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Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy Yuzhen Wang*, Li Lina, Li Xu, Zhai Yang, Zhang Qian, Jing Zhou, Li Suoni Department of Internal Medicine, Shaanxi Provincial Tumor Hospital, Xi’an, 710061, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2019 Accepted 20 September 2019 Available online xxx
Arctigenin is the active content of arctium lappa that present anti-cancer abilities in various carcinomas. However, its role and underlying mechanism in drug-resistant colorectal cancer cells has not been addressed. The present study used SW480 and SW620 to established cisplatin-resistant colorectal cancer cell lines, and explored the impact of arctigenin on these cells. Arctigenin at 100 mM significantly inhibited cell proliferation of cisplatin treated ReSW480 and ReSW620 cells as compared with cells treated with cisplatin alone. Arctigenin elevates cell apoptosis, up-regulated pro-apoptotic protein cleaved-caspase-3 and caspase-9 expression level in cisplatin treated ReSW480 and ReSW620 cells. Additionally, arctigenin triggered autophagy and promoted LC3-II and p65 expression, while inhibited LC3-Iexpression. Arctigenin impeded the IC50 of not only cisplatin but also oxaliplatin, doxorubicin and Paclitaxel of ReSW480 and ReSW620 cells. More importantly, the mRNA expression of multi drug resistance 1 (MDR1) and protein expression of pgp were significantly inhibited by arctigenin administration. Taken together, arctigenin has the potential in sensitize colorectal cancer cells by activating autophagy, which induced cell apoptosis and inhibited cell growth. Our study revealed that arctigenin has the potential for colorectal cancer treatment and may be useful in adjuvant chemotherapy. © 2019 Elsevier Inc. All rights reserved.
Keywords: Arctigenin Cisplatin Colorectal cancer Autophagy SW480 SW620
1. Introduction Colorectal cancer (CRC) is the third most common malignancy around the world [1]. Nowadays, the treatment for CRC mainly includes surgery, radiotherapy and chemotherapy [2]. At the early stage of CRC, surgery operations was prioritized with none risky parameters. Additionally, advanced CRC patients often faced with tumor recurrence and metastasis, and adjuvant chemotherapy was needed to prolong survival [3]. The occurrence of drug resistance in chemotherapy has become a formidable obstacle and a main cause of chemotherapeutic failure [4]. Therefore, it is acute to developing effective therapies to attenuate drug resistance and enhance chemo-sensitivity of CRC cells. In chemotherapy, malignant cells can develop resistance to most anticancer drugs, in which limiting the curative treatment effect [5]. Cisplatin is one of the most common used chemotherapy drug in cancer [6]. In the process of cisplatin chemotherapy, this drug forms complexes with DNA and inhibiting the synthesis of DNA and
RNA, therefore inducing cell apoptosis [7]. Unfortunately, rapid development of cisplatin resistance was often occurred in clinical chemotherapy of CRC [8]. In order to successfully use cisplatin in CRC treatment, it will be effective to develop novel factor that elevates the chemosensitivity of CRC cells to cisplatin. Arctium lappa, also known as greater burdock, is a well consumed vegetable and has been used in traditionally Chinese medicine [9]. Arctigenin is a member and bioactive lignin of Asteraceae family, which can be isolated from Arctium lappa seeds. It has been proved that arctigenin can be anti-inflammatory, antiviral and immunomodulatory [10]. Particularly, arctigenin was used in the treatment of several type of cancers. The anti-tumor function of arctigenin have been demonstrated in various human cancers, including breast cancer, colon cancer, prostate cancer, and so on [11,12]. Study have shown that arctigenin induce apoptosis, affect cell cycle and even the sensitivity of cancer cell to chemotherapy drugs in several cancers. A recent study addressed arctigenin inhibited etoposide resistance of HT-29 clone cancer under micro
* Corresponding author. Department of internal medicine, Shaanxi Provincial Tumor Hospital, No.309 Yanta West Road, Xi’an, 710061, Shaanxi, China E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.bbrc.2019.09.086 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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environmental stress [13]. However, the role and underlying mechanism of arctigenin on other chemotherapy drug resistance in CRC has not been addressed. Our study aims to prove the hypothesis that arctigenin can enhance the sensitivity of cisplatin resistant CRC cells by activating autophagy. To test our hypothesis, cisplatin resistant cell line ReSW480 and ReSW620 were established. Cellular response to arctigenin was evaluated using multiple assays. We also examined the effects of arctigenin on the activation of autophagy, and further explored the multi drug resistant of arctigenin treated ReSW480 and ReSW620 cells. 2. Material and methods 2.1. Cell lines and drugs The human colorectal can cancer cell line SW480 (ATCC® CCL228) and SW620 (ATCC® CCL-227) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Chemotherapy drugs (cisplatin, oxaliplatin, doxorubicin and paclitaxel) and arctigenin was supplied by sigma (St. Louis, MO, USA). SW480 and SW620 were cultured in Leibovitz’s L-15 medium containing 10% fetal bovine serum and 1% antibiotics (100 mg/mL streptomycin and 100 IU/mL penicillin, Gibco) in a cell incubator with 5% CO2 at 37 C. 2.2. Development of resistant CRC cell lines Establishment of resistant CRC cell lines followed previously described protocols [14,15]. Cisplatin-resistant cell line was established from normal SW480 and SW620 cells. The culture medium was first supplemented with 1 mg/L cisplatin. After the cells reached 90% confluence, we harvested and reseeded the cells, and cultured in medium with 4 mg/L cisplatin. Cells were treated with increasing concentration of cisplatin (4, 8, 12, 16, 20, 24 mg/L) for 6 month. Established cisplatin-resistant cell lines were stored at 80 C for further studies.
