National Sun Yat-sen University Institutional Repository:Item 987654321/39762

行政院國家科學委員會專題研究計畫 成果報告

子計畫二─探討新合成之抗癌試劑的抗癌效果與其分子作

用機轉(3/3)

中 華 民 國 94 年 11 月 17 日

DC-81-indole conjugate agents potently induce Chk2 activation, S phase arrest and apoptosis in colon cancer cells

Hui-Chiu Chang a, Li-Teh Liu a, Jeh-Jeng Wang b, Ming-Jung Wu b, and Wen-Chun Hung a

a

Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan and b School of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan

Abstract

DC-81 is an anti-tumor antibiotic produced by Streptomyces species. Our previous study demonstrated that DC-81-indole conjugate agents exerted potent cytotoxicity on various types of human cancer cells. In this study, we address whether DC-81-indole conjugate agents may activate checkpoint kinases (Chks) to induce growth arrest and apoptosis. The effects of DC-81-indole conjugate agents on proliferation, cell cycle progression, Chk activation, and apoptosis in COLO205 (harboring wild type p53) and SW480 (harboring mutant p53) human colon cancer cell lines were investigated. Our results demonstrated that DC-81-indole conjugate agent with a three carbon spacer between DC-81 and indole carboxylate moiety (I3) was more effective than

DC-81-indole conjugate agent with a four carbon spacer (I4) in the induction of

growth arrest and apoptosis. We also found that I3 stimulated Chk2, but not Chk1,

activation and induced cell cycle arrest at the S phase. p53 protein level was increased by I3 in COLO205 cells while the amount of mutant p53 protein in SW480 cells was

not affected. Up-regulation of p53 leaded to increase of Bax expression and apoptosis in COLO205 cells. However, I3 also triggered apoptosis at a lesser extent in SW480

cells via a p53- and Bax-independent manner. Taken together, these results suggest that DC-81-indole conjugate agents induce Chk2 activation and S phase arrest in human colon cancer cells and stimulate apoptosis in these cells via p53-dependent and -independent pathways.

1. Introduction

Pyrrolo[2, 1-c][1, 4]benzodiazepines (PBDs) are a group of naturally occurring anti-tumor antibiotics produced by Streptomyces species [1]. Well-known members of PBDs include DC-81, anthromycin and tomaymycin [2-4]. The cytotoxic effect of these antibiotics are believed to arise from the modification of DNA via their covalent binding ability to the N2 of the guanine residues in the minor groove of DNA, which leads to the inhibition of nucleic acid synthesis and the production of excision-dependent single and double strand breaks in cellular DNA [5].

Although PBDs exhibit high anti-tumor activity, they are cardiotoxic, which has precluded their continued clinical application [6]. Therefore, a number of PBD hybrid compounds have been designed and synthesized to provide highly sequence-selective DNA binding and anti-tumor activity [7-9]. We have previously synthesized various DC-81-indole conjugate agents and found that these compounds potently suppressed the growth of various types of human cancer cells [10]. However, the detailed mechanism of this growth-inhibitory action is unclear.

Recent studies indicate that DNA damages activate cellular responses that promote DNA repair, cell cycle arrest, and in some cases, induce apoptosis. Several protein kinases play important roles in the activation of cellular responses to DNA damage including ataxia-telangiectasia- mutated (ATM), ATR (ATM and rad3-related), and their kinase substrates checkpoint kinase 1 (Chk1) and Chk2 [11-14]. Chk1 is mainly

involved in the regulation of G2/M- and S-phase checkpoints and is expressed in the S and G2 phases of proliferating cells. In addition, Chk1 is essential for early embryonic development. In contrast to Chk1, Chk2 is a stable protein and is expressed throughout the cell cycle. In mammalian cells, both Chk1 and Chk2 pathways play a role in response to DNA damages and replication stress. In addition to these protein kinases, the p53 tumor suppressor protein also involved in the induction of either cell cycle arrest or apoptosis upon DNA damage [15]. In this study, we investigate whether Chks and p53 are involved in DC-81-induced anti-tumor action.

