Chemo-Sensitivity of Two-Dimensional Monolayer And

ANTICANCER RESEARCH 38 : 2101-2108 (2018) doi:10.21873/anticanres.12450

Chemo-sensitivity of Two-dimensional Monolayer and Three-dimensional Spheroid of Breast Cancer MCF-7 Cells to Daunorubicin, Docetaxel, and Arsenic Disulfide NAMI UEMATSU 1, YUXUE ZHAO 1,2 , ANNA KIYOMI 3, BO YUAN 4, KENJI ONDA 1, SACHIKO TANAKA 1, KENTARO SUGIYAMA 1, MUNETOSHI SUGIURA 3, NORIO TAKAGI 4, AKEMI HAYAKAWA 5 and TOSHIHIKO HIRANO 1

1Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan; 2Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing, P.R. China; 3Department of Drug Safety and Risk Management, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan; 4Department of Applied Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan; 5Organization for Promoting Life Science and Culture, Nagoya, and Department of Plastic and Reconstructive Surgery, Nagoya University of Graduate School of Medicine, Nagoya, Japan

Abstract. Background/Aim: Chemo-sensitivity of two- The standard treatments of breast cancer include surgical dimensional (2D) monolayers and three-dimensional (3D) resection, radiotherapy and chemotherapy, while those spheroids of human breast cancer MCF-7 cells were strategies have limited efficacy with toxicity and recurrence investigated. Materials and Methods: MCF-7 cells were of disease (1). Anti-estrogenic drugs and trastuzumab are cultured in monolayers or spheroids established using a thermo- effective for the treatment of breast cancer expressing reversible gelatin polymer, in the presence of daunorubicin, estrogen receptor and HER2, respectively. Whereas, breast docetaxel, or As 2S2. Cell proliferation was examined by a Cell cancer patients exhibiting low responses to these molecular- Counting Kit-8 assay. Results: Daunorubicin, docetaxel, and targeting drugs or patients without these target molecules As 2S2 dose-dependently decreased the MCF-7 cell proliferation should be treated with chemotherapeutic agents, which might in both 2D- and 3D-culture systems. The 3D spheroids were less cause serious toxicity. In addition, in many of these cases, sensitive to these agents than the 2D cultured cells. Verapamil, intrinsic and/or acquired drug resistance impose serious an inhibitor of P-glycoprotein, partially enhanced the problems for the success of the treatment of breast cancer (1). antiproliferative effects of the agents. DL-buthionine-(S, R)- To examine drug responses of breast cancer cells in vitro , sulfoximine significantly increased (p<0.05), while N-acetyl-L- studies using three dimensional (3D) cell culture systems to cysteine significantly inhibited the antiproliferative effects of generate multicellular tumor spheroids have drawn As 2S2 (p<0.003). Conclusion: The 3D spheroids showed less significant attention. Since solid tumor cells grow in vivo in sensitivity to the antiprolliferative efficacies of anticancer agents three dimensions, growing cancer cells in 3D-culture mimics than the 2D cultured cells. P-Glycoprotein is suggested to be the in vivo environment better than the traditional two- partially implicated in drug resistance. Reduction of cellular dimensional (2D) cell culture systems. Compared to 2D cell- glutathione level enhanced the As 2S2 cytotoxicity. culture system, the specific characteristics of 3D-cultured cancer cells include tight cell-cell interactions, cell- microenvironment interactions, and special cell internal and external structures (2, 3). Therefore, it has been suggested Correspondence to: Toshihiko Hirano, Department of Clinical that the 3D cultured breast cancer cells can provide more Pharmacology, School of Pharmacy, Tokyo University of Pharmacy accurate evidence for the chemo sensitivity of tumor cells and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel: +81 426765794, e-mail: [email protected] when we evaluate novel therapeutic drugs (4). Arsenic disulfide (As 2S2), as the main active component Key Words: Daunorubicin, docetaxel, arsenic disulfide, breast of realgar in traditional Chinese medicine, has been cancer MCF-7 cells, 2D monolayer, 3D spheroid. suggested to have therapeutic efficacies on several

