectively. 2.9. Mitochondria membrane potential analysis Mitochondrial membrane potential was measured by JC-1 stain. Cells were washed with PBS and incubated with 5 lg/ml JC-1 at 37 °C for 30 min. Cells were then washed twice with PBS and immediately assessed FACS. A 488 nm filter was used for the excitation of JC-1. Emission filters of 535 (FL-1) and 595 nm (FL-3) were used to quantify the population of mitochondria with green (JC-1 monomers) and red (JC-1 aggregates) fluorescence.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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2.10. Western blot The cells were washed three times with ice-cold phosphate buffer solution (PBS) and then lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Nadeoxycholate, 5 mg/ml aprotinin, 5 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation and denatured by boiling in Laemmli buffer. Equal amounts of protein samples were loaded per well and separated on SDSpolyacrylamide gels, and then electrophoretically transferred onto PVDF membranes. Following blocking with 5% non-fat milk at room temperature for 1 h, membranes were incubated with primary antibodies (1:500) at 4°°C overnight and then incubated with HRP-conjugated secondary antibodies (1:5000) for 2 h at room temperature. Detection was carried out using a LumiGLO chemiluminescent substrate system (KPL, Guildford, UK).
2.11. Immunohistochemical analysis Immunohistochemical analysis was performed on paraffinembedded colonic tissue sections (5 lm). Briefly, the sections were
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deparaffinised, rehydrated and washed in 1% PBS-Tween, and then they were treated with 2% hydrogen peroxide, blocked with 3% goat serum and incubated overnight at 4 °C with anti-BAX (1:100). The slides were then processed using GTVisinTM antimouse/anti-rabbit immunohistochemical analysis KIT according to the manufacturer’s instructions. The reaction was stopped by thorough washing in water and counter-stained with Harris’s haematoxylin. Images were obtained by using Olympus IX51 light microscope (Olympus, Japan). Settings for image acquisition were identical for control and experimental tissues. 2.12. TUNEL assay Terminal deoxynucleotidyl transferase-mediated dUTP nickend label-ing (TUNEL) staining was performed using Death detection kit from Vazyme Biotech Co., Ltd following the manufacturer’s instructions. Nuclei were stained with DAPI (10 mg/ml) for 1 min. Images were acquired using confocal microscopy (Leika, Germany). The images for TUNEL were acquired with 10 objectives. The parameters for fluorescence were as follows: FITC: excitation 488 nm, emission 540 nm; DAPI: excitation 405 nm, emission 488 nm.
Fig. 1. Andrographolide sensitizes HCT116/5-FUR cells to 5-FU-induced apoptosis. (A, B) HCT116 and HCT116/5-FUR cells were treated with indicated concentrations of 5-FU for 48 h. HCT116/5-FUR cells were pretreated with 10 lM Andro for 12 h followed by indicated doses of 5-FU for 36 h. Cell viability was tested by MTT assay. The data were expressed as mean ± SD obtained from 3 independent experiments. *P < 0.05, **P < 0.01 versus control group. (C, D) HCT116/5-FUR cells were pretreated with 10 lM Andro for 12 h followed by indicated doses of 5-FU for 36 h. Cells were then analyzed by Annexin V/PI staining. Data shown are representative of 3 independent experiments. Data are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01 versus control group. (E) HCT116/5-FUR cells were pretreated with 10 lM Andro and indicated doses of 5-FU for 24 h and expression of PARP and caspase-3 were analyzed by western blot. Data shown are representative of 3 independent experiments. Data are presented as means ± SD (n = 3). * P < 0.05, **P < 0.01 versus control group.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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2.13. Pull-down of Andrographolide-bind proteins HCT116/5-FUR cell lysates were incubated with biotin, Andrographolide-biotin or biotin-Andrographolide plus Andrographolide 4 °C overnight for 12 h. The lysates were pulled down with streptavidin-conjugated beads (Softlink soft release Avidin Resin, PROMEGA) at 4 °C for another 4 h. After an extensive wash with PBS, the beads were boiled in 2 loading buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 1% bromophenol blue, 20% glycerol and 2% b-mercaptoethanol). Then, the supernatants were collected and subject to western blot for BAX.
threshold cycle numbers were obtained using BioRad CFX Manager software. The program for amplification was 1 cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 95 °C for 10 s. The primer sequences used in this study were as follows: BAX forward, 50 -CCCGAGAGGTCTTTTTCCGAG-30 ; BAX reverse, 50 -CCAGCCCATGATGGTTCTGAT-30 ; GAPDH forward, 50 -G GTCTCCTCTGACTTCAACA-30 ; GAPDH reverse, 50 -AGC CAAATTCGTTGTCATAC-30 . The expression of BAX was normalized to GAPDH.
2.16. Cellular thermal shift assay 2.14. RNA interference RNA interference sequence for BAX: GACGAACUGGACAGUAACA (Genescript Co., Ltd, Nanjing, China) lipofectermin 3000 (Life Technology, USA). RNA interference was performed by using by lipofectamine 3000 as the manufacturer’s protocols (Life Technology, CA, USA). Briefly, 2 105 cells were seeded in 6-well plates and allowed to grow to 50% confluence. Then cells were transfected with BAX si-RNA or NC si-RNA. The cells were allowed to grow for another 18 h before collected for the following experiments.
HCT116/5FU-R cells were incubated with DMSO or Andrographolide (10 lM) for 3 h, then the cells were collected and subjected to Cellular Thermal Shift Assay (CESTA) assay [16]. Briefly, Incubated cells were equally divided into 10 parts, each part got heated for 3 min under different temperatures (43, 46, 49, 52, 55, 58, 61, 64, 67, 70 °C) , then the heated cells were kept into 80 °C for 12 h, transferred to room temperature for 5 min, and all the sequence was repeated one more time. After that, cell lysates were extracted by centrifugation at 20,000g, 20 min. BAX expression was detected by western blot.
2.15. Real-time PCR 2.17. Statistical analysis Real-time PCR was performed as follows: RNA samples were reversed to cDNA and subjected to quantitative PCR, which was performed with the BioRad CFX96 TouchTM Real-Time PCR Detection System (BioRad, USA) using iQTM SYBRÒ Green Supermix, and
Data are expressed as mean ± SD. Student’s t test and one-way ANOVA test were used for statistical analyses of the data. All statistical analyses were conducted using GraphPad Prism Software Ver-
Fig. 2. Andrographolide sensitizes HCT116/5-FUR cells to 5-FU chemotherapy in vivo. HCT116/5-FUR cells (2 106) were injected subcutaneously into the right flank of nude mice. Once the tumor reached 200 mm3, treatments were initiated as indicated in materials and methods. The body weight and tumor volume were measured daily after treatment begin (A, B). (C) Representative pictures of HCT116/5-FUR xenografts on day 24 after drug administration. (D) Tumor weight after 24 days of treatment. (E, F) Expression of PARP, Caspase-3 in tumor xenografts. (G) Histological examination of tumor xenografts stained with H&E (Scale bar: 100 lm). (H) Apoptosis of tumor cells were determined by TUNEL assay. Data are presented as means ± SD (n = 6). *P < 0.05, **P < 0.01 versus control group.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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sion 5.0 (GraphPad Software Inc., La Jolla, CA). Cases with P values of <0.05 were considered statistically significant.