3. Results

3.1. DC-81-indole conjugate agents induced S phase arrest and apoptosis in human colon cancer cells

Two DC-81-indole conjugate agents with a three (I3) or four (I4) carbon spacer

between DC-81 and indole carboxylate moiety (Fig. 1) were synthesized and the cytotoxicity of these two compounds on COLO205 (harboring wild type p53) and SW480 (harboring mutant p53) human colon cancer cells were tested. As shown in Fig. 2, I3 and I4 suppressed cell proliferation in a dose-dependent manner and I3 was

more potent in growth inhibition. Our results also indicated that COLO205 cells were more sensitive to the cytotoxicity of DC-81-indole conjugate agents than SW480 cells. We next addressed the underlying mechanism of the anti- tumor action of I3 and I4.

Flow cytometric analysis indicated that I3 and I4 might simultaneously affect cell

cycle progression and cell viability. Accumulation of cells at the S phase was obviously increased after drug treatment (Table 1). These data suggest that I3 and I4

of cells at the sub-G1 (apoptotic cells) was increased after drug treatment (Fig. 3A). In consistent with the results of MTT assays, the I3 was more potent than I4 in the

induction of apoptosis and p53 wild type COLO205 cells were more sensitive to these two DC-81-indole conjugate agents. Therefore, in addition to induce S phase arrest, I3

and I4 may stimulate apoptosis in colon cancer cells. To further verify this issue, we

extracted genomic DNA from control and drug-treated COLO205 cells and separated the extracted DNA samples by agarose gel electrophoresis. Our data indeed showed that DNA fragmentation (a typical feature of apoptosis) was seen in drug-treated COLO205 cells (Fig. 3B).

Because COLO205 cells are more sensitive to the cytotoxicity of DC-81-indole conjugate agents, it is possible that p53 plays a role in the induction of apoptosis by these agents. So, we studied the expression of p53 and Bax, a major downstream apoptotic effector of p53, in drug- treated cells. As shown in Fig. 4A, I3 and I4

up-regulated p53 and Bax protein level in a time-dependent fashion and I3 was more

potent in the induction of these two proteins. Conversely, the expression of mutant p53 and Bax protein in SW480 cells was not regulated by I3 and I4 (Fig. 4B).

Collectively, these results suggest that DC-81-indole conjugate agents induce apoptosis of cancer cells mainly via the p53/Bax pathway and are more effective on cancer cells harboring wild type p53. However, these agents also stimulate apoptosis via a p53-indepedent pathway because cancer cells harboring mutant p53 can still undergo apoptosis after drug treatment.

3.2. Activation of Chk2, but not Chk1, in colon cancer cells by I3

Because I3 potently induced S phase arrest, we tested whether this agent might

various types of DNA damage and are involved in the regulation of S phase checkpoint. Our results demonstrated that I3 did not change the Chk1 protein level in

colon cancer cells and Ser296, 317 and 345 of Chk1 were not phosphorylated after I3

treatment as detected by the phospho-specific Chk1 antibody (data not shown). On the contrary, we found that I3 could stimulate Chk2 activation. The amino-terminal

domain of Chk2 contains several Ser and Thr residues which are known to be phosphorylated by ATR or ATM kinase [19]. Our results demonstrated that I3

treatment increased the phosphorylation of Thr68 and 387, but not Ser19 (Fig. 5). Similar results were observed in I4-treated cells (data not shown). These data suggest

that DC-81-indole conjugate agents may induce Chk2 activation in colon cancer cells.

Disscussion

The interaction between PBDs and DNA is unique because these agents bind within the minor groove of DNA forming a covalent bond with the N2 amino group of guanine residues. The cytotoxic activity of PBDs is mainly attributed to their ability to form covalent DNA adducts. Previous investigations demonstrated that PBDs might exert their biological activity via several mechanisms. First, PBDs might interfere the interaction between endonuclease enzymes and DNA [20]. Second, PBDs might block transcription by inhibiting RNA polymerase [21]. Third, PBDs might stimulate excision-dependent single- and double- strand breaks of DNA [5]. PBDs have also been used to conjugate with other moieties such as EDTA, epoxide, polyamide and oligopyrrole to develop new PBD hybrid molecules which may exhibit more selective cross-linking and DNA-cleaving properties [7-9]. In this study, we show that DC-81-indole conjugate agents exert potent cytotoxicity on colon cancer cells and

provide the first evidence that these PBD hybrid molecules may stimulate Chk2 activation and induce S phase arrest and apoptosis.