2101 ANTICANCER RESEARCH 38 : 2101-2108 (2018) malignancies and the advantage of oral administration and Cell proliferation assay. Cell proliferation was analyzed using the 4 the relatively low toxicity (5-7). Increasing evidences show cell counting kit-8 assay. Totally 5×10 cells/well of MCF-7 cells were seeded into 48-well plates (cell density was 5×10 4 cells/500 that As 2S2 has the ability to induce apoptosis and to inhibit μl), and the cells were incubated at 37˚C in a humidified atmosphere growth of solid tumor cell lines. Apoptosis-inducing effects of 95% air and 5% CO 2 for 24 h without reagent. Then, of As S have been reported in human lymphoma cells (8), 2 2 daunorubicin, docetaxel, and As 2S2 were added to give each final cervical cancer cells (9), human hepatocellular carcinoma concentration shown in the figures. Then, the cells were incubated cells (10), and human osteosarcoma cells (11), respectively. at 37˚C in a humidified atmosphere of 95% air and 5% CO 2 for 72 The aim of the present study was to investigate the effects h. After incubation, 25 μl CCK-8 reagent was added into each well, followed by additional incubation for 3 h at 37˚C in a humidified of anticancer agents, daunorubicin, docetaxel, and As 2S2, on 2D- and 3D-cell cultured human breast cancer MCF-7 cells. atmosphere. The OD value of each well was measured by a Micro- plate reader (Corona MT P-32; Corona Co., Hitachi, Ibaraki, Japan) We compared the antiproliferative effects of anticancer at 570 nm. The cell proliferation rate was calculated according to agents between the 2D- and the 3D-cultured MCF-7 cells. the following equation: Cell viability rate=(OD sample value – OD We also tried to disclose the possible molecular mechanisms blank value)/(OD control value – OD blank value) ×100%. underlying drug resistance especially in the 3D-cultured cancer cells from the view point of P-glycoprotein function Statistical analysis. Data were presented as means±SEM. Statistical and cellular antioxidant activity. analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. For comparison of two groups, the data were analyzed Materials and Methods by Kolmogorov-Smirnov test, followed by the Student’s t-test or Mann–Whitney U- test. GraphPad Prism version 6.0 (GraphPad Reagents. MEM-alpha medium and fetal bovine serum were software Inc.) was used for the analyses, and the p- values less than purchased from Gibco BRL Co. (Grand Island, NY, USA). 0.05 were considered to be statistically significant. Each experiment Daunorubicin, docetaxel, and As 2S2 were obtained from Sigma was repeated independently at least three times. Chemical Co. (St. Louis, MO, USA). Stock solutions of daunorubicin and docetaxel were prepared at a concentration of 10 mmol/L with Results ethanol and diluted to working concentrations before use. Test- compound solutions were made at a concentration of 5 mmol/L with Anti-proliferative effects of anticancer drugs and As 2S2 on ethanol and diluted to working concentrations before use. As 2S2 was dissolved in 1N NaOH, and further adjusted to pH 7.35-7.45 using 2D- and 3D- cultured MCF-7 cells. MCF-7 cells were HCl to obtain a final concentration of 5 mM. Then, the solution was cultured in the presence of serial concentrations of anticancer filtrated with a 0.20 μm membrane filter (Advantec, Tokyo, Japan) drugs and As 2S2 for 72 h in both 2D- and 3D-cultured and stored at 4˚C until use. The working concentrations were systems. Then, cell counting was performed using the Kit-8 prepared after dilution with phosphate-buffered saline (PBS). Cell to assess the cell proliferation in both 2D and 3D cell culture Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories systems. As shown in Figure 1, daunorubicin dose- (Tokyo, Japan). DSeA-3D plate filled with thermo-reversible gelatin dependently inhibited proliferation of 2D-cultured MCF-7 polymer (TGP) was obtained from IFTL Inc., (Tokyo, Japan). All –7 other reagents were of the best available grade. cells with IC 50 values less than 10 M. Whereas, MCF-7 cells cultured in the 3D system showed relatively low Cell line and cell culture. The human breast cancer cell line MCF- responses to the antiproliferative effects of the drug with –5 7 was purchased from the American Type Culture Collection IC 50 values over than 10 M (Figure 1A). Docetaxel also (Manassas, VA, USA). MCF-7 cells were cultured in MEM-alpha inhibited proliferation of 2D-cultured MCF-7 cells at medium supplemented with penicillin, streptomycin and 10% fetal relatively high concentration with IC 50 values greater than bovine serum, and maintained as attached cells at 37˚C in 5% 10 –4 M. MCF-7 cells cultured in the 3D system showed carbon dioxide in a humidified atmosphere. Dye exclusion test using significantly low responses to the antiproliferative effects of trypan blue as a dye was used to determine number of living cells, and the living cell number was adjusted to 5×10 5 cells/ml for the the drug, as compared to the cells cultured in 2D system maintenance of the cells. (p< 0.01) (Figure 1B). As 2S2 decreased the MCF-7 cell proliferation cultured in 2D system, while MCF-7 cells 2D and 3D cell culture and drug treatment. MCF-7 cells were seeded cultured in the 3D system relatively showed resistance to the at a density of 10,000 cells per well in 500 μl of cell culture media suppressive effects of As 2S2 (Figure 1C). into 48-well plates (IWAKI micro-plates for 2D culture and DSeA- 3D micro-plates for 3D culture), followed by an incubation period of Influence of P-glycoprotein inhibitor verapamil on the 24 h. The 3D spheroids were established using TGP in the DSeA-3D antiproliferative efficacies of anticancer drugs and As S in micro-plates. Reagents, including vehicle for controls (cell culture 2 2 media) and different concentrations of the agents, were subsequently 2D- and 3D- cultured MCF-7 cells. One of the mechanisms added into the corresponding wells to adjust each reagent of drug resistance in cancer cells involves P-glycoprotein concentration. MCF-7 cells were then allowed to grow for 72 h in the function which has been considered as the major molecule presence of the agents, followed by cell proliferation assay. excreting several lipophilic anticancer drugs (12). Thus, we