3. Results 3.1. Andrographolide sensitizes the cytotoxic effect of 5-FU in HCT116/ 5-FUR cells First we examined the cytotoxic effect of 5-FU on HCT116 and HCT116 5-FU resistant cell line (HCT116/5-FUR) by using MTT assay. Our results showed that, compared with HCT116, HCT116/ 5-FUR cells exhibited a significantly resistance to 5-FU (Fig. 1A).
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The IC50 for HCT116 is 9 lM, while the IC50 for HCT116/5-FU R is 1000 lM. Andrographolide (10 lM) has no effect on HCT116/5FUR cell viability. To evaluate whether Andrographolide could sensitize 5-FU-induced cytotoxic effect, we treated HCT116/5-FUR with the combination of Andrographolide and 5-FU. As shown in Fig. 1B, when the cells were pretreated with Andrographolide (10 lM) for 12 h followed by indicated doses of 5-FU for 36 h, Andrographolide dramatically sensitized HCT116/5-FUR cells to 5-FU-induced cytotoxic effect. Apoptosis rate detected by Annexin V/PI staining also proved that Andrographolide pretreatment elevated 5-FU-induced cell apoptosis (Fig. 1C and D). Then we further examined the expression of apoptotic-related molecules induced
Fig. 3. Andrographolide enhanced protein expression level of BAX in vitro and in vivo. (A) HCT116/5-FUR cells were cultured with increasing concentrations of Andrographolide for 12 or 24 h. (B) HCT116/5-FUR cells were cultured with and without 10 lM Andrographolide in the presence or absence of 2 lg/ml actinomycin D (Act D) or 4 lM cycloheximide (CHX) for 24 h. (C, D) Expression of BAX in tumor xenografts after drug administration. (E) After treatment with 10 lM Andrographolide and indicated doses of 5-FU for 24 h, HCT116/5-FUR cells were stained with JC-1 to evaluate the mitochondrial membrane potential (MMP). Data shown are representative of 3 independent experiments. Data represented the mean ± SD (n = 3). *P < 0.05, **P < 0.01 versus control group.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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by Andrographolide and/or 5-FU treatment. As shown in Fig. 1E, PARP and Capase-9 were abundantly activated by Andrographolide and 5-FU combination. All these results suggest that Andrographolide significantly improved 5-FU sensitivity in colon cancer cells. 3.2. Andrographolide enhanced therapeutic effect of 5-FU in vivo To confirm whether Andrographolide can also sensitize the cytotoxic effect of 5-FU to 5-FU-resistant colorectal cancer in vivo, the effects of 5-FU alone or in combination with Andrographolide on the tumor growth of HCT116/5-FUR was tested in the subcutaneous xenograft model. The 5-FU treatment caused mice bodyweight decrease while co-treatment with Andrographolide and 5-FU or Andrographolide alone showed less effect on the bodyweight change, indicating the safety and low toxicity of this combination (Fig. 2A). 5-FU or Andrographolide alone has no inhibition on 5-FU-resistance tumor growth while their combination significantly suppressed its growth (Fig. 2B–D). Specifically, there is a 53% reduction of tumor volume and 56% reduction of tumor weight observed in Andrographolide and 5-FU co-treated mice compared to the control group. The same as the results got in vitro, western blot of PARP and caspase-3, H&E and tunnel staining all showed that the combination therapy significantly promoted the apoptosis of HCT116/5-FUR cells in vivo (Fig. 2E–G). 3.3. Andrographolide promoted BAX protein expression both in vitro and in vivo The above results have proved that Andrographolide combined with 5-FU can significantly induced apoptosis of HCT116/5-FUR cells. Next we wondered what the mechanism under this effect is. Occasionally, we found that Andrographolide can time and dose dependently elevate BAX protein levels in HCT116/5-FUR cells (Fig. 3A) while the mRNA level remains unchanged. And the transcription inhibitors CHX and Actinomycin D only partially suppressed the elevated level of BAX protein induced by Andrographolide (Fig. 3B). In addition, western blot analysis and immunohistochemistry of tumor samples from subcutaneous
xenograft model of HCT116/5-FUR cells in mice demonstrated that the expressions of BAX were highly elevated by the Andrographolide combination comparing to control or 5-FU alone (Fig. 3C and D). Our data here indicated that Andrographolide may promote BAX protein level via a post-transcriptional mechanism. As we know, the main function of BAX is to forming pores alone or with BAK within the mitochondrial outer membrane and enables the release of cytochrome c and Smac et al. which execute the apoptosis process [17]. With the increased of level of BAXinduced by Andrographolide, the membrane potential was collapsed dramatically with 5-FU treatment, indicating Andrographolide-induced BAX was biological functional (Fig. 3E).
3.4. Andrographolide directly targets BAX As we can see that Andrographolide may promote BAX protein level via preventing its degradation, we examined the possibility of Andrographolide directly binding with BAX. First, we designed a chemical probe biotin-labeled Andrographolide (biotinAndrographolide) as reported before [18]. By using this probe, we performed pull down assay in the total cell extracts from HCT116/5-FUR cells via streptavidin system. As shown in Fig. 4A and B, western blot analysis proved that biotinAndrographolide can dose-dependently bind with BAX and this bind can be competed away by high concentrations of unlabeled Andrographolide. In order to further confirm this bind, we employed the cellular thermal shift assay (CETSA) which can evaluate the bind of compound with target protein inside the cell based on ligand-induced stabilization of the target protein [16]. BAX started to degrade at 59 °C and almost disappeared at 61 °C in vehicle-treated cells while it delayed to 61–64 °C and disappeared at 70 °C in 10 lM Andrographolide-treated cells (Fig. 4C). Then we treated cells with five doses of Andrographolide and heated at 61 °C. The results showed a dose-pendent increase of the stability of BAX (Fig. 4D). All these results demonstrated that Andrographolide directly targets BAX.
