Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
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Synthesis, cytotoxicity, DNA binding and topoisomerase II inhibition of cassiarin A derivatives Urarika Luesakul a, Tanapat Palaga b, Kuakarun Krusong c, Nattaya Ngamrojanavanich a, Tirayut Vilaivan a, Songchan Puthong d, Nongnuj Muangsin a,⇑ a
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand d Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand b c
a r t i c l e
i n f o
Article history: Received 20 December 2013 Revised 17 March 2014 Accepted 25 April 2014 Available online xxxx Keywords: Cassiaria A Amonafide Selenium compound DNA-binding Topoisomerase II
a b s t r a c t Four series of cassiarin A derivatives with alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-substituted ethylene glycol (5a–5c) and selenium-containing (6a) side chains were synthesized. Their antitumor activities were evaluated against BT474, CHAGO, HepG2, KATO-III, SW620 and CaSki cancer cell lines. Preliminary results demonstrated that 5b had moderate activities against HepG2 and KATO-III cell lines, while 5c showed moderate to high cytotoxicity against most tested cell lines. In addition, 6a exhibited moderate cytotoxicity against cervical cells, CaSki. DNA-binding and ethidium bromide displacement experiments suggested that 5c and 5b binds to DNA via an intercalative mode, whereas 6a did not. However, the selenium-containing cassiarin A derivative 6a inhibited topoisomerase II with more than 95% inhibition at the concentration of 50 lM. These cassiarin A derivatives showed lower toxicity to normal cells, WI-38 than amonafide therefore they are potential lead compounds to be further developed as new anticancer agents. Ó 2014 Elsevier Ltd. All rights reserved.
Cancer is one of the major causes of death worldwide, accounted for 7.6 million deaths in 2007 and projected to continue rising.1 Many effective compounds used for treatment of cancer are natural products or related to them. The first plant-derived agents were used as clinical drugs, the so-called vinca alkaloids vinblastine and vincristine were isolated from the Madagascar periwinkle. Other well-known anticancer drugs approved for clinical use include etoposide from Mayapple (Podophyllum pellatum),2 doxorubicin from bacterium Streptomyces peucetius,3 and camptothecin from Camptotheca acuminata.4 In addition to these well-known examples, there are more than 3000 compounds which can be potentially useful for cancer treatment.5 Cassia siamea is a common plant in Southeast Asia. This herb has been used as food and for treatment of insomnia. The major chemical constituent extracted from C. siamea leaves and flowers is barakol (1).6 In 2007, Morita et al. discovered cassiarin A (2)—an aromatic alkaloid with a tricyclic skeleton—in C. siamea but is less abundant than barakol.7 In 2010, Thamyongkit and co-workers were interested in their potent antiplasmodial activity and transformed
⇑ Corresponding author. Tel./fax: +66 2 218 7635. E-mail addresses: [email protected], [email protected] (N. Muangsin).
barakol into cassiarin A. Due to the structural similarity between the tricyclic aromatic core of cassiarin A and the anticancer agent amonafide (Fig. 1), it was proposed that derivatives of cassiarin A may exhibit cytotoxicity against cancer cells.8 Cassiarin A derivatives including ester, ether, nitrogen-substituted and dehydroxy derivatives had been synthesized and evaluated for their antimalarial activities. Among them, the parent compound, cassiarin A showed more potent antimalarial activity and less toxicity to human cells, MCF-7.9 Amonafide is a naphthalimide analog that exhibits excellent antitumor activity and was tested in clinical trials for treatment of cancer. This compound binds to DNA by intercalation and also inhibits Topoisomerase II.10 Topoisomerase II is the key enzyme that regulates the topological state of DNA. It plays important roles in transcription and replication process by breaking and rejoining of phosphodiester backbone of DNA stands.11 However, amonafide has failed to enter clinical phase III12 because it is easily metabolized to N-acetyl-amonafide by enzyme N-acetyl transferase 2 (NAT2), resulting in unpredictable toxicity.13,14 Accordingly, many derivatives such as alkyl, aryl and alkylamino substituted amonafide analogues have been investigated. These compounds showed similar or more potent cytotoxicity than amonafide but could survive drug-metabolized enzymes. Moreover, DNA-binding studies
http://dx.doi.org/10.1016/j.bmcl.2014.04.107 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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Figure 1. Chemical structures of barakol, cassiarin A and amonafide.
