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J Pharmacol Sci 122, 153 – 157 (2013) Journal of Pharmacological Sciences© The Japanese Pharmacological Society

Short Communication

Cancer is currently a leading cause of death in through-out the world. Therefore there is an urgent need to develop anticancer agents. Most anticancer drugs work only after being absorbed into a cancer cell, the drugs having relatively low molecular weights and stable structure that can be easily incorporated via a diffusion system or transportation in the cell membrane. However, anticancer drugs that are easily absorbed are also often easily excreted from the cell, and the effect may be insuf-ficiently controlled. Also their use may be accompanied by severe adverse drug reactions and multiple drug resistances (1). Therefore, a new seed compounds for future development of anticancer drugs are required. We have synthesized a novel supramolecuar substance, [2] rotaxane (TRO-A0001) (Fig. 1A), which is a unique polymer compound devoid of a covalently bonded moiety (2 – 4).

In the therapy to control cancer cells through induction of apoptosis, it is considered important to focus on the mitochondria-mediated pathway, converging on caspase-3 and other proteases in the therapeutical means (5 – 7). The principal purpose of this study was to deter-mine whether this [2] rotaxane has bioactivity to inhibit the growth of cancer cells and whether its mechanism is related to action against caspase-3 or another protease.

[2] Rotaxane used in the present study is [2] [bis(2-(3,5-dimethylphenylcarbonyloxy)-ethyl) ammonium trifluoromethanesulfonate]-[dibenzo-24-crown-8] rotaxane (TRO-A0001). TRO-A0001 and bis(2-(3,5-dimethyl-phenylcarbonyloxy)-ethyl) ammonium trifluorometh-anesulfonate (NK7-40-2), which constitutes an axis part of TRO-A0001, were synthesized by Takata’s Lab (8). Dibenzo-24-crown 8-ether, which constitutes wheel part of TRO-A0001, was purchased from Tokyo Chemical Industry (Tokyo). These chemicals were dissolved in DMSO (Wako Pure Chemical Industries, Osaka).

The human Caucasian malignant melanoma cell line G361 and the human colon adenocarcinoma cell line

Influence of Novel Supramolecular Substance, [2] Rotaxane, on the Caspase Signaling Pathway in Melanoma and Colon Cancer Cells In VitroKazuki Hara1, Tatsuya Beppu1, Masahiko Kimura1, Yoshihiko Fujita2, Toshikazu Takata3, Kazuto Nishio2, and Nobufumi Ono1,*1Medicinal Informatics and Research Unit, Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma 8-19-1, Jonan-ku, Fukuoka 814-0180, Japan

2Department of Genome Biology, Kinki University School of Medicine, 377-21 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan

3Department of Organic and Polymeric Materials, Tokyo Institute of Technology, Ookayama 2-12-1, Meguro-ku, Tokyo 153-0063, Japan

Received October 23, 2012; Accepted April 15, 2013

Abstract. We studied the influence of novel supramolecular substance, [2] rotaxane (TRO-A0001), on caspase signaling and cell viability in cancer cell lines. TRO-A0001 suppressed concentration-dependently cell proliferation. Expression of the cleaved-form caspase-3 and PARP was significantly increased in cells exposed to TRO-A0001. The expression of Bax was increased by TRO-A0001. Furthermore, the down-regulation of Bax by siRNA resulted in growth activation significantly. The morphological analysis demonstrated that TRO-A0001 increased the levels of apoptotic cells in human cancer cell lines. These results suggest that TRO-A0001 induces apoptosis in cancer cells and holds potential as a new anti-tumor medicine.

Keywords: [2] rotaxane, supramolecular compound, apoptosis

*Corresponding author. ononob@fukuoka-u.ac.jpPublished online in J-STAGE on June 5, 2013doi: 10.1254/jphs.12244SC

154 K Hara et al

DLD-1 (European Collection of Cell Cultures, UK), mouse melanoma cell line B16/BL6, and mouse rectum carcinoma cell line Colon-26 (Institute of Development, Aging, and Cancer, Tohoku University, Japan) were used in this study. The cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 (Life Tech-nologies, Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and penicillin (50 units/ml)-streptomycin (50 g/ml) (Gibco) at 37°C in a humidified atmosphere of 5% CO2.

