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Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells Kosuke Inamura a, 1, Yuji Komizu a, 1, Mami Yamakuchi a, Seiichi Ishida b, Yoko Matsumoto a, Taku Matsushita a, * a b
Division of Applied Life Science, Graduate School of Engineering, Sojo University, Kumamoto-shi, Kumamoto, Japan Division of Pharmacology, National Institute of Health Sciences, Kawasaki-shi, Kanagawa, Japan
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a b s t r a c t
Article history: Received 6 December 2018 Accepted 15 December 2018 Available online xxx
Purpose: Cancer stem cells (CSCs), also known as tumor-initiating cells, are involved in tumor progression, metastasis, and drug resistance. Hybrid liposomes (HLs) are nano-sized liposomal particles that can be easily prepared by ultrasonicating a mixture of vesicular and micellar molecules in buffer solutions. In this study, we investigated the inhibitory effects of HL on the growth of CSC subpopulations in liver cancer cells (HepG2) in vitro. Methods: HLs composed of 90 mol% L-a-dimyristoylphosphatidylcholine and 10 mol% polyoxyethylene(23) dodecyl ether were prepared by sonication. Cell viability was determined by the trypan blue exclusion assay. In liver cancer cells, CSCs were identified by the presence of the cell surface marker proteins CD133 and EpCAM by flow cytometry. A soft agar colony formation assay was performed using HepG2 cells pretreated with HLs. Results: HLs selectively inhibited liver cancer cell growth without affecting normal hepatocytes. Additionally, HLs induced apoptosis of HepG2 cells by a"ctivating caspase-3. Notably, the CD133(þ)/ EpCAM(þ) CSC sub-population of liver cancer cells treated with HLs was reduced. Furthermore, HLs markedly decreased the number of colony-forming cells. Finally, we confirmed the fusion and accumulation of HLs into the cell membranes of CSCs using a fluorescently labeled lipid (NBDPC). Significant accumulation of HL/NBDPC into the CSCs (particularly EpCAM(þ) cells) occurred in a dose-dependent manner. Conclusion: These results suggest that HLs are a novel nanomedical therapeutic agent for targeting CSCs in liver cancer therapy. © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Cancer stem cell Doxorubicin Hybrid liposomes Liver cancer cell Nanomedicine
1. Introduction Cancer stem cells (CSCs), also known as tumor-initiating cells, were first identified in acute myeloid leukemia in the late 1990s [1]. In the 2000s, the presence of CSCs was reported in various tissues such as breast cancer, glioma, skin cancer, lung cancer, and colon cancer, among others [2,3]. CSCs exhibit not only properties of stem cells (asymmetric cell division), but also high drug resistance ability and high tumorigenicity, and thus are related to the poor prognosis of treatment such as treatment-resistance against conventional
* Corresponding author. Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto, 860-0082, Japan. E-mail address: [email protected] (T. Matsushita). 1 These authors contributed equally to this work.
anticancer drugs, malignant growth, and recurrence [4,5]. In liver cancer, the presence of CSCs has been reported, and several cell surface markers, including CD133, EpCAM (also known as CD326), CD13, CD44, and CD90 have been identified as CSC markers [6,7]. Chen et al. [8] reported that co-expression of CD133 and the EpCAM phenotype results in characteristics of cancer stemness, including high differentiation capacity, increased colonyformation ability, an appearance of drug-resistance to conventional anticancer drug (e.g. doxorubicin: DOX), in hepatocellular carcinoma cells. Regarding therapeutic strategies for targeting CSCs, many researchers are currently developing several CSCs targeted drugs [9,10]. For example, Napabucasin (BBI608) is a first-in-class cancer stemness inhibitor of solid cancers in the stomach, colorectal, and pancreas, among others, by inhibiting multiple pathways involved in cancer cell stemness: the signal transducing transcription
https://doi.org/10.1016/j.bbrc.2018.12.118 0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: K. Inamura et al., Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.118