reveal that they can interact with DNA by a similar mode of action of amonafide.15,16 Selenium is an essential trace mineral that is important for human health. Selenium compounds have been proven as potent anticarcinogenic agents.17,18 The mechanism of selenium action is unclear. Some selenium compounds effects on important tumor suppressor protein p53 activity and different selenium forms regulate p53 in the different ways.19–21 Selenomethionine and methyl-seleninic acid may modify p53 for DNA repair, while sodium selenite may modify p53 for apoptosis.19 Selenite acts as a topoisomerase II poison by inducing topoisomerase II cleavage complexes.22 In addition, selenium analogues of naphthalimide showed good cytotoxicity against many cell lines with IC50 ranging 10–20 lM.23 In this research, new analogues of amonafide were synthesized by modification of cassiarin A.8 Cassiarin A is a potential candidate
for development of anticancer agent due to (i) the molecule contains a tricyclic planar aromatic moiety which is an important feature of DNA intercatator,24 (ii) the molecule has a amonafide-like aromatic core but exhibited low toxicity against human cell,9 and (iii) it showed potent biological activities including antiplasmodial and antimalarial activities.7,9 In this work, the C7–OH position of cassiarin A was modified which can be classified into four different groups namely alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/aminosubstituted ethylene glycol (5a–5c) and selenium-containing (6a) as shown in Figure 2. The compounds were synthesized by acylation of cassiarin A or by nucleophilic substitution of appropriate electrophiles (alkyl tosylate for 5b and 5c and phenylselenyl chloride for 6a) with cassiarin A under basic conditions. The chemical structures were confirmed by spectroscopic methods (FT-IR, 1H NMR, 13C NMR and MALDI-TOF MS). All compounds were preliminarily evaluated for their cytotoxicity and active compounds were further explored for their possible functional mechanisms such as DNA-binding activity and topoisomerase II inhibition. Cytotoxicity of the cassiarin A derivatives were determined by MTT assay against six human cancer cell lines including breast carcinoma (BT474), lung carcinoma (CHAGO), liver carcinoma (HepG2), gastric carcinoma (KATO-III), colon carcinoma (SW620), cervical carcinoma (CaSki) cell lines comparison with amonafide as positive control. All compounds were subjected to preliminary screening for their cytotoxic activity at high concentration (100 lM) as shown in Figures 3 and S1 in Supplementary data and divided into two groups as active (less than 20% cell viability) and inactive (more than 20% cell viability). Subsequently, IC50
Figure 2. Cassiarin A derivatives with four groups of side chains: alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-substituted ethylene glycol (5a–5c) and seleniumcontaining (6a) side chains.
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Figure 4. Toxicity of amonafide, 5b, 5c and 6a to normal cell line, WI-38.
Figure 3. Preliminary screening of 5b, 5c and 6a at concentration 100 lM against six human cancer cell lines compared to amonafide as the reference.
values were determined for active compounds and these were classified into three categories as high (IC50 86
>15.3
Inactive
Inactive
Inactive
Inactive
Inactive
29.74 ± 0.03
70.0
—
37.80 ± 6.05
1.05 ± 0.05
0.32 ± 0.07
1.71 ± 0.14
9.24 ± 0.09
9.96 ± 0.10
78.9
8.2
IC50, as drug concentration that caused 50% reduced the viable cell population determined by MTT assay. Inactive is defined as % cell viability more than 20% at high concentration (100 lM).
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Ethidium bromide displacement assay: Ethidium bromide displacement assay has been widely used to confirm the intercalative binding mode between a compound and DNA. The principle of the assay based on the displacement of the intercalating dye, ethidium bromide (EtBr) from ct-DNA.34 The emission spectra of EtBr bound to ct-DNA in the absence and the presence of amonafide, 5b and 5c are given in Figure 6. Ethidium bromide is barely fluorescent in aqueous solution but exhibits intense fluorescence emission at 595 nm when bound to ct-DNA, due to the strong intercalation with DNA base pairs. If addition of the test compound to ct-DNA pretreated with EtBr in the ratio of EtBr/DNA equal to 1:2.5 in terms of base pairs results in quenching of the emission intensity, one can conclude that the compound can displace the EtBr from its intercalative site. The efficiency of the EtBr displacement was investigated by determining the Stern–Volmer constant (KSV) value.35 The data were plotted as Stern–Volmer equation: I0/ I = 1 + KSV[Q] where I0 and I denoted the fluorescence emission intensity in the absence and presence of the quencher, KSV is the Stern–Volmer quenching constant and Q is the concentration of the quencher (in this case, 5b, 5c and 6a). The compound 5c
Figure 5. Thermal denaturation profiles of ct-DNA (60 lM by nucleotide) in the absence and presence of amonafide, cassiarin A, 5b, 5c and 6a (30 lM) in 10 mM phosphate buffer pH 7.4 (A) Tm curves and (B) derivative curves of Tm at 260 nm.