Fig. 4. Andrographolide directly targets BAX. (A) HCT116/5-FUR cell lysates were incubated with biotin-Andrographolide (1, 3, 10 lM) or biotin followed by pull-down with streptavidin-agarose. The precipitates were resolved by SDS-PAGE, and detected by western blot for BAX protein. (B) HCT116/ 5FU-R cell lysates were incubated with biotinAndrographolide or biotin in the absence or presence of a 10-fold excess of Andrographolide, followed by pull-down with streptavidin-agarose. The precipitates were resolved by SDS-PAGE, and detected by western blot for BAX. (C) HCT116/5-FUR cells were incubated with DMSO or Andrographolide (10 lM) for 3 h, then the cells were collected and subjected to CESTA assay. (D) HCT116/ 5FU-R cells were incubated with Andrographolide (0, 0.1, 0.3, 1, 3, 10 lM) for 3 h, then the cells were collected and subjected to CESTA assay at the temperature of 61 °C. Data shown are representative of 3 independent experiments.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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3.5. Andrographolide enhanced 5-FU sensitivity dependent on BAX To confirm that Andrographolide enhance 5-FU sensitivity dependent on BAX, we knock down BAX in HCT116/5-FUR cells by siRNA. The effect of knock down was confirmed by western blot (Fig. 5A). In these BAX-knocked down HCT116/5-FUR cells, we detected the combination effect of Andrographolide and 5-FU with MTT and Annexin V/PI stain assay. As shown in Fig. 5B–E, in the siNC cells, BAX Andrographolide can still enhance the inhibitory effect of 5-FU while this effect was diminished in the si-BAX cells. 3.6. BAX expression was related to patient sensitivity to 5-FU-involved chemotherapy All the above results suggest that elevation of BAX expression in HCT116/5-FUR can restore the sensitivity of 5-FU. At the same
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time, we found that in the HCT116 cells with different sensitivity to 5-FU (HCT116/5-FUR1, HCT116/5-FUR2, HCT116/5-FUR), the expression of BAX positively correlated with the sensitivity (Fig. 6A and B). In order to confirm this phenomenon in clinic, we collected the samples from patients who were undergoing 5FU-involved chemotherapy and analyzed the correction between BAX expression and PFS. The patient with higher expression of BAX shows longer PFS (PFS, Progression-Free-Survival, which means the length of time during and after the treatment of a disease that a patient lives with the disease but it does not get worse. In a clinical trial, measuring the progression-free survival is one way to see how well a new treatment works.), the correlation coefficient is 0.2516 (Fig. 6C–E). Then we divided patients into two groups according to the BAX score (BAX low expression group and BAX high expression group). The PFS of BAX high expression group is relative longer than the BAX low expression group
Fig. 5. Andrographolide enhance 5-FU sensitivity dependent on BAX. (A) HCT116/5-FUR cells were transfected with si-NC or si-BAX for 36 h, then the level of BAX protein expression was detected by western blot analysis. (B, C) HCT116/ 5FU-R cells were transfected with si-NC or si-BAX for 36 h, then treated with 10 lM Andro for 12 h followed by indicated doses of 5-FU for 36 h. Cell viability was tested by MTT assay. The data were expressed as mean ± SD obtained from triplicate samples. *P < 0.05, **P < 0.01 versus control group. (D, E) After transfected with si-NC or si-BAX for 36 h, cells were treated with 10 lM Andro and indicated doses of 5-FU for 24 h, then analyzed by Annexin V/PI staining. Data shown are representative of 3 independent experiments. Data are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01 versus control group.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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Fig. 6. BAX expression was related to patient sensitivity to 5-FU-invovled chemotherapy. (A) The expression level of BAX in HCT116 cells with different sensitive to 5-FU (HCT116, HCT116/5-FUR1, HCT116/5-FUR2, HCT116/5-FUR) were detected by western blot. (B) Expression of BAX was detected in tumor xenografts of HCT116 cells and HCT116/5-FUR cells by IHC staining. (C) Representative IHC images of colorectal cancer tissues which before and after the treatment including 5-FU. (D, E, F) Relationship between BAX expression is significantly correlated and PFS of colorectal cancer patients.
(P = 0.134) (Fig. 6F). These results indicated that the expression of BAX was related to patient sensitivity to 5-FU-involved chemotherapy. 4. Discussion Up to now, 5-FU is still the most important chemotherapeutic drug used for the treatment of CRC, but still it is urgent to solve the problem of 5-FU resistance. Multiple mechanisms have been proposed to explain and several small molecules showed the ability to overcome 5-FU resistance. Overexpression of thymidylate synthase has been found to be associated with 5-FU resistance in colorectal cancer [19,20]. Glut1 expression was upregulated in 5-FU resistant cancer cells and exogenous overexpression of Glut1 facilitated colorectal cancer cells obtain resistance to 5-FU and the Glut1 specific inhibitor WZB117 can increase the sensitivity of 5-FU resistant cells to the drug [21]. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-FU cytotoxicity by elevating ER-b protein expression [22]. Our study here provides a novel mechanism for 5-Fu resistance in colon cancer cells probed by using a small natural compound Andrographolide. Andrographolide is a lactone diterpenoid originally isolated from the Chinese herb. In recent years, novel pharmacological effect of andrographolide has been identified, including anti-inflammation, anti-angiogenesis, and anti-cancer. In the present study, we demonstrated that Andrographolide could enhance sensitivity to 5-FU in 5-FU resistant CRC through directly targeting BAX. Apoptosis is one of the major mechanisms of cell death in response to chemotherapy drugs [23]. So defects in apoptotic pathways can promote cancer cell survival and also confer resistance to
chemotherapy [24,25]. BAX, a central cell death regulator, is an indispensable gateway to mitochondrial dysfunction and a major proapoptotic member of the B-cell lymphoma 2 (Bcl-2) family proteins that control apoptosis in normal and cancer cells [26,27]. Recent studies have shown that enhanced BAX expression levels correlate with a good response to chemotherapy in leukemia and low expression levels correlate with a poor prognosis in colorectal cancer [28,29]. Also, the down-regulation of BAX plays an important role in carcinogenesis and acquisition of resistance to 5-FU in colorectal cancer [30,31]. In our study, compared with the HCT116 cells, the levels of BAX in HCT116/5-FUR cells distinctly decreased. With the increase extent of drug resistance, the level of BAX presented gradually down regulated. What’s more, the study of clinical samples from patients who received 5-FUinvolved chemotherapy further confirmed these results. The clinical outcome showed that compared with the patients with low level of BAX, the patients with high level BAX have a longer PFS. So these results all revealed the relationship between downregulation of BAX and 5-FU resistance. Accumulating evidence suggests that BAX can serve as a promising direct target for cancer therapy [32,33]. A number of anticancer drugs in the clinic induce BAX activation indirectly to facilitate apoptosis while none of them can directly activate or bind to BAX. Usually, many anticancer agents induce cancer cell death via BAX-mediated apoptosis pathway, suggesting that direct activation or elevation of BAX expression may also provide useful agents for novel drug combinations. Several direct BAX activators have been identified to hold promise for cancer therapy with the advantages of specificity and the potential of overcoming chemoresistance [34,35]. However, no such activators have been applied to clinical.