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activator 3 and beta-catenin pathways [11,12]. However, the therapeutic effect in a Phase III clinical trial of patients with colorectal cancer has not been determined [13]. Thus, new drugs that effectively inhibit CSCs are needed for clinical treatments. Hybrid liposomes (HLs) are nano-sized liposomal particles that can be easily prepared by ultrasonicating a mixture of vesicular and micellar molecules in buffer solutions [14,15]. HLs composed of L-adimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) dodecyl ethers (C12(EO)23) have remarkable inhibitory effects on the growth of various cancer cells and cause apoptosis in vitro [16,17], in vivo in animal models [18,19], and clinical settings [20]. HLs are fused and accumulated in cancer cell membranes, and their apoptosis signals are passed through caspases-9, -3, and -8 [21]. We previously showed that HLs composed of 90 mol% DMPC and 10 mol% C12(EO)23 selectively inhibit cell growth and induce apoptosis of human liver cancer cells (HepG2, HuH7) by specifically accumulating HLs into the plasma membranes of cancer cells which have greater fluidity than those of normal hepatocytes [22]. However, no studies have examined the effect of HLs on CSCs. In this study, we investigated the inhibitory effects of HL on the growth and CSC populations of liver cancer cells (HepG2) in vitro. DOX is associated with drug resistance and the enrichment of CSCs in liver cancer cells [23,24] and was used as a negative control in this study. 2. Material and methods
After 48 h, DOX or HLs was added into each well and the plates were incubated for 48 h. The number of viable cells was determined by using the trypan blue exclusion method. 2.5. Apoptosis assay For apoptosis analysis, caspase-3 activity of the cells was analyzed at 12 and at 24 h after HL treatment by using the CellEvent™ Caspase 3/7 Green Detection Reagent (Invitrogen, Carlsbad, CA, USA). Caspase-3-activated cells were measured using a flow cytometer, CytoFLEX (Beckman Coulter, Brea, CA, USA), with a 488nm laser and FITC filter. Data were analyzed with CytExpert software. 2.6. CSC analysis Cells were resuspended in 0.5% BSA/HBSS and incubated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min. Next, the cells were stained with the following directly conjugated monoclonal antibodies (Miltenyi Biotec): antihuman CD133/2-PE, anti-human IgG2b-PE isotype, anti-human CD133/2-PE-Vio770, anti-human IgG2b-PE-Vio770 isotype, antihuman EpCAM-FITC, anti-human IgG1-FITC isotype, anti-human EpCAM-PE-Vio770, and anti-human IgG1-PE-Vio770 isotype, for 10 min at 4 C in the dark. Flow cytometry analysis was performed with a CytoFLEX and a 488-nm laser line. Data were analyzed using CytExpert software.
2.1. Cells and culture medium 2.7. Soft agar colony formation assay Liver cancer cells (HepG2 cells) were purchased from RIKEN Bio Resource Center (Tokyo, Japan). HepG2 cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and Penicillin-Streptomycin Solution (Wako). Human fetal hepatocytes (Hc cells) were obtained from DS Pharma Biomedical Co. (Osaka, Japan) with a certification of informed consent for research and were cultured in CS-C medium (DS Pharma Biomedical Co.). These cells were cultured in a 5% CO2 humidified incubator at 37 C. 2.2. Anti-cancer agent Doxorubicin hydrochloride (DOX) was purchased from Wako. DOX was dissolved in sterile water at a concentration of 10 mM (stock solution). 2.3. Preparation of hybrid liposomes HLs were prepared using the following method [25]. L-aDimyristoyl-phosphatidylcholine (DMPC) (NOF Co., Tokyo, Japan) and polyoxyethylene(23) dodecyl ether (C12(EO)23) (Sigma-Aldrich, St. Louis, MO, USA) were mixed in 5% glucose solution and sonicated with a sonicator (Ultrasonic-Cleaner-WT-200-M, Tokyo, Japan) at 45 C and 300 W, followed by filtration through a 0.20-mm filter. 2.4. Cell growth inhibition assay The inhibitory effects of DOX and HLs against HepG2 cells or Hc cells were measured by the trypan blue exclusion method. HepG2 cells were inoculated at 2 105 cells/dish in a 100-mm dish (Corning, Inc., Corning, NY, USA) and incubated at 37 C. After 48 h, DOX or HLs was added into each dish and incubated for 48 h. Hc cells were also inoculated at 5 103 cells/well in 24-well plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and incubated at 37 C.