For quantitative study of viable cells and proliferation, we used the MTT assay and analyzed the results with a microplate reader (Beckman Coulter, CA, USA). Cells (5.0 × 103 cells/well) were added to 96-well plates in RPMI (50 L) containing 10% FBS. After incubating for 12 h, cells were treated with various concentrations of TRO-A0001 (2.5, 5.0, and 10.0 M in 0.2% DMSO) and incubated for another 12 – 48 h. The control cultures were treated with 0.2% DMSO alone in RPMI. The

number of viable cells after treatment with different concentrations of TRO-A0001 was determined from the standard curve of the number of cells and absorbance value.

Western blotting methods were used for analyzing caspase-9, caspase-3, caspase-7, poly (ADP-ribose) polymerase (PARP), and Bax proteins (antibody pur-chased from Cell Signaling Technology, MA, USA). Cell lysates treated with TRO-A0001 for 24 h were centrifuged at 4°C for 15 min (14,000 × g), and superna-tants were used for protein samples. Equal amounts of protein samples (2.5 g) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA).

Bax and non-targeting control small interfering RNA (siRNA) were purchased from Cell Signaling Technology. Cells were seeded in 6-well culture plates and allowed to grow for 24 h (60% – 80% confluence) in

Fig. 1. Effect of TRO-A0001 on the growth in tumor cells. A) Chemical structure of [2] Rotaxane (TRO-A0001) and the struc-tural constituents of the wheel and axis portion of the molecule. B) Effect of TRO-A0001 on the growth of melanoma and colon cancer cell lines. Cells were exposed to TRO-A0001 of various concentrations (2.5, 5.0, 10.0 M), and control (0 M) cultures were treated with 0.2% DMSO in RPMI for 12 – 48 h. Data are expressed as the mean ± S.E.M. of three independent experiments performed in triplicate. *P < 0.01, compared with control group incubated for each time.

155Anti-tumor Effect of Rotaxane

complete medium. After incubating, cells were trans-fected for 24 – 48 h using TransIT-siQUEST Transfec-tion Reagent (Mirus, Madison, WI, USA) with 100 nM control or Bax siRNA. After transfection, cells were harvested and seeded in 96-well culture plates to measure cell viability as described below.

Terminal Deoxynucleotidyl Transferase-dUTP Nick End Labeling (TUNEL) assay showed apoptotic cells. To examine cell death caused by TRO-A0001, we used the Click-iT TUNEL Alexa Fluor 594 Imaging Assay (Life Technologies, Invitrogen, Carlsbad, CA, USA). TUNEL-positive cells stained red and nuclei stained blue with Hoechest 33342. To provide a positive TUNEL group, we added DNase to another control group. Photo-graphs were obtained using a fluorescent microscope (Keyence, Osaka).

Results were analyzed by one-way analysis of vari-ance, followed by the Tukey–Kramer post hoc test to determine differences among groups. All values are

expressed as the mean ± S.E.M. P < 0.01 was considered statistically significant.

After incubation with TRO-A0001 for 48 h, viable cells were significantly decreased by TRO-A0001 concentra-tion-dependently (2.5 – 10.0 M) in all cancer cell lines (Fig. 1B). IC50 values after incubation with TRO-A0001 for 24 h were 2.58 M in G361, 7.81 M in DLD-1, 6.39 M in B16/BL6 (data not shown), and less than 2.5 M in Colon-26 cells.

Expression levels of caspases in the signaling pathway were measured as a marker of apoptosis. In the experi-ment to determine expression levels of caspases, cleaved caspase-3 and cleaved PARP were up-regulated by TRO-A0001 (2.5 – 10.0 M) in all cancer cells (Fig. 2: A – D). On the other hand, full length caspase-3 was not changed as compared with the control samples, and full length PARP was decreased in colon cancer cell lines (Fig. 2: C, D). The expression of full length caspase-3 was decreas-ing in melanoma cells, although full length PARP indi-

Fig. 2. Expression levels of caspase-9, -3, -7, PARP, and Bax determined by western blotting. A) melanoma G361, B) B16/BL6, C) colon carcinoma DLD-1, and D) Colon-26 cells. Cells were exposed to TRO-A0001 (5.0, 10.0 M) and control (0 M) cultures were treated with 0.2% DMSO in RPMI for 24 h. β-actin was used as the internal control for protein loading and transfer efficiency.