cytometer (Becton Dickinson, San Jose, CA, USA). 2.5. Western blot Total proteins were extracted from cells using ice-cold RIPA buffer. The bicinchoninic acid assay was performed to determine the protein concentration. Equal amounts of proteins (20 mg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories), and blocked in 5% non-fat milk for 1 h, followed by incubation with primary antibodies against p65, LC3-I, LC3-II, Pgp and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 C overnight. After three 10-min washes in phosphate-buffered saline, the membranes were incubated with a secondary antibody at room temperature for 1 h. The protein bands were visualized by enhanced chemiluminescence detection and analyzed by Image-Pro Plus software. The relative protein expression was normalized to b-actin. 2.6. Rh123 detection Cells were washed twice with PBS and then incubated in fresh culture medium containing fluorescent dye Rh123 for 2 h at a concentration of 0.5 mg/mL. The cells were harvested and washed twice to remove extracellular Rh123, then suspended and kept in the dark. The mean fluorescence intensity (MFI) of Rh123 was determined by flow cytometry (Becton Dickinson, San Diego, CA, USA). 2.7. Statistical analysis Statistical analyses were conducted using SPSS 22.0 software. Data are presented as the mean ± standard deviation of three experiments. Differences between groups of two were evaluated by ttest. Differences between larger groups were analyzed by one-way analysis of variance, followed by Dunnett’s test. P values less than 0.05 were considered significant.
2.3. Cell proliferation assay
3. Results
Cell proliferation was detected using the MTT method. Briefly, SW480 or SW620 cells were seeded in 96-well plates with different concentrations of arctigenin (50, 100, 150, 200 mM) for 24 days. After adding 20 mL of MTT to each well for 4 h, the formazan products were dissolved with acid-isopropanol. The absorbance at a wavelength of 450 nm was detected by a SpectraMax Microplate Spectrophotometer (Molecular Devices LLC, Sunnyvale, CA, USA), and cell proliferation rate was calculated. For IC50 detection, cells were seeded in 96-well plates with increasing concentrations of chemotherapy drugs (cisplatin, oxaliplatin, doxorubicin and paclitaxel). After 72 h incubation, MTT method was used and absorbance (450 nm) was detected. Then IC50 was analyzed using SPSS 22.0 software.
3.1. Arctigenin inhibits cell proliferation of cisplatin induced ReSW480 and ReSW620
2.4. Flow cytometry Cell apoptosis was assessed via flow cytometry using an Annexin V-FITC and propidium iodide kit (Sigma, St. Louis, USA). Cells were first washed with phosphate-buffered saline and resuspended with the kit’s binding buffer. The cells then were incubated with 10 mL of Annexin V-FITC and 5 mL of propidium iodide for 15 min in the dark. Fluorescence was detected using a flow
Colorectal cancer cell SW480 and SW620 were first treated with increasing concentration of cisplatin to establish drug resistant cell lines. After 6 months of drug treatment, cells exhibited stable growth in 20 mg/L or 24 mg/L cisplatin, and cisplatin resistant cell line ReSW480 and ReSW620 were established (Fig. 1A and B). To evaluate the cytotoxicity of arctigenin in colorectal cancer cells, we detected cell proliferation of SW480 and SW620 cells treated with different concentration of arctigenin (50, 100, 150, 200 mM) for 24 h. Cell proliferation rate was detected by MTT method. As showed in Fig. 1C and D, 50 and 100 mM arctigenin showed no significant effect on cell proliferation of SW480 and SW620 cells, whereas 150 and 200 mM dramatically decreased cell proliferation of both SW480 and SW620. In order to explore the effect of arctigenin on cisplatin resistant colorectal cancer cells, ReSW480 and ReSW620 were treated by 20 mg/L cisplatin with or without 100 mM arctigenin. MTT results showed that arctigenin or cisplatin alone didn’t affect the proliferation rate of ReSW480 or ReSW620. Meanwhile, arctigenin significantly inhibited cell proliferation of cisplatin treated ReSW480 and ReSW620 cells (Fig. 1 E and F).