We evaluated the tumorigenicity of the cells by conducting a soft agar colony formation assay. First, 350 mL of 0.5% agar medium (1% agar solution: 2 DMEM ¼ 1:1) was added to a 48 well-plate (Sumitomo Bakelite Co., Ltd.) and solidified as Base agar. Next, 50 mL of top agar was prepared by mixing 0.5% agar medium with cell suspension at a 2:1 ratio for semi-solidity and was layered on the base agar. Cells were inoculated at a density of 1000 cells/well and cultured for 9 days in atmosphere of 5% CO2 and 95% air at 37 C. After 9 days of culture, viable colonies in the top agar medium were stained with 5 mM Calcein-AM solution (Dojindo, Kumamoto, Japan). Photographs of the stained colonies were acquired under a fluorescence microscope (EVOS FL; Thermo Fisher Scientific Inc., Waltham, MA, USA). The number of colonies was analyzed from photographs by using Image J software (National Institutes of Health, Bethesda, MD, USA). Furthermore, stained colonies with diameters of more than 80 mm, which corresponded to more than 20 cells and 5700 mm2 of area, were defined as tumorigenic colonies [26]. Tumorigenic colonies were those formed by the proliferation of a single cell. 2.8. Accumulation of HLs in the cell membrane analyzed by flow cytometry The accumulation of HLs labeled with a fluorescence probe (1palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-amino] dodecanoyl]-sn- glycero-3-phosphocholine (NBDPC; Avanti Polar Lipids, Inc., Alabaster, AL, USA) in the membrane of HepG2 cells was evaluated by flow cytometry analysis on a CytoFLEX with a 488-nm laser line. Cells (2 105 cells/100-mm dish) were cultured in a 5% CO2-humidified incubator at 37 C for 48 h. The cells were treated with HL ([DMPC] ¼ 100.0 mM, [C12(EO)23] ¼ 11.2 mM, [NBDPC] ¼ 1.1 mM) coupled with a fluorescence-labeled lipid (HL/ NBDPC) for 1 h. Accumulation of HL/NBDPC into the cells was measured by flow cytometry using the FITC channel with a 525/40
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bandpass filter. Furthermore, we examined the accumulation of HL/ NBDPC in the population of CD133/EpCAM-stained cells. The percentage of HL/NBDPC accumulation was analyzed for the 4 sections (CD133(þ)/EpCAM(þ), CD133(þ)/EpCAM(), CD133()/EpCAM(þ), and CD133()/EpCAM()) of the cell populations. 2.9. Statistical analysis The results are presented as the mean ± the standard error (SE). Data were statistically analyzed using Student's t-test. Values of p < 0.05 were considered to represent a statistically significant difference. 3. Results and discussion First, we examined the effect of DOX or HLs on the growth of liver cancer HepG2 cells and normal hepatocyte Hc cells. As shown in Fig. 1, DOX inhibited the growth of HepG2 (Fig. 1A) and Hc (Fig. 1B) cells in a dose-dependent manner. In contrast, treatment with HLs inhibited HepG2 cell growth in a dose-dependent manner in the concentration range of 50e200 mM (Fig. 1C). However, HLs did not inhibit Hc cells in the same concentration range (Fig. 1D). These results indicate that HLs selectively inhibited the growth of HepG2 cells without affecting normal hepatocytes in the concentration range of 50e200 mM. To confirm the caspase dependency of HL-induced apoptosis of HepG2 cells, we performed a caspase-3/-7 intracellular activity assay [22] to detect caspase-3 activation. The results are shown in Fig. S1. High activities of caspase-3 in the cells were observed in time- and dose-dependence manners, indicating that HLs induced apoptosis of HepG2 cells by activating caspase-
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3. Thus, marked inhibitory effects of HLs on the growth of HepG2 cells may be attained by inducing apoptosis via caspase-3 activation. It is well-known that cancer stem/progenitor cells are highly enriched in CD133(þ)/EpCAM(þ) populations among liver cancer cells [23,24]. Therefore, we examined the effect of HLs on CD133(þ)/EpCAM(þ) CSC populations among liver cancer cells. As shown in Fig. 2A, DOX increased the CD133(þ)/EpCAM(þ) population in HepG2 cells in a dose-dependent manner, indicating chemo-resistance in these cells. Notably, reduced CD133(þ)/ EpCAM(þ) CSC sub-populations of liver cancer cells treated with HLs were observed in a dose-dependent manner (Fig. 2B). These results indicate that HLs selectively inhibited CD133(þ)/EpCAM(þ) populations in HepG2 cells. Cancer cell stemness properties, including the colony formation capacity, were evaluated. We confirmed the effect of HLs on colony formation capacity in the soft agar colony formation assay. DOXpretreated cells induced the formation of larger and greater numbers of tumor colonies than non-treated cells (control) (Fig. 3A, left). DOX also showed a tendency to increase the number of colonies forming cells in the concentration range of 0.001e0.05 mM (Fig. 3B). This result suggests that DOX treatment enriched CSCs (particularly CD133(þ)/EpCAM(þ)) among HepG2 cells. In contrast, HL-pretreated cells induced lower numbers of tumor colonies than non-treated cells (control) in a dose-dependent manner (Fig. 4A, right). HLs significantly decreased the number of colony-forming cells in a dose-dependent manner (Fig. 3C, p < 0.05). These results revealed that HL suppresses colony-forming ability, which is a characteristic of CSCs. We next examined the fusion and accumulation of HLs including NBDPC (HL/NBDPC) in the cell membranes of each
Fig. 1. Inhibitory effects of DOX and HLs on the growth of HepG2 cells or Hc cells for 48 h HepG2 cells were treated with DOX (A) or HLs (C) for 48 h. Hc cells were treated with DOX (B) or HLs (D) for 48 h. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the controls.