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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Our study here found out that Andrographolide could elevate BAX level in 5-FU resistant cells. This elevated BAX contributed to the increase apoptosis induced by 5-FU. When knocked down BAX by siRNA, the effect of Andrographolide and 5-FU combination on apoptosis was diminished, confirming the BAX-dependent mechanism. All these results further suggested that BAX could be one therapy target in the combined treatment for cancer. Our research provided a clue for new indications with a drug already in clinical use. Actually, we are carrying out a clinical trial for the combined use of Xeloda and Xi-Yan-Ping injection (Andrographolide dosage form) in colorectal cancer patient. Previously, Andrographolide has been shown to induce apoptosis in cancer cells or sensitizes cancer cells to chemotherapy [36– 38]. Mechanism study showed that BAX-mediated apoptosis was involved in the therapy effect of Andrographolide, however, no evidence has proved BAX is a direct target of Andrographolide. Interestingly, in the present study, we found Andrographolide could directly bind to BAX, enhancing its stability without affecting its mRNA level. Based on these results, BAX may be a molecular target of Andrographolide in 5-FU resistant CRC. In summary, it could be concluded from these studies that, Andrographolide synergizes the cytotoxic effects of 5-FU in colon cancer cells via binding to BAX and triggering mitochondria mediated apoptosis in vivo and vitro. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing low level of BAX protein. Nevertheless, due to the modest effects achieved in terms of the re-sensitization of resistant colon cancer cells to 5-FU in this study, we are still trying to find more combinations between Andrographolide and other chemotherapeutics including apoptosis inducer for the extrinsic pathway, autophagic cell death inducer et al. to fully overcome drug-resistance. Conflict of interest The authors declared no conflict of interest. Author contributions W.W., W.G., and L.L. performed the experiments. W.W., W.L. and W.G. analyzed the data and prepared the figures. Z.F., J.G. and Y. Su provided expertise and the mouse lines. W.G., Q.X., Y. Sun. and Y.G. conceived, directed the study and wrote the manuscript. All authors discussed the results and commented on the manuscript. Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 91429308, 81402938, 81572389), Key Personnel of Jiangsu Province (RC2011170), Jiangsu Province Clinical Science and Technology Projects (Clinical Research Center, BL2012008) and Priority Academic Program Development of Jiangsu Higher Education Institutions. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA Cancer J. Clin. 65 (2) (2015) 87–108. [2] B. Chibaudel, C. Tournigand, T. Andre, A. de Gramont, Therapeutic strategy in unresectable metastatic colorectal cancer, Ther. Adv. Med. Oncol. 4 (2) (2012) 75–89. [3] B. Gustavsson, G. Carlsson, D. Machover, N. Petrelli, A. Roth, H.J. Schmoll, K.M. Tveit, F. Gibson, A review of the evolution of systemic chemotherapy in the management of colorectal cancer, Clin. Colorectal Cancer 14 (1) (2015) 1–10. [4] D.B. Longley, D.P. Harkin, P.G. Johnston, 5-Fluorouracil: mechanisms of action and clinical strategies, Nat. Rev. Cancer 3 (5) (2003) 330–338.
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Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression Weicheng Wang a,1, Wenjie Guo b,1, Lele Li a,1, Zan Fu a, Wen Liu b, Jian Gao b, Yongqian Shu a, Qiang Xu b,⇑, Yang Sun b,⇑, Yanhong Gu a,⇑ a b
Department of Oncology, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China
a r t i c l e
i n f o
Article history: Received 27 July 2016 Accepted 22 September 2016 Available online xxxx Keywords: Andrographolide BAX 5-FU-resistant Human colorectal cancer
a b s t r a c t 5-FU is the first line therapy for colorectal cancer, however, treatment effect is often hampered by the development of drug resistance or toxicity at high doses. Andrographolide is a natural diterpenoid from Andrographis paniculata which has anti-bacterial, anti-antiviral and anti-inflammation activities. In the current study, we test the hypothesis that Andrographolide reverses 5-FU resistance in colorectal cancer and examine the underlying mechanism. In vitro and vivo studies indicated that Andrographolide treatment significantly re-sensitizes HCT116/5-FUR cells (HCT116 cells which are 5-FU resistant) to cytotoxicity of 5-FU. Mechanism analysis showed that Andrographolide/5-FU co-treatment elevated apoptosis level of HCT116/5-FUR cells with highly increased level of BAX. By using biotin-Andrographolide pull down and cellular thermal shift assay, we found out that Andrographolide can directly target to BAX. Andrographolide-BAX interaction prevented BAX degradation, enhancing mitochondria-mediated apoptosis thus reversed 5-FU resistance while BAX silence diminished this effect. Further, by analyzing patient samples who received 5-FU involved chemotherapy, we found that expression level of BAX is correlated with PFS. Our results here provide a novel combination treatment strategy, especially for patients with 5FU-resistant tumors expressing low level of BAX. Meanwhile, we also proposed that BAX expression may be a predicted and prognosis marker of 5-FU involved chemotherapy. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Colorectal cancer (CRC) is one of the most commonly diagnosed solid tumors worldwide. Colorectal cancer is the second leading cause of cancer deaths in the United States and the third most common malignant neoplasm worldwide [1]. Current therapies for the treatment of CRC mainly comprise 5-Fluorouracil (5-FU)based chemotherapies that are used individually or in combination with oxaliplatin or anti-angiogenic agents, and/or anti-epidermal growth factor agents [2,3]. As a false substrate to thymidylate synthase enzyme that incorporates into DNA and RNA, 5-FU treatment causes the growth restrain and apoptosis of cancer cells [4]. Although CRC incidence rates have declined somewhat, current
⇑ Corresponding authors at: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, China (Q. Xu and Y. Sun). Department of Oncology, The First Affiliated Hospital with Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China (Y. Gu). E-mail addresses: [email protected] (Q. Xu), [email protected] (Y. Sun), [email protected] (Y. Gu). 1 Contributed equally to this work.
therapies are associated with significant side effects, high expense and recurrence rates can be as high as 50–60%, primarily due to the development of acquired resistance to 5-FU-based chemotherapeutics, as more than 15% of patients are resistant to 5-FU-based chemotherapies [5,6]. Thus, resistance to 5-FU has been a major obstacle in advanced colorectal cancer chemotherapy and confines its use in clinical practice. Thus novel and safe treatment strategies which can help overcome chemoresistance and enhance CRC response to 5-FU-based chemotherapies are desperately needed. Andrographolide, a natural diterpenoid from Andrographis paniculata, has been used as a herbal medicine in China for thousands of years. Andrographolide has been reported to exert antibacterial, antiviral, anti-inflammation and neuroprotective activities [7–9]. Our previous studies have proved that Andrographolide can alleviate colitis and suppress colitis-associated colon cancer [10,11]. Recent studies showed that Andrographolide alone can induce apoptosis of different cancer cells via regulating pro-apoptotic Bcl-2 family members [12]. Another group has reported that Andrographolide can inhibit tumor growth in mice although at a relatively high dose (about 200 mg/kg) [13]. What’s more, Andrographolide can potentiate the cytotoxic effect of various
http://dx.doi.org/10.1016/j.bcp.2016.09.024 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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chemotherapy drugs in different cell lines via enhancing apoptosis pathways or via suppression of pro-survival autophagy [14,15]. However, in colorectal cancer, no investigations about the effects and mechanism of Andrographolide on CRC 5-FU resistance have been reported. In the present study, we investigated the effect of Andrographolide in combination with 5-FU on various growth regulatory parameters and extensive characterization of underlying mechanisms in a 5-FU-resistant CRC cell line in vivo and vitro. Our data firstly reveal that the bind and up-regulation of BAX expression by Andrographolide is one of the principle mechanisms for resensitizing 5-FU-resistant CRC cells to 5-FU. At the same time, we found that BAX expression level maybe a prognosis marker for 5FU-based treatment. These findings highlight the potential possibility of using this natural, safe and relatively inexpensive compound as potential adjunctive treatments in improving the overall treatment response of patients with CRC in future.