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 1. Arctigenin inhibits cell proliferation of cisplatin induced ReSW480 and ReSW620. (A and B) The time course of establishment of cisplatin-resistant SW480 and SW620 cell. Three independent experiments were established simultaneously. (C and D) SW480 and SW620 cell were treated with different concentrations of arctigenin (50, 100, 150, 200 mM). Cell proliferation was measured by MTT method. *P < 0.05 versus 0 mM group. (E and F) Control group, ReSW480 or ReSW620 cells; arctigenin group, ReSW480 or ReSW620 treated with 100 mM arctigenin; cisplatin group, ReSW480 or ReSW620 treated with 20 mg/L cisplatin; cisplatin þ arctigenin group, ReSW480 or ReSW620 treated 20 mg/L cisplatin and 100 mM arctigenin. *P < 0.05 versus control group.
3.2. Arctigenin elevates cell apoptosis of cisplatin induced ReSW480 and ReSW620 To further investigate the effect of arctigenin in cisplatinresistant colorectal cancer cells, cell apoptosis of ReSW480 and ReSW620 was measured by flow cytometry. As shown in Fig. 2A, cell apoptosis rate of ReSW480 and ReSW620 was similar in control, arctigenin and cisplatin group. Additionally, cell apoptosis was significantly elevated in cisplatin þ arctigenin group as compared with cisplatin group (Fig. 2B and C). Consistently, the expression of cell apoptosis protein cleaved-caspase-3 and caspase9 were significantly boosted in cisplatin þ arctigenin group as compared with cisplatin group (Fig. 2D and E). 3.3. Arctigenin triggers autophagy in cisplatin induced ReSW480 and ReSW620 To explore whether autophagy induced by arctigenin was involved, hallmark of autophagy was evaluated. Western blot results showed LC3-II and p65 protein expression was slightly but not significantly increased in arctigenin group as compared with control group. Moreover, an obvious increase of in LC3-II and p65
protein expression were observe in cisplatin þ arctigenin group as compared with cisplatin group. Additionally, LC3-Iwas slightly decreased in arctigenin group as compared with control group. More importantly, arctigenin significantly inhibited the expression level of LC3-I in cisplatin incubated ReSW480 and ReSW620 cells, thus the ratio of LC3-II/LC3-I was elevated in cisplatin þ arctigenin group as compared with cisplatin group (Fig. 3A and B). 3.4. Arctigenin inhibits multidrug resistance of cisplatin-resistant colorectal cancer cells To investigate the effect of arctigenin on sensitivity of cisplatinresistant colorectal cancer cells, IC50 of cisplatin were detected. As showed in Fig. 4A, IC50 of cisplatin was significantly increased in ReSW480 cells as compared with normal SW480 cells, and dramatically decreased after arctigenin administration. Furthermore, the sensitivity of arctigenin treated ReSW480 to other common chemotherapy drugs such as oxaliplatin, doxorubicin and paclitaxel were tested. The IC50 of oxaliplatin, doxorubicin and paclitaxel increased significantly in ReSW480 group as compared with SW480 group, whereas arctigenin treated ReSW480 cells remain sensitive to these three agents with lower IC50 (Fig. 4B, C
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 2. Arctigenin elevates cell apoptosis of cisplatin induced ReSW480 and ReSW620. Control group, ReSW480 or ReSW620 cells; arctigenin group, ReSW480 or ReSW620 treated with 100 mM arctigenin; cisplatin group, ReSW480 or ReSW620 treated with 20 mg/L cisplatin; cisplatin þ arctigenin group, ReSW480 or ReSW620 treated 20 mg/L cisplatin and 100 mM arctigenin. (A, B and C) Cell apoptosis was measured by flow cytometry method. (D and E) Protein expression of cleaved-caspase-3 and caspase-9 were detected by western blot. Relative expression level was normalized to b-actin. *P < 0.05 versus control group.
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 3. Arctigenin triggers autophagy in cisplatin induced ReSW480 and ReSW620. (A and B) Protein expression of LC3-I, LC3-II and p65 were detected by western blot. Relative expression level was normalized to b-actin. P < 0.05 versus control group.
and D). The mRNA expression of MDR1 and protein expression of Pgp were examined to further confirm the effect arctigenin on multidrug resistance. Both MDR1 mRNA expression and Pgp protein expression were significantly increased in ReSW480 group as compared with SW480 group, while dramatically impeded by administration of arctigenin (Fig. 4E, F and G). Additionally, arctigenin reversed the decline of Rh123 MFI in ReSW480 cells (Fig. 4H). Similar results were also observed in SW620 cells (Fig. 4IP).