156 K Hara et al

cated a different expression in G361 cells (Fig. 2: A, B). These results suggest that TRO-A0001 induced injury or death of melanoma and colon cancer cells by activating an apoptotic pathway to impair nuclear function.

Expression levels of Bax as a key for cellular induced apoptosis through the mitochondrial pathway was mea-sured. The levels of Bax were increased by TRO-A0001, compared with the controls. Bax increases the mem-brane’s permeability, which leads to the increase of cytochrome c from mitochondria, activation of caspase-9, and initiation of the caspase activation pathway for apoptosis. Caspase-9 levels in human cancer cells were remarkably decreased in the 10.0-M samples compared to the control, but the different result in the expression

levels of cleaved caspase-9 was shown in G361 cells (Fig. 2: A, C). Furthermore, the expression levels of caspase-7 were different between each cell.

To confirm that the down-regulation of Bax blocked TRO-A0001-induced apoptosis, we used Bax siRNA transfection methods and examined the influence on cell viability. Our results showed that transfection of Bax siRNA significantly improved cell viability compared to control siRNA after 36-h treatment with TRO-A0001 in DLD-1, B16/BL6, and Colon-26 cells, although G361 cells did not show a significant improvement in viability (Fig. 3A). In G361, as shown Fig. 2A, expression levels of Bax were extremely low in the control samples (0 M), so we thought the Bax siRNA transfection did

Fig. 3. Effect of TRO-A0001 on the cell growth. A) Effect of Bax knockdown on apoptosis. Cells were transfected with siRNA (control or Bax) for 24 h. After transfection, cells were treated with TRO-A0001 in various concentrations (0.5 – 10.0 M), and control (0 M) cultures were treated with 0.2% DMSO in RPMI for 36 h. Positive control cultures were treated with camptothecin (CPT) (10.0 M). Data are expressed as the mean ± S.E.M. of three independent experiments performed in triplicate. *P < 0.05, between the control siRNA group and the Bax siRNA group. B) TUNEL imaging assay of TRO-A0001 in human cancer G361 (a) and DLD-1 (b) cells. Cells were treated with TRO-A0001 (10.0 M), and control (0 M) cultures were treated with 0.2% DMSO in RPMI for 12 h. To provide a positive TUNEL group, we added DNase to another control group. TUNEL-positive cells stained red and nuclei stained blue with Hoechest 33342. Magnification × 400. Bar = 50 m.

157Anti-tumor Effect of Rotaxane

not influence the viability of G361 cells treated with TRO-A0001. Altogether, these results suggested that the down-regulation of Bax was also one of the anti-apoptotitc events in the TRO-A0001-induced cell death.

TUNEL imaging assay showed a significant increase in the number of apoptotic cells after TRO-A0001 treat-ment (Fig. 3B). The numbers of TUNEL-positive cells were higher in the TRO-A0001-treated groups than in the control group.

Caspases play an important role in regulating apoptotic signal transmission. Caspases are synthesized as rela-tively inactive zymogens and are activated by cleavage via upstream proteases in an intracellular cascade (9). The initiator caspases are capable of cleaving and acti-vating downstream effector caspases, such as caspase-3, -6, and -7, and activation results in apoptosis (10). Apoptosis is a regulated suicide program. Induction of apoptosis is mediated either through death receptors or at the mitochondrial level, and both systems can activate caspase-3 (11, 12).