2. Methods 2.1. Chemicals and reagents Primary antibodies against BAX, BCL2 and Actin, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against Biotin, PARP, Caspase-3, GAPDH antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipofectamine 3000 and JC-1 was bought from Life Technology (Carlsbad, CA, USA). TUNEL assay kits were bought from Vazyme Biotech Co., Ltd (Nanjing, China). Aneexin V/PI staining kit were bought from Beyotime Company (Nantong, China). Softlink Soft Release Avidin Resin was purchased from PROMEGA (Madison, WI, USA). GTVisinTM anti-mouse/anti-rabbit immunohistochemical analysis KIT was purchased from Gene Company (Shanghai, China). 5-FU was bought from KingYork Group Co. Ltd. (Tianjin, China). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Cell culture The HCT116 human colon cancer cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The 5-FU-resistant HCT116 (HCT116/5-FUR) cell line was induced in our lab. Cells were respectively cultured in DMEM culture medium (Gibco) supplemented with 10% FBS (fetal bovine serum, Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin, under a humidified 5% CO2 atmosphere at 37 °C in incubator.
2.3. Induction of 5-FU-resistant HCT116 cell line 5-FU-resistant HCT116 variants (HCT116/5-FU R1, HCT116/5FU R2 and HCT116/5-FU R) of each cell line were derived from each original parental (PT) cell line (HCT116) by utilizing serial passage in the presence of increasing 5-FU concentrations (continuous exposure). Initially, cells were treated with 5-FU (10 lM) for 72 h. The media and dead cells were removed and cells were allowed to recover for a further 72 h and then treated with higher concentration of 5-FU. This development period was carried out for approximately 6 months and finally we got the HCT116/5-FU R subline while HCT116/5-FU R1, HCT116/5-FU R2 were subline with partly resistant to 5-FU during this process. HCT116/5-FU R subline was then maintained continuously in the presence of 1 mM 5-FU for a further 3 months to make it stable.
2.4. Animals Nude mice (B6 background), 6–8 weeks of age, were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). They were maintained with free access to pellet food and water in plastic cages at 21 ± 2 °C and kept on a 12 h light/dark cycle. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, the United States) and the related ethical regulations of our university. All experimental protocols were approved by Ethic Committee of Nanjing University. All efforts were made to reduce the number of animals used and to minimize animals’ suffering. 2.5. Human tissue samples Human tissue samples were got from Department of Oncology, The First Affiliated Hospital with Nanjing Medical University. The study was approved by the Ethic Committee of Nanjing University and Nanjing Medical University and the methods and experimental protocols were carried out in accordance with the approved guidelines in Nanjing University. Written informed consent was obtained from all donors involved. 2.6. Xenograft model HCT116/5-FUR cells (2 106) were injected subcutaneously into the right flank of nude mice. Once the tumor reached 200 mm3, treatments were initiated as follows: group 1, PBS (vehicle); group 2, Andrographolide at 25 mg/kg of body weight once daily, ip; group 3, 5-FU at 25 mg/kg of body weight every 3 days, ip; group 4, combination of Andrographolide and 5-FU. Drugs were administered on days 0–24. The body weight of mice was measured daily. The body weight and tumor volume were measured daily after treatment begin and tumor volumes were calculated using the formula V = ab2/2. After 24 days, mice were sacrificed and solid tumors were separated. 2.7. Cell viability assay The cells were plated at a density of approximately 4 103 viable cells per well in 96-well plates. Various concentrations of compound were used to treat cells in triplicates. After incubation for the indicated time, MTT assay was performed to measure cell viability by a 96-well plate reader (Elx 800, BIOTECH). 2.8. Cell apoptosis assay Cells were incubated with Annexin V/PI at room temperature for 15 min in dark and then analyzed by FACS Calibur flow cytometry (Becton Dickinson). Annexin V+/PI and Annexin V+/PI+ cells were considered as apoptotic cells in the early and late phase, respectively. 2.9. Mitochondria membrane potential analysis Mitochondrial membrane potential was measured by JC-1 stain. Cells were washed with PBS and incubated with 5 lg/ml JC-1 at 37 °C for 30 min. Cells were then washed twice with PBS and immediately assessed FACS. A 488 nm filter was used for the excitation of JC-1. Emission filters of 535 (FL-1) and 595 nm (FL-3) were used to quantify the population of mitochondria with green (JC-1 monomers) and red (JC-1 aggregates) fluorescence.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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2.10. Western blot The cells were washed three times with ice-cold phosphate buffer solution (PBS) and then lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Nadeoxycholate, 5 mg/ml aprotinin, 5 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation and denatured by boiling in Laemmli buffer. Equal amounts of protein samples were loaded per well and separated on SDSpolyacrylamide gels, and then electrophoretically transferred onto PVDF membranes. Following blocking with 5% non-fat milk at room temperature for 1 h, membranes were incubated with primary antibodies (1:500) at 4°°C overnight and then incubated with HRP-conjugated secondary antibodies (1:5000) for 2 h at room temperature. Detection was carried out using a LumiGLO chemiluminescent substrate system (KPL, Guildford, UK).
2.11. Immunohistochemical analysis Immunohistochemical analysis was performed on paraffinembedded colonic tissue sections (5 lm). Briefly, the sections were
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deparaffinised, rehydrated and washed in 1% PBS-Tween, and then they were treated with 2% hydrogen peroxide, blocked with 3% goat serum and incubated overnight at 4 °C with anti-BAX (1:100). The slides were then processed using GTVisinTM antimouse/anti-rabbit immunohistochemical analysis KIT according to the manufacturer’s instructions. The reaction was stopped by thorough washing in water and counter-stained with Harris’s haematoxylin. Images were obtained by using Olympus IX51 light microscope (Olympus, Japan). Settings for image acquisition were identical for control and experimental tissues. 2.12. TUNEL assay Terminal deoxynucleotidyl transferase-mediated dUTP nickend label-ing (TUNEL) staining was performed using Death detection kit from Vazyme Biotech Co., Ltd following the manufacturer’s instructions. Nuclei were stained with DAPI (10 mg/ml) for 1 min. Images were acquired using confocal microscopy (Leika, Germany). The images for TUNEL were acquired with 10 objectives. The parameters for fluorescence were as follows: FITC: excitation 488 nm, emission 540 nm; DAPI: excitation 405 nm, emission 488 nm.