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Figure 2. Effects of a P-glycoprotein inhibitor verapamil on the antiproliferative efficacies of anticancer drugs and As 2S2 in 2D-cultured Figure 1. Comparative study for the antiproliferative effects of human breast cancer MCF-7 cells. Cell proliferation in the 2D-cultured anticancer drugs and As 2S2 on 2D (open circle)- or 3D (closed circle)- MCF-7 cells in the presence of serial concentrations of anticancer cultured human breast cancer MCF-7 cells. Cell proliferation in the 2D- drugs and As 2S2 without (open circle) or with (closed circle) 10 μM or 3D-cultured MCF-7 cells in the presence of serial concentrations of verapamil were estimated with the Cell Counting Kit-8 assay anticancer drugs and As 2S2 were estimated with the Cell Counting Kit- procedures, and the percentages of the proliferated cells were plotted. 8 assay procedures, and the percentages of the proliferated cells were A, effects of daunorubicin; B, effects of docetaxel; C, effects of As 2S2. plotted. A, effects of daunorubicin; B, effects of docetaxel; C, effects of Statistically significant difference was observed in the daunorubicin As 2S2. Statistically significant differences were observed between the effects between cells cultured in the presence and absence of verapamil 2D- or 3D-cultured cells with p-values of *0.05 and *0.01, respectively. with p-value of *0.05.

2103 ANTICANCER RESEARCH 38 : 2101-2108 (2018) examined the influence of P-glycoprotein inhibitor verapamil on the antiproliferative efficacies of anticancer drugs and As 2S2 in 2D- and 3D- cultured MCF-7 cells (Figures 2 and 3). MCF-7 cells were cultured in 2D- or 3D-system with serial concentrations of anticancer drugs in the presence or absence of 10 μM verapamil, and the proliferated cells were analyzed by Cell Counting Kit-8 assays. Verapamil partially inhibited the antiproliferative efficacy of daunorubicin, docetaxel, and As 2S2 in 2D cultured MCF-7 cells, though the effects on docetaxel and As 2S2 were not statistically significant (Figure 2A-C). In contrast, verapamil showed little or no effect on the antiproliferative efficacies of anticancer drugs and As 2S2 in 3D-cultured MCF-7 cells (Figure 3A-C). A statistically significant effect of verapamil was observed only when the drug was used in combination with 4 μM As 2S2 in 3D-cultured MCF-7 cells (Figure 3C).