In this study, TRO-A0001 caused cell death character-ized by activation of the caspase signal pathway (Figs. 1B, 2). Activated caspase-3 is responsible for the proteo-lytic degradation of PARP, which occurs at the onset of apoptosis. PARP cleavage indicates the initiation of nuclear DNA damage, resulting in DNA fragmentation and the characteristic changes observed in apoptosis. PARP (116 kDa) is a nuclear enzyme that helps cells to maintain their viability so they can repair their DNA, while cleavage of PARP (89 kDa) facilitates cellular disassembly (13 – 15). Our expression analysis of cleaved caspase-3 suggested that TRO-A0001 activated caspase-3 and signaled to the downstream to mediate cleavage of PARP. The expression of cleaved PARP was increased in all cancer cells, resulting in apoptosis. The results on the expressions of caspase-9 and caspase-7 may be due to different properties in each cell line. Also, many TUNEL-positive cells were detected in TRO-A0001 (10.0 M)-treated human melanoma and colon cancer cells (Fig. 3B). Thus, cells with high levels of caspase-3 activation exhibited cell death driven by TRO-A0001, and the mechanism of cell death considered a lead to apoptosis. Furthermore, as shown in Fig. 3A, the knock-down of Bax markedly increased cell viability, in contrast with the anti-proliferative effect induced by TRO-A0001 in DLD-1, B16/BL6 and Colon-26 cells, indicating that siRNA has a tendency to cause the recovery of G361 cells.

In the exposure to each constituent element of TRO-A0001, dibenzo-24-crown-8-ether and NK7-40-2 did not affect cell viability (data not shown). It is suggested that the individual rotaxane structure has influenced in the growth of cells. The supra-molecular substance has an

inner-molecular movement such as a shuttling behavior on axis quantum mechanically. Then, the anti-prolifera-tive effect may participate in the specific molecular behavior in TRO-A0001.

In conclusion, [2] rotaxane, TRO-A0001, is able to induce apoptosis through caspase signaling activation in tumor cells and may be a useful anti-tumor drug.

References

1 Jabir N R, Tabrez S, Ashraf GM, Shakil S, Damanhouri GA, Kamal MA. Nanotechnology-based approaches in anticancer research. Int J Nanomedicine. 2012;7:4391–4408.

2 Nakazon o K, Takata T. Neutralization of a sec-ammonium group unusually stabilized by the “rotaxane effect”: synthesis, structure, and dynamic nature of a “free” sec-amine/crown ether-type rotaxane. Chemistry. 2010;16:13783–13794.

3 Tachiba na Y, Kawasaki H, Kihara N, Takata T. Sequential O- and N-acylation protocol for high-yield preparation and modification of rotaxanes: synthesis, functionalization, structure, and inter-component interaction of rotaxanes. J Org Chem. 2006;71:5093–5104.

4 Makita Y, Kihara N, Takata, T. Quantitative active transport in [2]rotaxane using a one-shot acylation reaction toward the linear molecular motor. J Org Chem. 2008;73:9245–9250.

5 Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell. 2002;9:459–470.

6 Alshatw i AA. Catechin hydrate suppresses MCF-7 proliferation through TP53/Caspase-mediated apoptosis. J Exp Clin Cancer Res. 2010;29:167.

7 Chakrab orty D, Bishayee K, Ghosh S, Biswas R, Kumar MS, Rahman KBA. [6]-Gingerol induces caspase 3 dependent apop-tosis and autophagy in cancer cells: Drug-DNA interaction and expression of certain signal genes in HeLa cells. Eur J Pharma-col. 2012;694:20–29.

8 Kihara N, Koike Y, Takata T. Effect of steric barrier on the shuttling of rotaxane having crown ether wheel. Chem Lett. 2007;36:208–209.

9 Earnsha w WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem. 1999;68:383–424.

10 Krajew ska M, Rosenthal RE, Mikolajczyk J, Stennicke HR, Wiesenthal T, Mai J, et al. Early processing of Bid and caspase-6, -8, -10, -14 in the canine brain during cardiac arrest and resusci-tation. Exp Neurol. 2004;189:261–279.

11 Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309–1312.

12 Ashken azi A, Dixit VM. Death receptors: signaling and modula-tion. Science. 1998;281:1305–1308.

13 Stenni cke HR, Salvesen GS. Properties of the caspases. Biochim Biophys Acta. 1998;1387:17–31.

14 Yang H L, Chen CS, Chang WH, Lu FJ, Lai YC, Chen CC, et al. Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata. Cancer Lett. 2006:231;215–227.

15 Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell. 1997;89:175–184.