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Fig. 2. Flow cytometry analysis of CSC populations of CD133þ/EpCAMþ in liver cancer cells treated with DOX or HLs for 48 h. (A) Expression of CD133 and EpCAM by DOX treatment for 48 h. (B) Percentage of CD133(þ)/EpCAM(þ) cells (CSCs) by DOX treatment for 48 h. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the control. (C) Expression of CD133 and EpCAM by HL treatment for 48 h. (D) Percentage of CD133(þ)/EpCAM(þ) cells (CSCs) by HL treatment for 48 h. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the control.
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Fig. 3. HLs reduce the number of colony-forming cells for HepG2 cells. (A) Fluorescence micrographs of colony-forming cells (blue) by pretreatment with DOX (left) or HLs (right) in soft agar medium. The threshold of the colony was set to over 5700 mm2 and shown in blue in the photograph (non-colony is shown in red). Scale bars shown 200 mm. Inhibitory effect of DOX (B) and HLs (C) on the number of colony-forming cells. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the controls. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
population in HepG2 cells by flow cytometry. As shown in Fig. S2, increased accumulation of HL/NBDPC into HepG2 was observed in a dose-dependent manner. After treatment of each cell population with 200 mM HL/NBDPC, NBDPC(þ) cells were gated for CD133(þ)/EpCAM(), CD133(þ)/EpCAM(þ), CD133()/ EpCAM(þ), and CD133()/EpCAM() cells (Fig. 4A). This result indicates that HL/NBDPC accumulated more in CD133(þ)/ EpCAM(þ) and CD133()/EpCAM(þ) cells than in CD133(þ)/ EpCAM() and CD133()/EpCAM() cells. Furthermore, significant accumulation of HL/NBDPC in CD133(þ)/EpCAM(þ) cells (particularly EpCAM(þ) cells) compared to other populations occurred in a dose-dependent manner (Fig. 4B). These results suggest that HLs selectively accumulated in CD133(þ)/EpCAM(þ) populations in HepG2 cells. Although the mechanistic details remain unclear, specific accumulation of HLs in the CSCs may be related to the difference in membrane physical properties among the CSC subpopulations, well-differentiated cancer cells, and normal cells, as described below. We reported that HLs distinguished between membranes of liver cancer cells and normal cells because of differences in membrane fluidity, and then fused and accumulated preferentially in the membranes of liver cancer cells [22]. Other studies also reported that the membranes of normal stem cells are more fluid
than those of well-differentiated cells [27]. This suggests that HLs accumulate in the more fluid membrane of CSCs than that of nonCSCs. We are currently investigating the membrane fluidity of these cells. Thus, our findings demonstrate that HLs are potential nanomedicine anti-cancer agent that targets co-expression of the membrane with the CD133 and EpCAM phenotypes (CSC-like cells) for liver cancer cells. In conclusion, we clearly demonstrated that HLs composed of DMPC and C12(EO)23 showed inhibitory effects towards the growth of CSC populations of HepG2 cells in vitro. The notable findings are as follows: (a) HLs selectively inhibited the growth of HepG2 cells without affecting normal hepatocytes through apoptosis via caspase-3. (b) HLs selectively inhibited CD133(þ)/EpCAM(þ) populations in HepG2 cells. (c) HLs suppressed the colony-forming ability of HepG2 cells. (d) HLs significantly accumulated in CD133(þ)/EpCAM(þ) populations in HepG2 cells. These results suggest that HLs are a novel nanomedical therapeutic agent for targeting CSCs in liver cancer therapy. Conflicts of interest The authors have no conflicts of interest to declare.
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Fig. 4. Flow cytometry analysis of CSC populations of CD133þ/EpCAMþ in HepG2 cells treated with HLs including fluorescently labeled lipids (NDBPC) for 1 h. (A) Flow cytometry analysis of each cell population treated with HL/NBDPC at 200 mM. HL/NBDPC (þ) cells were gated for each cell populations: (i):CD133(þ)/EpCAM(), (ii): CD133(þ)/EpCAM(þ), (iii): CD133()/EpCAM(), (iv): CD133()/EpCAM(). Data represent the mean ± SE of the indicated group (n ¼ 3). (B) Percentage of accumulation of HL/NBDPC for each cell population.
Author contributions KI, YK, and MY performed the research and analyzed the data. KI, YK, SI, YM, and TM designed the research. KI, YK, and TM wrote the manuscript. YM contributed the reagents. All authors read and approved the final manuscript.
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This work was supported by JSPS KAKENHI Grant Number JP16K18304.