Influence of reactive oxygen species-modifiers on the antiproliferative efficacies of anticancer drugs and As 2S2 on 2D- and 3D- cultured MCF-7 cells. Anticancer agents such as arsenic compounds have been reported to show suppressive effects on cancer cells via increase in cellular toxic oxygen species ls (13). Thus, we examined proliferation of 2D- and 3D- cultured MCF-7 cells treated with daunorubicin, docetaxel, or As 2S2 in the presence of glutathione modifier ascorbic acid, DL-buthionine-(S,R)-sulfoximine, and N-acetyl-L-cysteine. In 2D-cultured MCF-7 cells, ascorbic acid at 125 μM, DL- buthionine-(S, R)-sulfoximine at 100 μM, or N-acetyl-L- cysteine at 10 mM showed no influence on cell proliferation. In the presence of As 2S2, co-administration of ascorbic acid had no effect (Figure 4A), whereas DL-buthionine-(S, R)- sulfoximine significantly promoted the suppressive efficacy of 4-16 μM As 2S2 (Figure 4B, C) ( p= 0.0004 and 0.0001). In contrast, N-acetyl-L-cysteine significantly reduced the suppressive efficacy of 16 μM As 2S2 (p= 0.0123) (Figure 4D). In 3D-cultured MCF-7 cells, similar tendencies were observed on the additive effects of ascorbic acid, DL- buthionine-(S, R)-sulfoximine, or N-acetyl-L-cysteine (Figure 5). N-acetyl-L-cysteine itself significantly promoted cell proliferation ( p= 0.0028) (Figure 5), and significantly inhibited the antiproliferative effects of 8 and 16 μM As 2S2, compared to control ( p= 0.0028 and 0.0001) (Figure 5C, D). In the presence of As 2S2, co-administration of ascorbic acid had no effect (Figure 5A-D), whereas DL-buthionine-(S, R)- sulfoximine significantly promoted the suppressive efficacy of Figure 3. Effects of verapamil on the antiproliferative efficacies of 4-16 μM As S (Figure 5B, C) ( p= 0.0001). In contrast, N- 2 2 anticancer drugs and As 2S2 in 3D-cultured human breast cancer MCF- acetyl-L-cysteine significantly reduced the suppressive efficacy 7 cells. Cell proliferation in the presence of serial concentrations of of 16 μM As 2S2 (Figure 5D). anticancer drugs and As 2S2 without (open circle) or with (closed circle) 10 μM verapamil were estimated with the Cell Counting Kit-8 assay Discussion procedures, and the percentages of the proliferated cells were plotted. A, effects of daunorubicin; B, effects of docetaxel; C, effects of As 2S2. Statistically significant difference was observed in the As 2S2 effects It has been known that 3D cultured cancer cells sometimes between cells cultured in the presence and absence of verapamil with show different drug responses, as compared to 2D cultured p-value of *0.05.

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Figure 4. Effects of reactive-oxygen modifiers on the antiproliferative efficacies of As 2S2 in 2D-cultured human breast cancer MCF-7 cells. Cell proliferation in the presence of serial concentrations of As 2S2 with or without reactive-oxygen modifiers, ascorbic acid (AA) at 125 μM, DL- buthionine-(S, R)-sulfoximine (BSO) at 100 μM, and N-acetyl-L-cysteine(NAC) at 10 mM were estimated with the Cell Counting Kit-8 assay procedures, and the percentages of the proliferated cells were indicated. A: Effects of reactive-oxygen modifier alone; B: effects of 4 μM As 2S2; C: effects of 8 μM As 2S2; D: effects of 16 μM As 2S2. In A, control means vehicle alone, while in B-D, control means each concentration of As 2S2 alone. Statistically significant difference was observed in the cell proliferation (%) between control cells and cells in the presence of each reactive- oxygen modifier with p-values of *0.0123, ***0.0004, and ****0.0001, respectively.