equation expressed as A = ebc where A is UV absorbance of ct-DNA, in the range of 0.38–0.39, e = extinction coefficient at 260 nm (6600 M 1 cm 1) and b is the cuvette path length (1 cm).28,29 The Tm values of the ct-DNA were found to be 70.7, 70.7 and 70.0 °C for free DNA, with cassiarin A and with 6a, respectively. The insignificant change of Tm indicated that cassiarin A and 6a did not interact with DNA by intercalation or groove binding, which should result in stabilization of the DNA duplex. On the other hand, amonafide and 5b can stabilize the DNA duplex as shown by small Tm increase to 78.9 °C (DTm = 8.2 °C) and 75.7 °C (DTm = 4.8 °C), for amonafide and 5b, respectively. Importantly, the compound 5c caused a significant increase of the Tm of ct-DNA (Tm >86 °C and DTm >15.3 °C) which is comparable to common organic intercalator such as ethidium bromide (DTm = 13 °C). The large DTm indicated that 5c strongly interacted with and stabilized the double helix of DNA. It is well known that many anti-tumor agents containing positively charged group such as dimethylamino-ethyl showed the excellent cytotoxic activity.30–32 The terminal nitrogen of dimethylamino-ethyl group of 5c which has pKa values around 9 should be readily protonated and electrostatically bound to the anionic phosphate group of DNA. This favorable electrostatic interaction precedes subsequent intercalative binding by the aromatic core of cassiarin A that results in the increase of Tm of the ct-DNA.15,33 This experiment suggested that compounds 5b and 5c can interact with ct-DNA, while compound 6a cannot. There are three types of interaction between a DNA-binding compound and double stranded DNA including electrostatic interactions with the phosphate groups, binding to minor or major grooves of DNA and intercalation between the DNA based pairs. In order to confirm the intercalation ability of these compounds, ethidium bromide displacement assay was studied using fluorescence spectrophotometry.
Figure 6. Emission spectra of EtBr (10 lM) bound to ct-DNA, 25 lM base pairs in the presence of compound (A) amonafide, (B) 5b and (C) 5c (0–70 lM) in 10 mM phosphate buffer pH 7.4. The arrow shows the direction of change of fluorescence intensity of EtBr bound to ct-DNA upon increasing amounts of tested compound. Inserts show Stern–Volmer curve for quenching of ct-DNA.
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showed the highest KSV value of 2.1 104 M 1, while the 5b and amonafide showed smaller values of 4.3 103 and 1.5 104 M 1, respectively, which is comparable to DNA-intercalator such as acridine derivative (KSV = 1.5 104 M 1).36 From these results, it can be concluded that the compounds 5b and 5c bind to ct-DNA via the intercalation mode. Furthermore, the apparent binding constants (Kapp) can be calculated using the equation; Kapp[compound] = KEtBr[EtBr], where KEtBr = 1.0 107 M 1 and [EtBr] = 10 lM.34 Compound 5c also showed the highest apparent binding constant (2.1 106 M 1), which confirmed that the intercalative binding occurs between compound 5c and ct-DNA. The DNA-binding studies correlate well with the cytotoxicity to cancer cell lines, the strongest DNA binding compound (5c) is the most cytotoxic compound. As 6a showed only weak DNA binding, its moderate cytotoxicity against CaSki cell line may arise from a different mode of action. Many reports showed that selenium compound could play multi-roles such as cancer prevention, DNA damage or topoisomerase II poison.22,23 Subsequently, compound 6a was further explored for other plausible mechanisms of action such as the inhibition of topoisomerase II. Topoisomerase II inhibition: Topoisomerase II is the key cellular enzyme that regulates the topological state of the DNA in cells. It plays an important role in replication and transcription process by breaking and rejoining of phosphodiester backbone of DNA stands.11 Amonafide is a known DNA intercalator and can inhibit topoisomerase II by stabilizing the enzyme–DNA complex.10 In this work, we also determined the ability of compound 5b, 5c and 6a to inhibit topoisomerase II decatenation of kDNA.37 Both forms of kDNA including (i) catenated kDNA (C) and (ii) nicked open circular decatenated kDNA (NOC) which is formed by the action of topoisomerase II were monitored. Catenated kDNA appears at the top and cannot enter into the gel because of its size, while decatenated product can readily move into the gel.38 The results shown in Figure 7 demonstrated that at a concentration of 100 lM, the selenium-containing compound 6a displayed a strong topoisomerase II inhibitory activity with more than 95% inhibition. The compound 5c exhibited a weak inhibitory effect (30.1%), whereas compound 5b did not inhibit topoisomerase II. The positive control, amonafide, showed a moderate activity of 61.8%. Moreover, 6a inhibited topoisomerase II with 31.5%, 75.7% and 95.7% inhibition 10, 30 and 50 lM, respectively. In conclusion, cassiarin A derivatives were synthesized and their cytotoxicity against six cancer cell lines (BT474, CHAGO, HepG2, KATO-III, SW620 and CaSki) were investigated. The preliminary results demonstrated that alkanoyl (3a–3d), aroyl (4a–4d) and ethyleneglycol (5a) derivatives showed low cytotoxic activity against all tested cell lines. Two hydroxy/amino-substituted ethylene
Figure 7. Inhibitory effect of the tested compounds on human DNA topoisomarase II decatenation. Lane 1, decatenated kDNA marker; lane 2, kDNA; lane 3, kDNA + 4 units of Topo II; lanes 4–7, kDNA + 4 units of Topo II + amonafide, 5b, 5c and 6a (100 lM); lanes 8–10, 6a at 10, 30 and 50 lM.