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Appendix A. Supplementary data [11]
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Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells Kosuke Inamura a, 1, Yuji Komizu a, 1, Mami Yamakuchi a, Seiichi Ishida b, Yoko Matsumoto a, Taku Matsushita a, * a b
Division of Applied Life Science, Graduate School of Engineering, Sojo University, Kumamoto-shi, Kumamoto, Japan Division of Pharmacology, National Institute of Health Sciences, Kawasaki-shi, Kanagawa, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2018 Accepted 15 December 2018 Available online xxx
Purpose: Cancer stem cells (CSCs), also known as tumor-initiating cells, are involved in tumor progression, metastasis, and drug resistance. Hybrid liposomes (HLs) are nano-sized liposomal particles that can be easily prepared by ultrasonicating a mixture of vesicular and micellar molecules in buffer solutions. In this study, we investigated the inhibitory effects of HL on the growth of CSC subpopulations in liver cancer cells (HepG2) in vitro. Methods: HLs composed of 90 mol% L-a-dimyristoylphosphatidylcholine and 10 mol% polyoxyethylene(23) dodecyl ether were prepared by sonication. Cell viability was determined by the trypan blue exclusion assay. In liver cancer cells, CSCs were identified by the presence of the cell surface marker proteins CD133 and EpCAM by flow cytometry. A soft agar colony formation assay was performed using HepG2 cells pretreated with HLs. Results: HLs selectively inhibited liver cancer cell growth without affecting normal hepatocytes. Additionally, HLs induced apoptosis of HepG2 cells by a"ctivating caspase-3. Notably, the CD133(þ)/ EpCAM(þ) CSC sub-population of liver cancer cells treated with HLs was reduced. Furthermore, HLs markedly decreased the number of colony-forming cells. Finally, we confirmed the fusion and accumulation of HLs into the cell membranes of CSCs using a fluorescently labeled lipid (NBDPC). Significant accumulation of HL/NBDPC into the CSCs (particularly EpCAM(þ) cells) occurred in a dose-dependent manner. Conclusion: These results suggest that HLs are a novel nanomedical therapeutic agent for targeting CSCs in liver cancer therapy. © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Cancer stem cell Doxorubicin Hybrid liposomes Liver cancer cell Nanomedicine
1. Introduction Cancer stem cells (CSCs), also known as tumor-initiating cells, were first identified in acute myeloid leukemia in the late 1990s [1]. In the 2000s, the presence of CSCs was reported in various tissues such as breast cancer, glioma, skin cancer, lung cancer, and colon cancer, among others [2,3]. CSCs exhibit not only properties of stem cells (asymmetric cell division), but also high drug resistance ability and high tumorigenicity, and thus are related to the poor prognosis of treatment such as treatment-resistance against conventional
* Corresponding author. Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto, 860-0082, Japan. E-mail address: [email protected] (T. Matsushita). 1 These authors contributed equally to this work.
anticancer drugs, malignant growth, and recurrence [4,5]. In liver cancer, the presence of CSCs has been reported, and several cell surface markers, including CD133, EpCAM (also known as CD326), CD13, CD44, and CD90 have been identified as CSC markers [6,7]. Chen et al. [8] reported that co-expression of CD133 and the EpCAM phenotype results in characteristics of cancer stemness, including high differentiation capacity, increased colonyformation ability, an appearance of drug-resistance to conventional anticancer drug (e.g. doxorubicin: DOX), in hepatocellular carcinoma cells. Regarding therapeutic strategies for targeting CSCs, many researchers are currently developing several CSCs targeted drugs [9,10]. For example, Napabucasin (BBI608) is a first-in-class cancer stemness inhibitor of solid cancers in the stomach, colorectal, and pancreas, among others, by inhibiting multiple pathways involved in cancer cell stemness: the signal transducing transcription
https://doi.org/10.1016/j.bbrc.2018.12.118 0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: K. Inamura et al., Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.118