Chk2 is the mammalian homolog of the Saccharomyces cerevisiae Rad53 and

Schizosaccharomyces pombe Cds1 kinases [22]. This kinase is implicated in the DNA

damage signaling pathway and can be activated by phosphorylation by ATM or ATR upon different types of stresses. For example, ionizing radiation activates ATM to phosphorylate Chk2 at Thr68 and this phosphorylation is necessary for Chk2 activation [19]. Thr68 can also be phosphorylated by ATR in vitro. In addition, ATM can phosphorylate Ser19 and Thr33/35 [19]. Recent studies demonstrate that Chk2 is a tumor suppressor that may regulate apoptosis in both an ATM-dependent and -independent manner [23]. A number of substrates for Chk2 including p53, CDC25A, E2F1 and BRCA1 have been identified [24]. Among these substrates, p53 may be an important mediator for Chk2-induced apoptosis. In response to DNA damage, Chk2 phosphorylates p53 at the Ser20 residue [25, 26]. Ser20 comprises part of the binding site for MDM2 and phosphorylation of this residue interferes the binding between p53 and MDM2 and attenuates MDM2-mediated degradation of p53. Increased protein level and activity of p53 then activate the downstream effectors such as Bax, p21WAF1, and GADD45 and cause growth arrest and apoptosis. Indeed, we find that DC-81-indole conjugate agents up-regulate wild type p53 and Bax protein levels and potently induce apoptosis in COLO205 cells. However, it is noteworthy that these conjugate agents can also induce apoptosis via a p53- independent pathway. The detailed mechanism of this pathway is currently unknown. Collectively, we conclude that PBD hybrid molecules may stimulate Chk2 activation and apoptosis in colon cancer cells and may be a novel class of anti-tumor agents for the therapy of this malignancy.

Figure Legends

Fig. 1. Chemical structure of DC-81-indole conjugate agents I3 and I4.

Fig. 2. I3 and I4 showed potent cytotoxic activity on human colon cancer cells. Cells

were incubated with various concentrations of I3 and I4 for 24 h and harvested for

analysis of cell viability by a MTT-based assay as described in the Materials and methods. COLO205 cells treated with I3 (◆) or I4 (▲); SW480 cells treated with I3

(●) or I4 (■).

Fig. 3. Induction of apoptosis by I3 and I4 in human colon cancer cells. (A) Cells were

treated with 0.01μg/ml of I3 and I4 for 24 h and harvested for flow cytometric

analysis as described in Materials and methods. Percentages of cells in the sub-G1 phase from three independent experiments were calculated and expressed as mean± SD. (B) Genomic DNA was also extracted and separated by agarose gel electrophoresis to clarify the DNA fragmentation in drug-treated cells.

Fig. 4. I3 and I4 increased the p53 and Bax protein expression in COLO205, but not

SW480 cells. (A) COLO205 cells were treated with 0.01μg/ml of I3 and I4 for

different times and cellular proteins were extracted. Immunoblotting was performed to investigate the protein level of p53 and Bax in cells. Actin was used as an internal control to verify the equal loading of each lane. (B) SW480 cells were treated and the protein level of p53, Bax and actin were analyzed as described above.

Fig. 5. I3 stimulated Chk2 phosphorylation in COLO205 cells. COLO205 cells were

incubated with 0.01μg/ml of I3 for 8 h and protein level of Chk2 was investigated by

immunoblotting. The phosphorylation status of Chk2 was analyzed by probing the blots with various phospho-specific Chk2 antibodies.

Table 1

Effect of I3 and I4 on cell cycle distribution in colon cancer cells

COLO205 SW480 G0/G1 S G2/M G0/G1 S G2/M Treatment Control 45.6% 37.3% 17.1% 55.3% 37.1% 7.5% I3 (0.01μg/ml) 29.1% 52.5% 18.6% 35.8% 52.2% 12.0% I4 (0.01μg/ml) 37.0% 43.2% 19.8% 46.9% 42.2% 10.9%

Cells were treated with drugs for 24 h and harvested for cell cycle analysis by flow cytometry. Three independent experiments demonstrated similar results and data from a typical assay were shown.

Fig. 1

Fig. 3