5
glycol derivatives (5b and 5c) showed moderate to strong cytotoxicity against several cell lines including HepG2, KATO-III and SW620 cell lines (with IC50 values around 10–30 lM). Moreover, the selenium-containing derivative (6a) of cassiarin A exhibited moderate cytotoxicity against cervical cell line line, CaSki. The compound 5c with positively charged dimethylamino-ethyl side chain displayed a strong DNA intercalating property, as demonstrated by its ability to stabilize ct-DNA and by ethidium bromide displacement assay. The compound 5b, but not 6a, also acts through intercalative DNA binding mode. The kDNA decatenation experiments showed that 6a strongly inhibited topoisomerase II. This suggests that a plausible explanation for cytotoxicity of the selenium-containing cassiarin A derivative 6a. All three active compounds showed lower toxicity to normal cell line (WI-38) than amonafide, which made them a good candidate for new anticancer agents. The evaluation of cytotoxicity was based on the reduction of MTT dye by viable cells to give purple formazan products, which can be measured spectrophotometrically at 540 nm. One hundred microliters of cancer cells were seeded into 96-well plates at 5 103 cell/well and incubated at 37 °C. After 24 h, 1 ll of each compound (200, 100, 10 and 1 lM) was mixed into triplicate wells and incubated as above for 3 days. Then, 10 ll of MTT solution (5 mg/ml) was added and incubated again for 4 h. After removal of media and solubilization of forman crystals in 150 lL of DMSO, the absorbance was measured at 540 nm. The cytotoxicity activity was measured as % cell viability. The IC50 values were calculated using software GraphPad Prism 5 (GraphPad. Software Inc.), using a nonlinear regression of ‘log(inhibitor) versus response’: Y = bottom + (top bottom)/(1 + 10^((X Log IC50))). Thermal denaturation of ct-DNA: The thermal denaturation experiments were performed to study the effect of each compound on the stability of ct-DNA by determination of melting temperature (Tm) value. The ct-DNA was prepared in phosphate buffer (pH 7.4). The Tm of ct-DNA were measured using CARY 100 Bio UV–visible spectrophotometer with a temperature control attachment. Absorption at 260 nm of DNA complements was measured with rate 1 °C/min. Ethidium bromide displacement assay: DNA binding studies were performed with fluorescent intercalator displacement assay using Ethidium bromide (EtBr) as intercalator.34 The experiments were performed by adding the compound (0–70 lM) into a solution of ct-DNA (50 lM base pairs) and ethidium bromide (10 lM) in phosphate buffer (pH 7.4). The fluorescence spectra (kex = 520 nm) were recorded at room temperature. The data were plotted as Stern–Volmer equation: I0/I = 1 + KSVQ where I0 and I denoted the fluorescence emission intensity (at kem = 595 nm) in the absence and presence of the quencher, KSV is the Stern–Volmer quenching constant and Q is the concentration of the quencher.35 The apparent binding constant (Kapp) was calculated from the equation KEtBr[EtBr] = Kapp[compound], where KEtBr = 1.0 107 M 1, [EtBr] = ethidium bromide concentration and [compound] is the compound concentration at 50% reduction of fluorescence intensity of complex EtBr-ct-DNA was observed.39 The kDNA decatenation assay was performed in order to study the ability of topoisomerase II decationation of kDNA by using amonafide as a positive control. Stock solutions of the cassiarin A derivatives (10 mM) were prepared in DMSO. The reaction mixture contain 250 ng kDNA (TopoGEN), 4 units of topo IIa (USB Affymetrix) and the indicated concentrations of compounds were incubated for 1 h at 37 °C in 10 reaction buffer (USB Affymetrix) in final volume of 20 lL. The reaction was stopped by adding 3 lL of 6 loading dye (USB Affymetrix). Samples were electrophoresed in 1% SDS in TBE buffer for 90 min at 75 V. The gel was stained with ethidium bromide and photographed under a UV transilluminator.
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Acknowledgment The authors gratefully acknowledge funding from the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University to N.M. (RES560530064-FW). Supplementary data
15. 16. 17. 18. 19. 20. 21. 22.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04. 107.