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activator 3 and beta-catenin pathways [11,12]. However, the therapeutic effect in a Phase III clinical trial of patients with colorectal cancer has not been determined [13]. Thus, new drugs that effectively inhibit CSCs are needed for clinical treatments. Hybrid liposomes (HLs) are nano-sized liposomal particles that can be easily prepared by ultrasonicating a mixture of vesicular and micellar molecules in buffer solutions [14,15]. HLs composed of L-adimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) dodecyl ethers (C12(EO)23) have remarkable inhibitory effects on the growth of various cancer cells and cause apoptosis in vitro [16,17], in vivo in animal models [18,19], and clinical settings [20]. HLs are fused and accumulated in cancer cell membranes, and their apoptosis signals are passed through caspases-9, -3, and -8 [21]. We previously showed that HLs composed of 90 mol% DMPC and 10 mol% C12(EO)23 selectively inhibit cell growth and induce apoptosis of human liver cancer cells (HepG2, HuH7) by specifically accumulating HLs into the plasma membranes of cancer cells which have greater fluidity than those of normal hepatocytes [22]. However, no studies have examined the effect of HLs on CSCs. In this study, we investigated the inhibitory effects of HL on the growth and CSC populations of liver cancer cells (HepG2) in vitro. DOX is associated with drug resistance and the enrichment of CSCs in liver cancer cells [23,24] and was used as a negative control in this study. 2. Material and methods
After 48 h, DOX or HLs was added into each well and the plates were incubated for 48 h. The number of viable cells was determined by using the trypan blue exclusion method. 2.5. Apoptosis assay For apoptosis analysis, caspase-3 activity of the cells was analyzed at 12 and at 24 h after HL treatment by using the CellEvent™ Caspase 3/7 Green Detection Reagent (Invitrogen, Carlsbad, CA, USA). Caspase-3-activated cells were measured using a flow cytometer, CytoFLEX (Beckman Coulter, Brea, CA, USA), with a 488nm laser and FITC filter. Data were analyzed with CytExpert software. 2.6. CSC analysis Cells were resuspended in 0.5% BSA/HBSS and incubated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min. Next, the cells were stained with the following directly conjugated monoclonal antibodies (Miltenyi Biotec): antihuman CD133/2-PE, anti-human IgG2b-PE isotype, anti-human CD133/2-PE-Vio770, anti-human IgG2b-PE-Vio770 isotype, antihuman EpCAM-FITC, anti-human IgG1-FITC isotype, anti-human EpCAM-PE-Vio770, and anti-human IgG1-PE-Vio770 isotype, for 10 min at 4 C in the dark. Flow cytometry analysis was performed with a CytoFLEX and a 488-nm laser line. Data were analyzed using CytExpert software.
2.1. Cells and culture medium 2.7. Soft agar colony formation assay Liver cancer cells (HepG2 cells) were purchased from RIKEN Bio Resource Center (Tokyo, Japan). HepG2 cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and Penicillin-Streptomycin Solution (Wako). Human fetal hepatocytes (Hc cells) were obtained from DS Pharma Biomedical Co. (Osaka, Japan) with a certification of informed consent for research and were cultured in CS-C medium (DS Pharma Biomedical Co.). These cells were cultured in a 5% CO2 humidified incubator at 37 C. 2.2. Anti-cancer agent Doxorubicin hydrochloride (DOX) was purchased from Wako. DOX was dissolved in sterile water at a concentration of 10 mM (stock solution). 2.3. Preparation of hybrid liposomes HLs were prepared using the following method [25]. L-aDimyristoyl-phosphatidylcholine (DMPC) (NOF Co., Tokyo, Japan) and polyoxyethylene(23) dodecyl ether (C12(EO)23) (Sigma-Aldrich, St. Louis, MO, USA) were mixed in 5% glucose solution and sonicated with a sonicator (Ultrasonic-Cleaner-WT-200-M, Tokyo, Japan) at 45 C and 300 W, followed by filtration through a 0.20-mm filter. 2.4. Cell growth inhibition assay The inhibitory effects of DOX and HLs against HepG2 cells or Hc cells were measured by the trypan blue exclusion method. HepG2 cells were inoculated at 2 105 cells/dish in a 100-mm dish (Corning, Inc., Corning, NY, USA) and incubated at 37 C. After 48 h, DOX or HLs was added into each dish and incubated for 48 h. Hc cells were also inoculated at 5 103 cells/well in 24-well plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and incubated at 37 C.