Fig. 1. Andrographolide sensitizes HCT116/5-FUR cells to 5-FU-induced apoptosis. (A, B) HCT116 and HCT116/5-FUR cells were treated with indicated concentrations of 5-FU for 48 h. HCT116/5-FUR cells were pretreated with 10 lM Andro for 12 h followed by indicated doses of 5-FU for 36 h. Cell viability was tested by MTT assay. The data were expressed as mean ± SD obtained from 3 independent experiments. *P < 0.05, **P < 0.01 versus control group. (C, D) HCT116/5-FUR cells were pretreated with 10 lM Andro for 12 h followed by indicated doses of 5-FU for 36 h. Cells were then analyzed by Annexin V/PI staining. Data shown are representative of 3 independent experiments. Data are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01 versus control group. (E) HCT116/5-FUR cells were pretreated with 10 lM Andro and indicated doses of 5-FU for 24 h and expression of PARP and caspase-3 were analyzed by western blot. Data shown are representative of 3 independent experiments. Data are presented as means ± SD (n = 3). * P < 0.05, **P < 0.01 versus control group.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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2.13. Pull-down of Andrographolide-bind proteins HCT116/5-FUR cell lysates were incubated with biotin, Andrographolide-biotin or biotin-Andrographolide plus Andrographolide 4 °C overnight for 12 h. The lysates were pulled down with streptavidin-conjugated beads (Softlink soft release Avidin Resin, PROMEGA) at 4 °C for another 4 h. After an extensive wash with PBS, the beads were boiled in 2 loading buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 1% bromophenol blue, 20% glycerol and 2% b-mercaptoethanol). Then, the supernatants were collected and subject to western blot for BAX.
threshold cycle numbers were obtained using BioRad CFX Manager software. The program for amplification was 1 cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 95 °C for 10 s. The primer sequences used in this study were as follows: BAX forward, 50 -CCCGAGAGGTCTTTTTCCGAG-30 ; BAX reverse, 50 -CCAGCCCATGATGGTTCTGAT-30 ; GAPDH forward, 50 -G GTCTCCTCTGACTTCAACA-30 ; GAPDH reverse, 50 -AGC CAAATTCGTTGTCATAC-30 . The expression of BAX was normalized to GAPDH.
2.16. Cellular thermal shift assay 2.14. RNA interference RNA interference sequence for BAX: GACGAACUGGACAGUAACA (Genescript Co., Ltd, Nanjing, China) lipofectermin 3000 (Life Technology, USA). RNA interference was performed by using by lipofectamine 3000 as the manufacturer’s protocols (Life Technology, CA, USA). Briefly, 2 105 cells were seeded in 6-well plates and allowed to grow to 50% confluence. Then cells were transfected with BAX si-RNA or NC si-RNA. The cells were allowed to grow for another 18 h before collected for the following experiments.
HCT116/5FU-R cells were incubated with DMSO or Andrographolide (10 lM) for 3 h, then the cells were collected and subjected to Cellular Thermal Shift Assay (CESTA) assay [16]. Briefly, Incubated cells were equally divided into 10 parts, each part got heated for 3 min under different temperatures (43, 46, 49, 52, 55, 58, 61, 64, 67, 70 °C) , then the heated cells were kept into 80 °C for 12 h, transferred to room temperature for 5 min, and all the sequence was repeated one more time. After that, cell lysates were extracted by centrifugation at 20,000g, 20 min. BAX expression was detected by western blot.
2.15. Real-time PCR 2.17. Statistical analysis Real-time PCR was performed as follows: RNA samples were reversed to cDNA and subjected to quantitative PCR, which was performed with the BioRad CFX96 TouchTM Real-Time PCR Detection System (BioRad, USA) using iQTM SYBRÒ Green Supermix, and
Data are expressed as mean ± SD. Student’s t test and one-way ANOVA test were used for statistical analyses of the data. All statistical analyses were conducted using GraphPad Prism Software Ver-
Fig. 2. Andrographolide sensitizes HCT116/5-FUR cells to 5-FU chemotherapy in vivo. HCT116/5-FUR cells (2 106) were injected subcutaneously into the right flank of nude mice. Once the tumor reached 200 mm3, treatments were initiated as indicated in materials and methods. The body weight and tumor volume were measured daily after treatment begin (A, B). (C) Representative pictures of HCT116/5-FUR xenografts on day 24 after drug administration. (D) Tumor weight after 24 days of treatment. (E, F) Expression of PARP, Caspase-3 in tumor xenografts. (G) Histological examination of tumor xenografts stained with H&E (Scale bar: 100 lm). (H) Apoptosis of tumor cells were determined by TUNEL assay. Data are presented as means ± SD (n = 6). *P < 0.05, **P < 0.01 versus control group.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
W. Wang et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
sion 5.0 (GraphPad Software Inc., La Jolla, CA). Cases with P values of <0.05 were considered statistically significant.
3. Results 3.1. Andrographolide sensitizes the cytotoxic effect of 5-FU in HCT116/ 5-FUR cells First we examined the cytotoxic effect of 5-FU on HCT116 and HCT116 5-FU resistant cell line (HCT116/5-FUR) by using MTT assay. Our results showed that, compared with HCT116, HCT116/ 5-FUR cells exhibited a significantly resistance to 5-FU (Fig. 1A).
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The IC50 for HCT116 is 9 lM, while the IC50 for HCT116/5-FU R is 1000 lM. Andrographolide (10 lM) has no effect on HCT116/5FUR cell viability. To evaluate whether Andrographolide could sensitize 5-FU-induced cytotoxic effect, we treated HCT116/5-FUR with the combination of Andrographolide and 5-FU. As shown in Fig. 1B, when the cells were pretreated with Andrographolide (10 lM) for 12 h followed by indicated doses of 5-FU for 36 h, Andrographolide dramatically sensitized HCT116/5-FUR cells to 5-FU-induced cytotoxic effect. Apoptosis rate detected by Annexin V/PI staining also proved that Andrographolide pretreatment elevated 5-FU-induced cell apoptosis (Fig. 1C and D). Then we further examined the expression of apoptotic-related molecules induced
Fig. 3. Andrographolide enhanced protein expression level of BAX in vitro and in vivo. (A) HCT116/5-FUR cells were cultured with increasing concentrations of Andrographolide for 12 or 24 h. (B) HCT116/5-FUR cells were cultured with and without 10 lM Andrographolide in the presence or absence of 2 lg/ml actinomycin D (Act D) or 4 lM cycloheximide (CHX) for 24 h. (C, D) Expression of BAX in tumor xenografts after drug administration. (E) After treatment with 10 lM Andrographolide and indicated doses of 5-FU for 24 h, HCT116/5-FUR cells were stained with JC-1 to evaluate the mitochondrial membrane potential (MMP). Data shown are representative of 3 independent experiments. Data represented the mean ± SD (n = 3). *P < 0.05, **P < 0.01 versus control group.