23. 24. 25.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis, cytotoxicity, DNA binding and topoisomerase II inhibition of cassiarin A derivatives Urarika Luesakul a, Tanapat Palaga b, Kuakarun Krusong c, Nattaya Ngamrojanavanich a, Tirayut Vilaivan a, Songchan Puthong d, Nongnuj Muangsin a,⇑ a
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand d Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand b c
a r t i c l e
i n f o
Article history: Received 20 December 2013 Revised 17 March 2014 Accepted 25 April 2014 Available online xxxx Keywords: Cassiaria A Amonafide Selenium compound DNA-binding Topoisomerase II
a b s t r a c t Four series of cassiarin A derivatives with alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-substituted ethylene glycol (5a–5c) and selenium-containing (6a) side chains were synthesized. Their antitumor activities were evaluated against BT474, CHAGO, HepG2, KATO-III, SW620 and CaSki cancer cell lines. Preliminary results demonstrated that 5b had moderate activities against HepG2 and KATO-III cell lines, while 5c showed moderate to high cytotoxicity against most tested cell lines. In addition, 6a exhibited moderate cytotoxicity against cervical cells, CaSki. DNA-binding and ethidium bromide displacement experiments suggested that 5c and 5b binds to DNA via an intercalative mode, whereas 6a did not. However, the selenium-containing cassiarin A derivative 6a inhibited topoisomerase II with more than 95% inhibition at the concentration of 50 lM. These cassiarin A derivatives showed lower toxicity to normal cells, WI-38 than amonafide therefore they are potential lead compounds to be further developed as new anticancer agents. Ó 2014 Elsevier Ltd. All rights reserved.
Cancer is one of the major causes of death worldwide, accounted for 7.6 million deaths in 2007 and projected to continue rising.1 Many effective compounds used for treatment of cancer are natural products or related to them. The first plant-derived agents were used as clinical drugs, the so-called vinca alkaloids vinblastine and vincristine were isolated from the Madagascar periwinkle. Other well-known anticancer drugs approved for clinical use include etoposide from Mayapple (Podophyllum pellatum),2 doxorubicin from bacterium Streptomyces peucetius,3 and camptothecin from Camptotheca acuminata.4 In addition to these well-known examples, there are more than 3000 compounds which can be potentially useful for cancer treatment.5 Cassia siamea is a common plant in Southeast Asia. This herb has been used as food and for treatment of insomnia. The major chemical constituent extracted from C. siamea leaves and flowers is barakol (1).6 In 2007, Morita et al. discovered cassiarin A (2)—an aromatic alkaloid with a tricyclic skeleton—in C. siamea but is less abundant than barakol.7 In 2010, Thamyongkit and co-workers were interested in their potent antiplasmodial activity and transformed
⇑ Corresponding author. Tel./fax: +66 2 218 7635. E-mail addresses: [email protected], [email protected] (N. Muangsin).
barakol into cassiarin A. Due to the structural similarity between the tricyclic aromatic core of cassiarin A and the anticancer agent amonafide (Fig. 1), it was proposed that derivatives of cassiarin A may exhibit cytotoxicity against cancer cells.8 Cassiarin A derivatives including ester, ether, nitrogen-substituted and dehydroxy derivatives had been synthesized and evaluated for their antimalarial activities. Among them, the parent compound, cassiarin A showed more potent antimalarial activity and less toxicity to human cells, MCF-7.9 Amonafide is a naphthalimide analog that exhibits excellent antitumor activity and was tested in clinical trials for treatment of cancer. This compound binds to DNA by intercalation and also inhibits Topoisomerase II.10 Topoisomerase II is the key enzyme that regulates the topological state of DNA. It plays important roles in transcription and replication process by breaking and rejoining of phosphodiester backbone of DNA stands.11 However, amonafide has failed to enter clinical phase III12 because it is easily metabolized to N-acetyl-amonafide by enzyme N-acetyl transferase 2 (NAT2), resulting in unpredictable toxicity.13,14 Accordingly, many derivatives such as alkyl, aryl and alkylamino substituted amonafide analogues have been investigated. These compounds showed similar or more potent cytotoxicity than amonafide but could survive drug-metabolized enzymes. Moreover, DNA-binding studies
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Figure 1. Chemical structures of barakol, cassiarin A and amonafide.