We evaluated the tumorigenicity of the cells by conducting a soft agar colony formation assay. First, 350 mL of 0.5% agar medium (1% agar solution: 2 DMEM ¼ 1:1) was added to a 48 well-plate (Sumitomo Bakelite Co., Ltd.) and solidified as Base agar. Next, 50 mL of top agar was prepared by mixing 0.5% agar medium with cell suspension at a 2:1 ratio for semi-solidity and was layered on the base agar. Cells were inoculated at a density of 1000 cells/well and cultured for 9 days in atmosphere of 5% CO2 and 95% air at 37 C. After 9 days of culture, viable colonies in the top agar medium were stained with 5 mM Calcein-AM solution (Dojindo, Kumamoto, Japan). Photographs of the stained colonies were acquired under a fluorescence microscope (EVOS FL; Thermo Fisher Scientific Inc., Waltham, MA, USA). The number of colonies was analyzed from photographs by using Image J software (National Institutes of Health, Bethesda, MD, USA). Furthermore, stained colonies with diameters of more than 80 mm, which corresponded to more than 20 cells and 5700 mm2 of area, were defined as tumorigenic colonies [26]. Tumorigenic colonies were those formed by the proliferation of a single cell. 2.8. Accumulation of HLs in the cell membrane analyzed by flow cytometry The accumulation of HLs labeled with a fluorescence probe (1palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-amino] dodecanoyl]-sn- glycero-3-phosphocholine (NBDPC; Avanti Polar Lipids, Inc., Alabaster, AL, USA) in the membrane of HepG2 cells was evaluated by flow cytometry analysis on a CytoFLEX with a 488-nm laser line. Cells (2 105 cells/100-mm dish) were cultured in a 5% CO2-humidified incubator at 37 C for 48 h. The cells were treated with HL ([DMPC] ¼ 100.0 mM, [C12(EO)23] ¼ 11.2 mM, [NBDPC] ¼ 1.1 mM) coupled with a fluorescence-labeled lipid (HL/ NBDPC) for 1 h. Accumulation of HL/NBDPC into the cells was measured by flow cytometry using the FITC channel with a 525/40
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bandpass filter. Furthermore, we examined the accumulation of HL/ NBDPC in the population of CD133/EpCAM-stained cells. The percentage of HL/NBDPC accumulation was analyzed for the 4 sections (CD133(þ)/EpCAM(þ), CD133(þ)/EpCAM(), CD133()/EpCAM(þ), and CD133()/EpCAM()) of the cell populations. 2.9. Statistical analysis The results are presented as the mean ± the standard error (SE). Data were statistically analyzed using Student's t-test. Values of p < 0.05 were considered to represent a statistically significant difference. 3. Results and discussion First, we examined the effect of DOX or HLs on the growth of liver cancer HepG2 cells and normal hepatocyte Hc cells. As shown in Fig. 1, DOX inhibited the growth of HepG2 (Fig. 1A) and Hc (Fig. 1B) cells in a dose-dependent manner. In contrast, treatment with HLs inhibited HepG2 cell growth in a dose-dependent manner in the concentration range of 50e200 mM (Fig. 1C). However, HLs did not inhibit Hc cells in the same concentration range (Fig. 1D). These results indicate that HLs selectively inhibited the growth of HepG2 cells without affecting normal hepatocytes in the concentration range of 50e200 mM. To confirm the caspase dependency of HL-induced apoptosis of HepG2 cells, we performed a caspase-3/-7 intracellular activity assay [22] to detect caspase-3 activation. The results are shown in Fig. S1. High activities of caspase-3 in the cells were observed in time- and dose-dependence manners, indicating that HLs induced apoptosis of HepG2 cells by activating caspase-
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3. Thus, marked inhibitory effects of HLs on the growth of HepG2 cells may be attained by inducing apoptosis via caspase-3 activation. It is well-known that cancer stem/progenitor cells are highly enriched in CD133(þ)/EpCAM(þ) populations among liver cancer cells [23,24]. Therefore, we examined the effect of HLs on CD133(þ)/EpCAM(þ) CSC populations among liver cancer cells. As shown in Fig. 2A, DOX increased the CD133(þ)/EpCAM(þ) population in HepG2 cells in a dose-dependent manner, indicating chemo-resistance in these cells. Notably, reduced CD133(þ)/ EpCAM(þ) CSC sub-populations of liver cancer cells treated with HLs were observed in a dose-dependent manner (Fig. 2B). These results indicate that HLs selectively inhibited CD133(þ)/EpCAM(þ) populations in HepG2 cells. Cancer cell stemness properties, including the colony formation capacity, were evaluated. We confirmed the effect of HLs on colony formation capacity in the soft agar colony formation assay. DOXpretreated cells induced the formation of larger and greater numbers of tumor colonies than non-treated cells (control) (Fig. 3A, left). DOX also showed a tendency to increase the number of colonies forming cells in the concentration range of 0.001e0.05 mM (Fig. 3B). This result suggests that DOX treatment enriched CSCs (particularly CD133(þ)/EpCAM(þ)) among HepG2 cells. In contrast, HL-pretreated cells induced lower numbers of tumor colonies than non-treated cells (control) in a dose-dependent manner (Fig. 4A, right). HLs significantly decreased the number of colony-forming cells in a dose-dependent manner (Fig. 3C, p < 0.05). These results revealed that HL suppresses colony-forming ability, which is a characteristic of CSCs. We next examined the fusion and accumulation of HLs including NBDPC (HL/NBDPC) in the cell membranes of each
Fig. 1. Inhibitory effects of DOX and HLs on the growth of HepG2 cells or Hc cells for 48 h HepG2 cells were treated with DOX (A) or HLs (C) for 48 h. Hc cells were treated with DOX (B) or HLs (D) for 48 h. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the controls.