4. Discussion The experiments performed in this paper were designed to provide a novel understanding of the effect of arctigenin on chemotherapy sensitivity of cisplatin resistant human colorectal cancer cell SW480 and SW620. Chemotherapy of malignant cancer has been significantly improved in the recent years. Besides the conventional chemotherapeutic drug cisplatin, several new drugs such as oxaliplatin, doxorubicin and paclitaxel have been developed and used in chemotherapy for colorectal cancer [16]. Additionally, several Chinese medicine have been proved to be useful in the treatment of CRC [17]. The individual or combination of these drugs and medicines enhanced the overall survival of CRC patients [18]. However, drug resistance developed in these treatments are still affect the long-term survival of cancers, and has become a vital research focus in CRC studies. Historically, consumption of arctium lappa was noticed to have healthy benefits against multiple illness including CRC [19]. Study have shown that combined therapy of traditional Chinese medicine and chemotherapy in post-operative cancer patients relieved the toxicity and resistance generated [20]. Since the effects of arcigenin on CRC have been reported, investigations of the effects and underlying mechanism of arcigenin on chemotherapy is needed. In this study, we established cisplatin resisted CRC cell line, ReSW480 and ReSW620. The results demonstrated that cell proliferation was decreased and cell apoptosis was increased of cisplatin stimulated ReSW480 and ReSW620, which didn’t affect by cisplatin stimulation alone due to its drug resistance ability. It has been reported that arctigenin induces cell apoptosis in
different cancers, therefore exhibit a antitumor effect [21]. Autophagy related cell apoptosis have been observed in the treatment of cancers [22]. A previous study indicated a significant role of arctigenin in regulating autophagy [23]. Thus, we evaluate the protein expression level of LC3-II, LC3-Iand p65, the marker of autophagy [24]. The results addressed a slightly change of autophagy in arctigenin treated ReSW480 and ReSW620 cells. This results was different to Maxwell’s study, which indicated arctigenin significantly trigger autophagy response in ER-positive human breast cancer cells, suggested autophagy may act dissimilar in different cancer cells or in resistant cancer cells [25]. Further research should be performed to explore the effect and correlation between arctigenin and autophagy in various cancers and resistant cancer cells. Our results further showed arctigenin dramatically triggered autophagy of cisplatin stimulated ReSW480 and ReSW620, which potentially boosted cell apoptosism and suggested that arctigenin may enhance the sensitivity of CRC cells by trigger autophagy induced apoptosis. The occurrence of multidrug resistance (MDR) is a formidable obstacle of chemotherapy [26]. In many cases, a resistant cancer cell line may be insensitive to various chemotherapy drugs, which is the developing of MDR [27]. Our results showed cisplatin resistant cell lines, ReSW480 and ReSW620, were also insensitive to oxaliplatin, doxorubicin and paclitaxel besides cisplatin, which suggested the occurrence of MDR in ReSW480 and ReSW620 cells. MDR1 is a critical MDR regulated gene, it encoded Pgp, which then control the development of MDR [28]. The significant inhibition of IC50, the elevation of MDR1 mRNA expression and Pgp protein expression indicated arctigenin impeded MDR in ReSW480 and ReSW620 cells. In summary, our findings demonstrated a significant role of arctigenin in elevating sensitivity of cisplatin-resistant CRC cells through activating autophagy response and inhibiting MDR. Given its effects, arctigenin holds promise to use in extend long-term efficacy of chemotherapy in CRC.
Conflicts of interest The authors declare that they have no conflict of interest.