reveal that they can interact with DNA by a similar mode of action of amonafide.15,16 Selenium is an essential trace mineral that is important for human health. Selenium compounds have been proven as potent anticarcinogenic agents.17,18 The mechanism of selenium action is unclear. Some selenium compounds effects on important tumor suppressor protein p53 activity and different selenium forms regulate p53 in the different ways.19–21 Selenomethionine and methyl-seleninic acid may modify p53 for DNA repair, while sodium selenite may modify p53 for apoptosis.19 Selenite acts as a topoisomerase II poison by inducing topoisomerase II cleavage complexes.22 In addition, selenium analogues of naphthalimide showed good cytotoxicity against many cell lines with IC50 ranging 10–20 lM.23 In this research, new analogues of amonafide were synthesized by modification of cassiarin A.8 Cassiarin A is a potential candidate
for development of anticancer agent due to (i) the molecule contains a tricyclic planar aromatic moiety which is an important feature of DNA intercatator,24 (ii) the molecule has a amonafide-like aromatic core but exhibited low toxicity against human cell,9 and (iii) it showed potent biological activities including antiplasmodial and antimalarial activities.7,9 In this work, the C7–OH position of cassiarin A was modified which can be classified into four different groups namely alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/aminosubstituted ethylene glycol (5a–5c) and selenium-containing (6a) as shown in Figure 2. The compounds were synthesized by acylation of cassiarin A or by nucleophilic substitution of appropriate electrophiles (alkyl tosylate for 5b and 5c and phenylselenyl chloride for 6a) with cassiarin A under basic conditions. The chemical structures were confirmed by spectroscopic methods (FT-IR, 1H NMR, 13C NMR and MALDI-TOF MS). All compounds were preliminarily evaluated for their cytotoxicity and active compounds were further explored for their possible functional mechanisms such as DNA-binding activity and topoisomerase II inhibition. Cytotoxicity of the cassiarin A derivatives were determined by MTT assay against six human cancer cell lines including breast carcinoma (BT474), lung carcinoma (CHAGO), liver carcinoma (HepG2), gastric carcinoma (KATO-III), colon carcinoma (SW620), cervical carcinoma (CaSki) cell lines comparison with amonafide as positive control. All compounds were subjected to preliminary screening for their cytotoxic activity at high concentration (100 lM) as shown in Figures 3 and S1 in Supplementary data and divided into two groups as active (less than 20% cell viability) and inactive (more than 20% cell viability). Subsequently, IC50
Figure 2. Cassiarin A derivatives with four groups of side chains: alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-substituted ethylene glycol (5a–5c) and seleniumcontaining (6a) side chains.
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Figure 4. Toxicity of amonafide, 5b, 5c and 6a to normal cell line, WI-38.
Figure 3. Preliminary screening of 5b, 5c and 6a at concentration 100 lM against six human cancer cell lines compared to amonafide as the reference.
values were determined for active compounds and these were classified into three categories as high (IC50 86
>15.3
Inactive
Inactive
Inactive
Inactive
Inactive
29.74 ± 0.03
70.0
—
37.80 ± 6.05
1.05 ± 0.05
0.32 ± 0.07
1.71 ± 0.14
9.24 ± 0.09
9.96 ± 0.10
78.9
8.2
IC50, as drug concentration that caused 50% reduced the viable cell population determined by MTT assay. Inactive is defined as % cell viability more than 20% at high concentration (100 lM).
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Ethidium bromide displacement assay: Ethidium bromide displacement assay has been widely used to confirm the intercalative binding mode between a compound and DNA. The principle of the assay based on the displacement of the intercalating dye, ethidium bromide (EtBr) from ct-DNA.34 The emission spectra of EtBr bound to ct-DNA in the absence and the presence of amonafide, 5b and 5c are given in Figure 6. Ethidium bromide is barely fluorescent in aqueous solution but exhibits intense fluorescence emission at 595 nm when bound to ct-DNA, due to the strong intercalation with DNA base pairs. If addition of the test compound to ct-DNA pretreated with EtBr in the ratio of EtBr/DNA equal to 1:2.5 in terms of base pairs results in quenching of the emission intensity, one can conclude that the compound can displace the EtBr from its intercalative site. The efficiency of the EtBr displacement was investigated by determining the Stern–Volmer constant (KSV) value.35 The data were plotted as Stern–Volmer equation: I0/ I = 1 + KSV[Q] where I0 and I denoted the fluorescence emission intensity in the absence and presence of the quencher, KSV is the Stern–Volmer quenching constant and Q is the concentration of the quencher (in this case, 5b, 5c and 6a). The compound 5c
Figure 5. Thermal denaturation profiles of ct-DNA (60 lM by nucleotide) in the absence and presence of amonafide, cassiarin A, 5b, 5c and 6a (30 lM) in 10 mM phosphate buffer pH 7.4 (A) Tm curves and (B) derivative curves of Tm at 260 nm.