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Fig. 2. Flow cytometry analysis of CSC populations of CD133þ/EpCAMþ in liver cancer cells treated with DOX or HLs for 48 h. (A) Expression of CD133 and EpCAM by DOX treatment for 48 h. (B) Percentage of CD133(þ)/EpCAM(þ) cells (CSCs) by DOX treatment for 48 h. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the control. (C) Expression of CD133 and EpCAM by HL treatment for 48 h. (D) Percentage of CD133(þ)/EpCAM(þ) cells (CSCs) by HL treatment for 48 h. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the control.
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Fig. 3. HLs reduce the number of colony-forming cells for HepG2 cells. (A) Fluorescence micrographs of colony-forming cells (blue) by pretreatment with DOX (left) or HLs (right) in soft agar medium. The threshold of the colony was set to over 5700 mm2 and shown in blue in the photograph (non-colony is shown in red). Scale bars shown 200 mm. Inhibitory effect of DOX (B) and HLs (C) on the number of colony-forming cells. Data represent the mean ± SE (n ¼ 4). *Significant difference (p < 0.05) compared to the controls. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
population in HepG2 cells by flow cytometry. As shown in Fig. S2, increased accumulation of HL/NBDPC into HepG2 was observed in a dose-dependent manner. After treatment of each cell population with 200 mM HL/NBDPC, NBDPC(þ) cells were gated for CD133(þ)/EpCAM(), CD133(þ)/EpCAM(þ), CD133()/ EpCAM(þ), and CD133()/EpCAM() cells (Fig. 4A). This result indicates that HL/NBDPC accumulated more in CD133(þ)/ EpCAM(þ) and CD133()/EpCAM(þ) cells than in CD133(þ)/ EpCAM() and CD133()/EpCAM() cells. Furthermore, significant accumulation of HL/NBDPC in CD133(þ)/EpCAM(þ) cells (particularly EpCAM(þ) cells) compared to other populations occurred in a dose-dependent manner (Fig. 4B). These results suggest that HLs selectively accumulated in CD133(þ)/EpCAM(þ) populations in HepG2 cells. Although the mechanistic details remain unclear, specific accumulation of HLs in the CSCs may be related to the difference in membrane physical properties among the CSC subpopulations, well-differentiated cancer cells, and normal cells, as described below. We reported that HLs distinguished between membranes of liver cancer cells and normal cells because of differences in membrane fluidity, and then fused and accumulated preferentially in the membranes of liver cancer cells [22]. Other studies also reported that the membranes of normal stem cells are more fluid
than those of well-differentiated cells [27]. This suggests that HLs accumulate in the more fluid membrane of CSCs than that of nonCSCs. We are currently investigating the membrane fluidity of these cells. Thus, our findings demonstrate that HLs are potential nanomedicine anti-cancer agent that targets co-expression of the membrane with the CD133 and EpCAM phenotypes (CSC-like cells) for liver cancer cells. In conclusion, we clearly demonstrated that HLs composed of DMPC and C12(EO)23 showed inhibitory effects towards the growth of CSC populations of HepG2 cells in vitro. The notable findings are as follows: (a) HLs selectively inhibited the growth of HepG2 cells without affecting normal hepatocytes through apoptosis via caspase-3. (b) HLs selectively inhibited CD133(þ)/EpCAM(þ) populations in HepG2 cells. (c) HLs suppressed the colony-forming ability of HepG2 cells. (d) HLs significantly accumulated in CD133(þ)/EpCAM(þ) populations in HepG2 cells. These results suggest that HLs are a novel nanomedical therapeutic agent for targeting CSCs in liver cancer therapy. Conflicts of interest The authors have no conflicts of interest to declare.
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Fig. 4. Flow cytometry analysis of CSC populations of CD133þ/EpCAMþ in HepG2 cells treated with HLs including fluorescently labeled lipids (NDBPC) for 1 h. (A) Flow cytometry analysis of each cell population treated with HL/NBDPC at 200 mM. HL/NBDPC (þ) cells were gated for each cell populations: (i):CD133(þ)/EpCAM(), (ii): CD133(þ)/EpCAM(þ), (iii): CD133()/EpCAM(), (iv): CD133()/EpCAM(). Data represent the mean ± SE of the indicated group (n ¼ 3). (B) Percentage of accumulation of HL/NBDPC for each cell population.
Author contributions KI, YK, and MY performed the research and analyzed the data. KI, YK, SI, YM, and TM designed the research. KI, YK, and TM wrote the manuscript. YM contributed the reagents. All authors read and approved the final manuscript.
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Acknowledgements [9]
This work was supported by JSPS KAKENHI Grant Number JP16K18304.
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Appendix A. Supplementary data [11]
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