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by Andrographolide and/or 5-FU treatment. As shown in Fig. 1E, PARP and Capase-9 were abundantly activated by Andrographolide and 5-FU combination. All these results suggest that Andrographolide significantly improved 5-FU sensitivity in colon cancer cells. 3.2. Andrographolide enhanced therapeutic effect of 5-FU in vivo To confirm whether Andrographolide can also sensitize the cytotoxic effect of 5-FU to 5-FU-resistant colorectal cancer in vivo, the effects of 5-FU alone or in combination with Andrographolide on the tumor growth of HCT116/5-FUR was tested in the subcutaneous xenograft model. The 5-FU treatment caused mice bodyweight decrease while co-treatment with Andrographolide and 5-FU or Andrographolide alone showed less effect on the bodyweight change, indicating the safety and low toxicity of this combination (Fig. 2A). 5-FU or Andrographolide alone has no inhibition on 5-FU-resistance tumor growth while their combination significantly suppressed its growth (Fig. 2B–D). Specifically, there is a 53% reduction of tumor volume and 56% reduction of tumor weight observed in Andrographolide and 5-FU co-treated mice compared to the control group. The same as the results got in vitro, western blot of PARP and caspase-3, H&E and tunnel staining all showed that the combination therapy significantly promoted the apoptosis of HCT116/5-FUR cells in vivo (Fig. 2E–G). 3.3. Andrographolide promoted BAX protein expression both in vitro and in vivo The above results have proved that Andrographolide combined with 5-FU can significantly induced apoptosis of HCT116/5-FUR cells. Next we wondered what the mechanism under this effect is. Occasionally, we found that Andrographolide can time and dose dependently elevate BAX protein levels in HCT116/5-FUR cells (Fig. 3A) while the mRNA level remains unchanged. And the transcription inhibitors CHX and Actinomycin D only partially suppressed the elevated level of BAX protein induced by Andrographolide (Fig. 3B). In addition, western blot analysis and immunohistochemistry of tumor samples from subcutaneous
xenograft model of HCT116/5-FUR cells in mice demonstrated that the expressions of BAX were highly elevated by the Andrographolide combination comparing to control or 5-FU alone (Fig. 3C and D). Our data here indicated that Andrographolide may promote BAX protein level via a post-transcriptional mechanism. As we know, the main function of BAX is to forming pores alone or with BAK within the mitochondrial outer membrane and enables the release of cytochrome c and Smac et al. which execute the apoptosis process [17]. With the increased of level of BAXinduced by Andrographolide, the membrane potential was collapsed dramatically with 5-FU treatment, indicating Andrographolide-induced BAX was biological functional (Fig. 3E).
3.4. Andrographolide directly targets BAX As we can see that Andrographolide may promote BAX protein level via preventing its degradation, we examined the possibility of Andrographolide directly binding with BAX. First, we designed a chemical probe biotin-labeled Andrographolide (biotinAndrographolide) as reported before [18]. By using this probe, we performed pull down assay in the total cell extracts from HCT116/5-FUR cells via streptavidin system. As shown in Fig. 4A and B, western blot analysis proved that biotinAndrographolide can dose-dependently bind with BAX and this bind can be competed away by high concentrations of unlabeled Andrographolide. In order to further confirm this bind, we employed the cellular thermal shift assay (CETSA) which can evaluate the bind of compound with target protein inside the cell based on ligand-induced stabilization of the target protein [16]. BAX started to degrade at 59 °C and almost disappeared at 61 °C in vehicle-treated cells while it delayed to 61–64 °C and disappeared at 70 °C in 10 lM Andrographolide-treated cells (Fig. 4C). Then we treated cells with five doses of Andrographolide and heated at 61 °C. The results showed a dose-pendent increase of the stability of BAX (Fig. 4D). All these results demonstrated that Andrographolide directly targets BAX.
Fig. 4. Andrographolide directly targets BAX. (A) HCT116/5-FUR cell lysates were incubated with biotin-Andrographolide (1, 3, 10 lM) or biotin followed by pull-down with streptavidin-agarose. The precipitates were resolved by SDS-PAGE, and detected by western blot for BAX protein. (B) HCT116/ 5FU-R cell lysates were incubated with biotinAndrographolide or biotin in the absence or presence of a 10-fold excess of Andrographolide, followed by pull-down with streptavidin-agarose. The precipitates were resolved by SDS-PAGE, and detected by western blot for BAX. (C) HCT116/5-FUR cells were incubated with DMSO or Andrographolide (10 lM) for 3 h, then the cells were collected and subjected to CESTA assay. (D) HCT116/ 5FU-R cells were incubated with Andrographolide (0, 0.1, 0.3, 1, 3, 10 lM) for 3 h, then the cells were collected and subjected to CESTA assay at the temperature of 61 °C. Data shown are representative of 3 independent experiments.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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3.5. Andrographolide enhanced 5-FU sensitivity dependent on BAX To confirm that Andrographolide enhance 5-FU sensitivity dependent on BAX, we knock down BAX in HCT116/5-FUR cells by siRNA. The effect of knock down was confirmed by western blot (Fig. 5A). In these BAX-knocked down HCT116/5-FUR cells, we detected the combination effect of Andrographolide and 5-FU with MTT and Annexin V/PI stain assay. As shown in Fig. 5B–E, in the siNC cells, BAX Andrographolide can still enhance the inhibitory effect of 5-FU while this effect was diminished in the si-BAX cells. 3.6. BAX expression was related to patient sensitivity to 5-FU-involved chemotherapy All the above results suggest that elevation of BAX expression in HCT116/5-FUR can restore the sensitivity of 5-FU. At the same
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time, we found that in the HCT116 cells with different sensitivity to 5-FU (HCT116/5-FUR1, HCT116/5-FUR2, HCT116/5-FUR), the expression of BAX positively correlated with the sensitivity (Fig. 6A and B). In order to confirm this phenomenon in clinic, we collected the samples from patients who were undergoing 5FU-involved chemotherapy and analyzed the correction between BAX expression and PFS. The patient with higher expression of BAX shows longer PFS (PFS, Progression-Free-Survival, which means the length of time during and after the treatment of a disease that a patient lives with the disease but it does not get worse. In a clinical trial, measuring the progression-free survival is one way to see how well a new treatment works.), the correlation coefficient is 0.2516 (Fig. 6C–E). Then we divided patients into two groups according to the BAX score (BAX low expression group and BAX high expression group). The PFS of BAX high expression group is relative longer than the BAX low expression group
Fig. 5. Andrographolide enhance 5-FU sensitivity dependent on BAX. (A) HCT116/5-FUR cells were transfected with si-NC or si-BAX for 36 h, then the level of BAX protein expression was detected by western blot analysis. (B, C) HCT116/ 5FU-R cells were transfected with si-NC or si-BAX for 36 h, then treated with 10 lM Andro for 12 h followed by indicated doses of 5-FU for 36 h. Cell viability was tested by MTT assay. The data were expressed as mean ± SD obtained from triplicate samples. *P < 0.05, **P < 0.01 versus control group. (D, E) After transfected with si-NC or si-BAX for 36 h, cells were treated with 10 lM Andro and indicated doses of 5-FU for 24 h, then analyzed by Annexin V/PI staining. Data shown are representative of 3 independent experiments. Data are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01 versus control group.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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Fig. 6. BAX expression was related to patient sensitivity to 5-FU-invovled chemotherapy. (A) The expression level of BAX in HCT116 cells with different sensitive to 5-FU (HCT116, HCT116/5-FUR1, HCT116/5-FUR2, HCT116/5-FUR) were detected by western blot. (B) Expression of BAX was detected in tumor xenografts of HCT116 cells and HCT116/5-FUR cells by IHC staining. (C) Representative IHC images of colorectal cancer tissues which before and after the treatment including 5-FU. (D, E, F) Relationship between BAX expression is significantly correlated and PFS of colorectal cancer patients.