equation expressed as A = ebc where A is UV absorbance of ct-DNA, in the range of 0.38–0.39, e = extinction coefficient at 260 nm (6600 M 1 cm 1) and b is the cuvette path length (1 cm).28,29 The Tm values of the ct-DNA were found to be 70.7, 70.7 and 70.0 °C for free DNA, with cassiarin A and with 6a, respectively. The insignificant change of Tm indicated that cassiarin A and 6a did not interact with DNA by intercalation or groove binding, which should result in stabilization of the DNA duplex. On the other hand, amonafide and 5b can stabilize the DNA duplex as shown by small Tm increase to 78.9 °C (DTm = 8.2 °C) and 75.7 °C (DTm = 4.8 °C), for amonafide and 5b, respectively. Importantly, the compound 5c caused a significant increase of the Tm of ct-DNA (Tm >86 °C and DTm >15.3 °C) which is comparable to common organic intercalator such as ethidium bromide (DTm = 13 °C). The large DTm indicated that 5c strongly interacted with and stabilized the double helix of DNA. It is well known that many anti-tumor agents containing positively charged group such as dimethylamino-ethyl showed the excellent cytotoxic activity.30–32 The terminal nitrogen of dimethylamino-ethyl group of 5c which has pKa values around 9 should be readily protonated and electrostatically bound to the anionic phosphate group of DNA. This favorable electrostatic interaction precedes subsequent intercalative binding by the aromatic core of cassiarin A that results in the increase of Tm of the ct-DNA.15,33 This experiment suggested that compounds 5b and 5c can interact with ct-DNA, while compound 6a cannot. There are three types of interaction between a DNA-binding compound and double stranded DNA including electrostatic interactions with the phosphate groups, binding to minor or major grooves of DNA and intercalation between the DNA based pairs. In order to confirm the intercalation ability of these compounds, ethidium bromide displacement assay was studied using fluorescence spectrophotometry.
Figure 6. Emission spectra of EtBr (10 lM) bound to ct-DNA, 25 lM base pairs in the presence of compound (A) amonafide, (B) 5b and (C) 5c (0–70 lM) in 10 mM phosphate buffer pH 7.4. The arrow shows the direction of change of fluorescence intensity of EtBr bound to ct-DNA upon increasing amounts of tested compound. Inserts show Stern–Volmer curve for quenching of ct-DNA.
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showed the highest KSV value of 2.1 104 M 1, while the 5b and amonafide showed smaller values of 4.3 103 and 1.5 104 M 1, respectively, which is comparable to DNA-intercalator such as acridine derivative (KSV = 1.5 104 M 1).36 From these results, it can be concluded that the compounds 5b and 5c bind to ct-DNA via the intercalation mode. Furthermore, the apparent binding constants (Kapp) can be calculated using the equation; Kapp[compound] = KEtBr[EtBr], where KEtBr = 1.0 107 M 1 and [EtBr] = 10 lM.34 Compound 5c also showed the highest apparent binding constant (2.1 106 M 1), which confirmed that the intercalative binding occurs between compound 5c and ct-DNA. The DNA-binding studies correlate well with the cytotoxicity to cancer cell lines, the strongest DNA binding compound (5c) is the most cytotoxic compound. As 6a showed only weak DNA binding, its moderate cytotoxicity against CaSki cell line may arise from a different mode of action. Many reports showed that selenium compound could play multi-roles such as cancer prevention, DNA damage or topoisomerase II poison.22,23 Subsequently, compound 6a was further explored for other plausible mechanisms of action such as the inhibition of topoisomerase II. Topoisomerase II inhibition: Topoisomerase II is the key cellular enzyme that regulates the topological state of the DNA in cells. It plays an important role in replication and transcription process by breaking and rejoining of phosphodiester backbone of DNA stands.11 Amonafide is a known DNA intercalator and can inhibit topoisomerase II by stabilizing the enzyme–DNA complex.10 In this work, we also determined the ability of compound 5b, 5c and 6a to inhibit topoisomerase II decatenation of kDNA.37 Both forms of kDNA including (i) catenated kDNA (C) and (ii) nicked open circular decatenated kDNA (NOC) which is formed by the action of topoisomerase II were monitored. Catenated kDNA appears at the top and cannot enter into the gel because of its size, while decatenated product can readily move into the gel.38 The results shown in Figure 7 demonstrated that at a concentration of 100 lM, the selenium-containing compound 6a displayed a strong topoisomerase II inhibitory activity with more than 95% inhibition. The compound 5c exhibited a weak inhibitory effect (30.1%), whereas compound 5b did not inhibit topoisomerase II. The positive control, amonafide, showed a moderate activity of 61.8%. Moreover, 6a inhibited topoisomerase II with 31.5%, 75.7% and 95.7% inhibition 10, 30 and 50 lM, respectively. In conclusion, cassiarin A derivatives were synthesized and their cytotoxicity against six cancer cell lines (BT474, CHAGO, HepG2, KATO-III, SW620 and CaSki) were investigated. The preliminary results demonstrated that alkanoyl (3a–3d), aroyl (4a–4d) and ethyleneglycol (5a) derivatives showed low cytotoxic activity against all tested cell lines. Two hydroxy/amino-substituted ethylene
Figure 7. Inhibitory effect of the tested compounds on human DNA topoisomarase II decatenation. Lane 1, decatenated kDNA marker; lane 2, kDNA; lane 3, kDNA + 4 units of Topo II; lanes 4–7, kDNA + 4 units of Topo II + amonafide, 5b, 5c and 6a (100 lM); lanes 8–10, 6a at 10, 30 and 50 lM.