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 4. Arctigenin inhibits multidrug resistance of cisplatin-resistant colorectal cancer cells. SW480 group, normal SW480 cells, ReSW480 group, cisplatin-resistant SW480 cells. ReSW480 þ arctigenin, cisplatin-resistant SW480 cells treated with 100 mM arctigenin. IC50 of cisplatin (A), oxaliplatin (B), doxorubicin (C) and paclitaxel (D) were measured by MTT method. (E) MDR1 mRNA expression level was measured by qRT-PCR method. (F and G) Protein expression of Pgp was detected by western blot method. (H) Rh123 MFI was measured by flow cytometry. *P < 0.05 versus SW480 group; #P < 0.05 versus ReSW480 group. SW620 group, normal SW620 cells, ReSW620 group, cisplatinresistant SW620 cells. ReSW620 þ arctigenin, cisplatin-resistant SW620 cells treated with 100 mM arctigenin. (IeP) *P < 0.05 versus SW620 group; *P < 0.05 versus SW620 group.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy Yuzhen Wang*, Li Lina, Li Xu, Zhai Yang, Zhang Qian, Jing Zhou, Li Suoni Department of Internal Medicine, Shaanxi Provincial Tumor Hospital, Xi’an, 710061, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2019 Accepted 20 September 2019 Available online xxx
Arctigenin is the active content of arctium lappa that present anti-cancer abilities in various carcinomas. However, its role and underlying mechanism in drug-resistant colorectal cancer cells has not been addressed. The present study used SW480 and SW620 to established cisplatin-resistant colorectal cancer cell lines, and explored the impact of arctigenin on these cells. Arctigenin at 100 mM significantly inhibited cell proliferation of cisplatin treated ReSW480 and ReSW620 cells as compared with cells treated with cisplatin alone. Arctigenin elevates cell apoptosis, up-regulated pro-apoptotic protein cleaved-caspase-3 and caspase-9 expression level in cisplatin treated ReSW480 and ReSW620 cells. Additionally, arctigenin triggered autophagy and promoted LC3-II and p65 expression, while inhibited LC3-Iexpression. Arctigenin impeded the IC50 of not only cisplatin but also oxaliplatin, doxorubicin and Paclitaxel of ReSW480 and ReSW620 cells. More importantly, the mRNA expression of multi drug resistance 1 (MDR1) and protein expression of pgp were significantly inhibited by arctigenin administration. Taken together, arctigenin has the potential in sensitize colorectal cancer cells by activating autophagy, which induced cell apoptosis and inhibited cell growth. Our study revealed that arctigenin has the potential for colorectal cancer treatment and may be useful in adjuvant chemotherapy. © 2019 Elsevier Inc. All rights reserved.
Keywords: Arctigenin Cisplatin Colorectal cancer Autophagy SW480 SW620
1. Introduction Colorectal cancer (CRC) is the third most common malignancy around the world [1]. Nowadays, the treatment for CRC mainly includes surgery, radiotherapy and chemotherapy [2]. At the early stage of CRC, surgery operations was prioritized with none risky parameters. Additionally, advanced CRC patients often faced with tumor recurrence and metastasis, and adjuvant chemotherapy was needed to prolong survival [3]. The occurrence of drug resistance in chemotherapy has become a formidable obstacle and a main cause of chemotherapeutic failure [4]. Therefore, it is acute to developing effective therapies to attenuate drug resistance and enhance chemo-sensitivity of CRC cells. In chemotherapy, malignant cells can develop resistance to most anticancer drugs, in which limiting the curative treatment effect [5]. Cisplatin is one of the most common used chemotherapy drug in cancer [6]. In the process of cisplatin chemotherapy, this drug forms complexes with DNA and inhibiting the synthesis of DNA and
RNA, therefore inducing cell apoptosis [7]. Unfortunately, rapid development of cisplatin resistance was often occurred in clinical chemotherapy of CRC [8]. In order to successfully use cisplatin in CRC treatment, it will be effective to develop novel factor that elevates the chemosensitivity of CRC cells to cisplatin. Arctium lappa, also known as greater burdock, is a well consumed vegetable and has been used in traditionally Chinese medicine [9]. Arctigenin is a member and bioactive lignin of Asteraceae family, which can be isolated from Arctium lappa seeds. It has been proved that arctigenin can be anti-inflammatory, antiviral and immunomodulatory [10]. Particularly, arctigenin was used in the treatment of several type of cancers. The anti-tumor function of arctigenin have been demonstrated in various human cancers, including breast cancer, colon cancer, prostate cancer, and so on [11,12]. Study have shown that arctigenin induce apoptosis, affect cell cycle and even the sensitivity of cancer cell to chemotherapy drugs in several cancers. A recent study addressed arctigenin inhibited etoposide resistance of HT-29 clone cancer under micro
* Corresponding author. Department of internal medicine, Shaanxi Provincial Tumor Hospital, No.309 Yanta West Road, Xi’an, 710061, Shaanxi, China E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.bbrc.2019.09.086 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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environmental stress [13]. However, the role and underlying mechanism of arctigenin on other chemotherapy drug resistance in CRC has not been addressed. Our study aims to prove the hypothesis that arctigenin can enhance the sensitivity of cisplatin resistant CRC cells by activating autophagy. To test our hypothesis, cisplatin resistant cell line ReSW480 and ReSW620 were established. Cellular response to arctigenin was evaluated using multiple assays. We also examined the effects of arctigenin on the activation of autophagy, and further explored the multi drug resistant of arctigenin treated ReSW480 and ReSW620 cells. 2. Material and methods 2.1. Cell lines and drugs The human colorectal can cancer cell line SW480 (ATCC® CCL228) and SW620 (ATCC® CCL-227) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Chemotherapy drugs (cisplatin, oxaliplatin, doxorubicin and paclitaxel) and arctigenin was supplied by sigma (St. Louis, MO, USA). SW480 and SW620 were cultured in Leibovitz’s L-15 medium containing 10% fetal bovine serum and 1% antibiotics (100 mg/mL streptomycin and 100 IU/mL penicillin, Gibco) in a cell incubator with 5% CO2 at 37 C. 2.2. Development of resistant CRC cell lines Establishment of resistant CRC cell lines followed previously described protocols [14,15]. Cisplatin-resistant cell line was established from normal SW480 and SW620 cells. The culture medium was first supplemented with 1 mg/L cisplatin. After the cells reached 90% confluence, we harvested and reseeded the cells, and cultured in medium with 4 mg/L cisplatin. Cells were treated with increasing concentration of cisplatin (4, 8, 12, 16, 20, 24 mg/L) for 6 month. Established cisplatin-resistant cell lines were stored at 80 C for further studies.