monolayer cells. Since the 3D cultured cancer cell models chemotherapeutic agents, daunorubicin and docetaxel, to enable cells to form structures that mimic tumors in vivo in inhibit MCF-7 cell proliferation. These observations were a more physiologically-relevant condition than traditional 2D consistent with those of other researchers (17-19), which monolayers (14-16), it provided a more reliable way to might be related with hypoxia and endocrine changes in 3D investigate the efficacies of anticancer drugs on breast cancer spheroids microenvironment. cells. Thus, in the present study, we compared the chemo Many kinds of gels have been used for 3D culture of sensitivities of 2D monolayers and 3D spheroids established cancer cells and tissues, which include spinner cultivation using TGP gel of breast cancer MCF-7 cells in vitro . We technique (20), alginate beans (21), agarose (22), soft demonstrated that, comparing with 2D monolayer, the 3D agarose, Matrigel (23), or collagen matrix gel (24). spheroid showed resistance against the efficacies of However, cytotoxic enzyme use is required for the

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Figure 5. Effects of reactive-oxygen modifiers on the antiproliferative efficacies of As 2S2 in 3D-cultured human breast cancer MCF-7 cells. Cell proliferation in the presence of serial concentrations of As 2S2 with or without reactive-oxygen modifiers, ascorbic acid (AA) at 125 μM, DL- buthionine-(S, R)-sulfoximine (BSO) at 100 μM, and N-acetyl-L-cysteine(NAC) at 10 mM were estimated, and the percentages of proliferated cells were indicated. A: Effects of reactive-oxygen modifier alone; B: effects of 4 μM As 2S2; C: effects of 8 μM As 2S2; D: effects of 16 μM As 2S2. In A, control means vehicle alone, while in B-D, control means each concentration of As 2S2 alone. Statistically significant difference was observed in the cell proliferation (%) between control cells and cells in the presence of each reactive-oxygen modifier with p-values of *0.0206, **0.0028, and ****0.0001, respectively.

culturing of cells or tissues in these culture systems. The toxicity of TGP is extremely low so that cells can be 3D culture system using thermo-reversible gelatin polymer cultured for long time in this gel system. Moreover, (TGP) (25) is simple and easy for culturing cells or tissues fibroblasts are hard to grow, while cancer cells can easily and collecting culture-superatant without damaging the proliferate, in the TGP gel. cells. TGP is a heat-reversible hydrogel constituted by There have been many reports describing that arsenic block-copolymer of hydrophilic polymer and temperature- compounds have antitumor potencies against breast cancer sensitive polymer. TGP has a unique property as a gel in cell lines (26-28). Among those, arsenic trioxide was known high temperature and a sol state in low temperature to suppress cell growth, inhibit cell migration, and induce reversibly, with a transition temperature of 22˚C. The cell apoptosis and cell-cycle arrest in breast cancer cell lines

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(29-32). Whereas, arsenic trioxide was reported to cause References severe adverse effects, especially hepatotoxicity (33, 34). 1 Recht A, Come SE, Henderson IC, Gelman RS, Silver B, Hayes As 2S2 was suggested to have much less toxicity as compared to arsenic trioxide, and has already been approved DF, Shulman LN and Harris JR: The sequencing of chemotherapy and radiation therapy after conservative surgery to be used clinically in the treatment of leukemia in China for early-stage breast cancer. N Engl J Med 334 : 1356-1361, (35, 36). Therefore, in the present study, we examined the 1996. anticancer effects of As 2S2 and the underlying mechanisms 2 Weigelt B, Ghajar CM and Bissell MJ: The need for complex involved in the drug resistance especially in 3D-cultured 3D culture models to unravel novel pathways and identify MCF-7 cells. As 2S2 decreased proliferation of MCF-7 cells accurate biomarkers in breast cancer. 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