(P = 0.134) (Fig. 6F). These results indicated that the expression of BAX was related to patient sensitivity to 5-FU-involved chemotherapy. 4. Discussion Up to now, 5-FU is still the most important chemotherapeutic drug used for the treatment of CRC, but still it is urgent to solve the problem of 5-FU resistance. Multiple mechanisms have been proposed to explain and several small molecules showed the ability to overcome 5-FU resistance. Overexpression of thymidylate synthase has been found to be associated with 5-FU resistance in colorectal cancer [19,20]. Glut1 expression was upregulated in 5-FU resistant cancer cells and exogenous overexpression of Glut1 facilitated colorectal cancer cells obtain resistance to 5-FU and the Glut1 specific inhibitor WZB117 can increase the sensitivity of 5-FU resistant cells to the drug [21]. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-FU cytotoxicity by elevating ER-b protein expression [22]. Our study here provides a novel mechanism for 5-Fu resistance in colon cancer cells probed by using a small natural compound Andrographolide. Andrographolide is a lactone diterpenoid originally isolated from the Chinese herb. In recent years, novel pharmacological effect of andrographolide has been identified, including anti-inflammation, anti-angiogenesis, and anti-cancer. In the present study, we demonstrated that Andrographolide could enhance sensitivity to 5-FU in 5-FU resistant CRC through directly targeting BAX. Apoptosis is one of the major mechanisms of cell death in response to chemotherapy drugs [23]. So defects in apoptotic pathways can promote cancer cell survival and also confer resistance to
chemotherapy [24,25]. BAX, a central cell death regulator, is an indispensable gateway to mitochondrial dysfunction and a major proapoptotic member of the B-cell lymphoma 2 (Bcl-2) family proteins that control apoptosis in normal and cancer cells [26,27]. Recent studies have shown that enhanced BAX expression levels correlate with a good response to chemotherapy in leukemia and low expression levels correlate with a poor prognosis in colorectal cancer [28,29]. Also, the down-regulation of BAX plays an important role in carcinogenesis and acquisition of resistance to 5-FU in colorectal cancer [30,31]. In our study, compared with the HCT116 cells, the levels of BAX in HCT116/5-FUR cells distinctly decreased. With the increase extent of drug resistance, the level of BAX presented gradually down regulated. What’s more, the study of clinical samples from patients who received 5-FUinvolved chemotherapy further confirmed these results. The clinical outcome showed that compared with the patients with low level of BAX, the patients with high level BAX have a longer PFS. So these results all revealed the relationship between downregulation of BAX and 5-FU resistance. Accumulating evidence suggests that BAX can serve as a promising direct target for cancer therapy [32,33]. A number of anticancer drugs in the clinic induce BAX activation indirectly to facilitate apoptosis while none of them can directly activate or bind to BAX. Usually, many anticancer agents induce cancer cell death via BAX-mediated apoptosis pathway, suggesting that direct activation or elevation of BAX expression may also provide useful agents for novel drug combinations. Several direct BAX activators have been identified to hold promise for cancer therapy with the advantages of specificity and the potential of overcoming chemoresistance [34,35]. However, no such activators have been applied to clinical.
Please cite this article in press as: W. Wang et al., Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.09.024
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Our study here found out that Andrographolide could elevate BAX level in 5-FU resistant cells. This elevated BAX contributed to the increase apoptosis induced by 5-FU. When knocked down BAX by siRNA, the effect of Andrographolide and 5-FU combination on apoptosis was diminished, confirming the BAX-dependent mechanism. All these results further suggested that BAX could be one therapy target in the combined treatment for cancer. Our research provided a clue for new indications with a drug already in clinical use. Actually, we are carrying out a clinical trial for the combined use of Xeloda and Xi-Yan-Ping injection (Andrographolide dosage form) in colorectal cancer patient. Previously, Andrographolide has been shown to induce apoptosis in cancer cells or sensitizes cancer cells to chemotherapy [36– 38]. Mechanism study showed that BAX-mediated apoptosis was involved in the therapy effect of Andrographolide, however, no evidence has proved BAX is a direct target of Andrographolide. Interestingly, in the present study, we found Andrographolide could directly bind to BAX, enhancing its stability without affecting its mRNA level. Based on these results, BAX may be a molecular target of Andrographolide in 5-FU resistant CRC. In summary, it could be concluded from these studies that, Andrographolide synergizes the cytotoxic effects of 5-FU in colon cancer cells via binding to BAX and triggering mitochondria mediated apoptosis in vivo and vitro. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing low level of BAX protein. Nevertheless, due to the modest effects achieved in terms of the re-sensitization of resistant colon cancer cells to 5-FU in this study, we are still trying to find more combinations between Andrographolide and other chemotherapeutics including apoptosis inducer for the extrinsic pathway, autophagic cell death inducer et al. to fully overcome drug-resistance. Conflict of interest The authors declared no conflict of interest. Author contributions W.W., W.G., and L.L. performed the experiments. W.W., W.L. and W.G. analyzed the data and prepared the figures. Z.F., J.G. and Y. Su provided expertise and the mouse lines. W.G., Q.X., Y. Sun. and Y.G. conceived, directed the study and wrote the manuscript. All authors discussed the results and commented on the manuscript. Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 91429308, 81402938, 81572389), Key Personnel of Jiangsu Province (RC2011170), Jiangsu Province Clinical Science and Technology Projects (Clinical Research Center, BL2012008) and Priority Academic Program Development of Jiangsu Higher Education Institutions. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA Cancer J. Clin. 65 (2) (2015) 87–108. [2] B. Chibaudel, C. Tournigand, T. Andre, A. de Gramont, Therapeutic strategy in unresectable metastatic colorectal cancer, Ther. Adv. Med. Oncol. 4 (2) (2012) 75–89. [3] B. Gustavsson, G. Carlsson, D. Machover, N. Petrelli, A. Roth, H.J. Schmoll, K.M. Tveit, F. Gibson, A review of the evolution of systemic chemotherapy in the management of colorectal cancer, Clin. Colorectal Cancer 14 (1) (2015) 1–10. [4] D.B. Longley, D.P. Harkin, P.G. Johnston, 5-Fluorouracil: mechanisms of action and clinical strategies, Nat. Rev. Cancer 3 (5) (2003) 330–338.
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