cytometer (Becton Dickinson, San Jose, CA, USA). 2.5. Western blot Total proteins were extracted from cells using ice-cold RIPA buffer. The bicinchoninic acid assay was performed to determine the protein concentration. Equal amounts of proteins (20 mg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories), and blocked in 5% non-fat milk for 1 h, followed by incubation with primary antibodies against p65, LC3-I, LC3-II, Pgp and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 C overnight. After three 10-min washes in phosphate-buffered saline, the membranes were incubated with a secondary antibody at room temperature for 1 h. The protein bands were visualized by enhanced chemiluminescence detection and analyzed by Image-Pro Plus software. The relative protein expression was normalized to b-actin. 2.6. Rh123 detection Cells were washed twice with PBS and then incubated in fresh culture medium containing fluorescent dye Rh123 for 2 h at a concentration of 0.5 mg/mL. The cells were harvested and washed twice to remove extracellular Rh123, then suspended and kept in the dark. The mean fluorescence intensity (MFI) of Rh123 was determined by flow cytometry (Becton Dickinson, San Diego, CA, USA). 2.7. Statistical analysis Statistical analyses were conducted using SPSS 22.0 software. Data are presented as the mean ± standard deviation of three experiments. Differences between groups of two were evaluated by ttest. Differences between larger groups were analyzed by one-way analysis of variance, followed by Dunnett’s test. P values less than 0.05 were considered significant.
2.3. Cell proliferation assay
3. Results
Cell proliferation was detected using the MTT method. Briefly, SW480 or SW620 cells were seeded in 96-well plates with different concentrations of arctigenin (50, 100, 150, 200 mM) for 24 days. After adding 20 mL of MTT to each well for 4 h, the formazan products were dissolved with acid-isopropanol. The absorbance at a wavelength of 450 nm was detected by a SpectraMax Microplate Spectrophotometer (Molecular Devices LLC, Sunnyvale, CA, USA), and cell proliferation rate was calculated. For IC50 detection, cells were seeded in 96-well plates with increasing concentrations of chemotherapy drugs (cisplatin, oxaliplatin, doxorubicin and paclitaxel). After 72 h incubation, MTT method was used and absorbance (450 nm) was detected. Then IC50 was analyzed using SPSS 22.0 software.
3.1. Arctigenin inhibits cell proliferation of cisplatin induced ReSW480 and ReSW620
2.4. Flow cytometry Cell apoptosis was assessed via flow cytometry using an Annexin V-FITC and propidium iodide kit (Sigma, St. Louis, USA). Cells were first washed with phosphate-buffered saline and resuspended with the kit’s binding buffer. The cells then were incubated with 10 mL of Annexin V-FITC and 5 mL of propidium iodide for 15 min in the dark. Fluorescence was detected using a flow
Colorectal cancer cell SW480 and SW620 were first treated with increasing concentration of cisplatin to establish drug resistant cell lines. After 6 months of drug treatment, cells exhibited stable growth in 20 mg/L or 24 mg/L cisplatin, and cisplatin resistant cell line ReSW480 and ReSW620 were established (Fig. 1A and B). To evaluate the cytotoxicity of arctigenin in colorectal cancer cells, we detected cell proliferation of SW480 and SW620 cells treated with different concentration of arctigenin (50, 100, 150, 200 mM) for 24 h. Cell proliferation rate was detected by MTT method. As showed in Fig. 1C and D, 50 and 100 mM arctigenin showed no significant effect on cell proliferation of SW480 and SW620 cells, whereas 150 and 200 mM dramatically decreased cell proliferation of both SW480 and SW620. In order to explore the effect of arctigenin on cisplatin resistant colorectal cancer cells, ReSW480 and ReSW620 were treated by 20 mg/L cisplatin with or without 100 mM arctigenin. MTT results showed that arctigenin or cisplatin alone didn’t affect the proliferation rate of ReSW480 or ReSW620. Meanwhile, arctigenin significantly inhibited cell proliferation of cisplatin treated ReSW480 and ReSW620 cells (Fig. 1 E and F).
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 1. Arctigenin inhibits cell proliferation of cisplatin induced ReSW480 and ReSW620. (A and B) The time course of establishment of cisplatin-resistant SW480 and SW620 cell. Three independent experiments were established simultaneously. (C and D) SW480 and SW620 cell were treated with different concentrations of arctigenin (50, 100, 150, 200 mM). Cell proliferation was measured by MTT method. *P < 0.05 versus 0 mM group. (E and F) Control group, ReSW480 or ReSW620 cells; arctigenin group, ReSW480 or ReSW620 treated with 100 mM arctigenin; cisplatin group, ReSW480 or ReSW620 treated with 20 mg/L cisplatin; cisplatin þ arctigenin group, ReSW480 or ReSW620 treated 20 mg/L cisplatin and 100 mM arctigenin. *P < 0.05 versus control group.
3.2. Arctigenin elevates cell apoptosis of cisplatin induced ReSW480 and ReSW620 To further investigate the effect of arctigenin in cisplatinresistant colorectal cancer cells, cell apoptosis of ReSW480 and ReSW620 was measured by flow cytometry. As shown in Fig. 2A, cell apoptosis rate of ReSW480 and ReSW620 was similar in control, arctigenin and cisplatin group. Additionally, cell apoptosis was significantly elevated in cisplatin þ arctigenin group as compared with cisplatin group (Fig. 2B and C). Consistently, the expression of cell apoptosis protein cleaved-caspase-3 and caspase9 were significantly boosted in cisplatin þ arctigenin group as compared with cisplatin group (Fig. 2D and E). 3.3. Arctigenin triggers autophagy in cisplatin induced ReSW480 and ReSW620 To explore whether autophagy induced by arctigenin was involved, hallmark of autophagy was evaluated. Western blot results showed LC3-II and p65 protein expression was slightly but not significantly increased in arctigenin group as compared with control group. Moreover, an obvious increase of in LC3-II and p65
protein expression were observe in cisplatin þ arctigenin group as compared with cisplatin group. Additionally, LC3-Iwas slightly decreased in arctigenin group as compared with control group. More importantly, arctigenin significantly inhibited the expression level of LC3-I in cisplatin incubated ReSW480 and ReSW620 cells, thus the ratio of LC3-II/LC3-I was elevated in cisplatin þ arctigenin group as compared with cisplatin group (Fig. 3A and B). 3.4. Arctigenin inhibits multidrug resistance of cisplatin-resistant colorectal cancer cells To investigate the effect of arctigenin on sensitivity of cisplatinresistant colorectal cancer cells, IC50 of cisplatin were detected. As showed in Fig. 4A, IC50 of cisplatin was significantly increased in ReSW480 cells as compared with normal SW480 cells, and dramatically decreased after arctigenin administration. Furthermore, the sensitivity of arctigenin treated ReSW480 to other common chemotherapy drugs such as oxaliplatin, doxorubicin and paclitaxel were tested. The IC50 of oxaliplatin, doxorubicin and paclitaxel increased significantly in ReSW480 group as compared with SW480 group, whereas arctigenin treated ReSW480 cells remain sensitive to these three agents with lower IC50 (Fig. 4B, C
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 2. Arctigenin elevates cell apoptosis of cisplatin induced ReSW480 and ReSW620. Control group, ReSW480 or ReSW620 cells; arctigenin group, ReSW480 or ReSW620 treated with 100 mM arctigenin; cisplatin group, ReSW480 or ReSW620 treated with 20 mg/L cisplatin; cisplatin þ arctigenin group, ReSW480 or ReSW620 treated 20 mg/L cisplatin and 100 mM arctigenin. (A, B and C) Cell apoptosis was measured by flow cytometry method. (D and E) Protein expression of cleaved-caspase-3 and caspase-9 were detected by western blot. Relative expression level was normalized to b-actin. *P < 0.05 versus control group.
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 3. Arctigenin triggers autophagy in cisplatin induced ReSW480 and ReSW620. (A and B) Protein expression of LC3-I, LC3-II and p65 were detected by western blot. Relative expression level was normalized to b-actin. P < 0.05 versus control group.
and D). The mRNA expression of MDR1 and protein expression of Pgp were examined to further confirm the effect arctigenin on multidrug resistance. Both MDR1 mRNA expression and Pgp protein expression were significantly increased in ReSW480 group as compared with SW480 group, while dramatically impeded by administration of arctigenin (Fig. 4E, F and G). Additionally, arctigenin reversed the decline of Rh123 MFI in ReSW480 cells (Fig. 4H). Similar results were also observed in SW620 cells (Fig. 4IP).
4. Discussion The experiments performed in this paper were designed to provide a novel understanding of the effect of arctigenin on chemotherapy sensitivity of cisplatin resistant human colorectal cancer cell SW480 and SW620. Chemotherapy of malignant cancer has been significantly improved in the recent years. Besides the conventional chemotherapeutic drug cisplatin, several new drugs such as oxaliplatin, doxorubicin and paclitaxel have been developed and used in chemotherapy for colorectal cancer [16]. Additionally, several Chinese medicine have been proved to be useful in the treatment of CRC [17]. The individual or combination of these drugs and medicines enhanced the overall survival of CRC patients [18]. However, drug resistance developed in these treatments are still affect the long-term survival of cancers, and has become a vital research focus in CRC studies. Historically, consumption of arctium lappa was noticed to have healthy benefits against multiple illness including CRC [19]. Study have shown that combined therapy of traditional Chinese medicine and chemotherapy in post-operative cancer patients relieved the toxicity and resistance generated [20]. Since the effects of arcigenin on CRC have been reported, investigations of the effects and underlying mechanism of arcigenin on chemotherapy is needed. In this study, we established cisplatin resisted CRC cell line, ReSW480 and ReSW620. The results demonstrated that cell proliferation was decreased and cell apoptosis was increased of cisplatin stimulated ReSW480 and ReSW620, which didn’t affect by cisplatin stimulation alone due to its drug resistance ability. It has been reported that arctigenin induces cell apoptosis in
different cancers, therefore exhibit a antitumor effect [21]. Autophagy related cell apoptosis have been observed in the treatment of cancers [22]. A previous study indicated a significant role of arctigenin in regulating autophagy [23]. Thus, we evaluate the protein expression level of LC3-II, LC3-Iand p65, the marker of autophagy [24]. The results addressed a slightly change of autophagy in arctigenin treated ReSW480 and ReSW620 cells. This results was different to Maxwell’s study, which indicated arctigenin significantly trigger autophagy response in ER-positive human breast cancer cells, suggested autophagy may act dissimilar in different cancer cells or in resistant cancer cells [25]. Further research should be performed to explore the effect and correlation between arctigenin and autophagy in various cancers and resistant cancer cells. Our results further showed arctigenin dramatically triggered autophagy of cisplatin stimulated ReSW480 and ReSW620, which potentially boosted cell apoptosism and suggested that arctigenin may enhance the sensitivity of CRC cells by trigger autophagy induced apoptosis. The occurrence of multidrug resistance (MDR) is a formidable obstacle of chemotherapy [26]. In many cases, a resistant cancer cell line may be insensitive to various chemotherapy drugs, which is the developing of MDR [27]. Our results showed cisplatin resistant cell lines, ReSW480 and ReSW620, were also insensitive to oxaliplatin, doxorubicin and paclitaxel besides cisplatin, which suggested the occurrence of MDR in ReSW480 and ReSW620 cells. MDR1 is a critical MDR regulated gene, it encoded Pgp, which then control the development of MDR [28]. The significant inhibition of IC50, the elevation of MDR1 mRNA expression and Pgp protein expression indicated arctigenin impeded MDR in ReSW480 and ReSW620 cells. In summary, our findings demonstrated a significant role of arctigenin in elevating sensitivity of cisplatin-resistant CRC cells through activating autophagy response and inhibiting MDR. Given its effects, arctigenin holds promise to use in extend long-term efficacy of chemotherapy in CRC.
Conflicts of interest The authors declare that they have no conflict of interest.
Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086
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Fig. 4. Arctigenin inhibits multidrug resistance of cisplatin-resistant colorectal cancer cells. SW480 group, normal SW480 cells, ReSW480 group, cisplatin-resistant SW480 cells. ReSW480 þ arctigenin, cisplatin-resistant SW480 cells treated with 100 mM arctigenin. IC50 of cisplatin (A), oxaliplatin (B), doxorubicin (C) and paclitaxel (D) were measured by MTT method. (E) MDR1 mRNA expression level was measured by qRT-PCR method. (F and G) Protein expression of Pgp was detected by western blot method. (H) Rh123 MFI was measured by flow cytometry. *P < 0.05 versus SW480 group; #P < 0.05 versus ReSW480 group. SW620 group, normal SW620 cells, ReSW620 group, cisplatinresistant SW620 cells. ReSW620 þ arctigenin, cisplatin-resistant SW620 cells treated with 100 mM arctigenin. (IeP) *P < 0.05 versus SW620 group; *P < 0.05 versus SW620 group.
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Please cite this article as: Y. Wang et al., Arctigenin enhances the sensitivity of cisplatin resistant colorectal cancer cell by activating autophagy, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.086