5
glycol derivatives (5b and 5c) showed moderate to strong cytotoxicity against several cell lines including HepG2, KATO-III and SW620 cell lines (with IC50 values around 10–30 lM). Moreover, the selenium-containing derivative (6a) of cassiarin A exhibited moderate cytotoxicity against cervical cell line line, CaSki. The compound 5c with positively charged dimethylamino-ethyl side chain displayed a strong DNA intercalating property, as demonstrated by its ability to stabilize ct-DNA and by ethidium bromide displacement assay. The compound 5b, but not 6a, also acts through intercalative DNA binding mode. The kDNA decatenation experiments showed that 6a strongly inhibited topoisomerase II. This suggests that a plausible explanation for cytotoxicity of the selenium-containing cassiarin A derivative 6a. All three active compounds showed lower toxicity to normal cell line (WI-38) than amonafide, which made them a good candidate for new anticancer agents. The evaluation of cytotoxicity was based on the reduction of MTT dye by viable cells to give purple formazan products, which can be measured spectrophotometrically at 540 nm. One hundred microliters of cancer cells were seeded into 96-well plates at 5 103 cell/well and incubated at 37 °C. After 24 h, 1 ll of each compound (200, 100, 10 and 1 lM) was mixed into triplicate wells and incubated as above for 3 days. Then, 10 ll of MTT solution (5 mg/ml) was added and incubated again for 4 h. After removal of media and solubilization of forman crystals in 150 lL of DMSO, the absorbance was measured at 540 nm. The cytotoxicity activity was measured as % cell viability. The IC50 values were calculated using software GraphPad Prism 5 (GraphPad. Software Inc.), using a nonlinear regression of ‘log(inhibitor) versus response’: Y = bottom + (top bottom)/(1 + 10^((X Log IC50))). Thermal denaturation of ct-DNA: The thermal denaturation experiments were performed to study the effect of each compound on the stability of ct-DNA by determination of melting temperature (Tm) value. The ct-DNA was prepared in phosphate buffer (pH 7.4). The Tm of ct-DNA were measured using CARY 100 Bio UV–visible spectrophotometer with a temperature control attachment. Absorption at 260 nm of DNA complements was measured with rate 1 °C/min. Ethidium bromide displacement assay: DNA binding studies were performed with fluorescent intercalator displacement assay using Ethidium bromide (EtBr) as intercalator.34 The experiments were performed by adding the compound (0–70 lM) into a solution of ct-DNA (50 lM base pairs) and ethidium bromide (10 lM) in phosphate buffer (pH 7.4). The fluorescence spectra (kex = 520 nm) were recorded at room temperature. The data were plotted as Stern–Volmer equation: I0/I = 1 + KSVQ where I0 and I denoted the fluorescence emission intensity (at kem = 595 nm) in the absence and presence of the quencher, KSV is the Stern–Volmer quenching constant and Q is the concentration of the quencher.35 The apparent binding constant (Kapp) was calculated from the equation KEtBr[EtBr] = Kapp[compound], where KEtBr = 1.0 107 M 1, [EtBr] = ethidium bromide concentration and [compound] is the compound concentration at 50% reduction of fluorescence intensity of complex EtBr-ct-DNA was observed.39 The kDNA decatenation assay was performed in order to study the ability of topoisomerase II decationation of kDNA by using amonafide as a positive control. Stock solutions of the cassiarin A derivatives (10 mM) were prepared in DMSO. The reaction mixture contain 250 ng kDNA (TopoGEN), 4 units of topo IIa (USB Affymetrix) and the indicated concentrations of compounds were incubated for 1 h at 37 °C in 10 reaction buffer (USB Affymetrix) in final volume of 20 lL. The reaction was stopped by adding 3 lL of 6 loading dye (USB Affymetrix). Samples were electrophoresed in 1% SDS in TBE buffer for 90 min at 75 V. The gel was stained with ethidium bromide and photographed under a UV transilluminator.
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Acknowledgment The authors gratefully acknowledge funding from the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University to N.M. (RES560530064-FW). Supplementary data
15. 16. 17. 18. 19. 20. 21. 22.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04. 107.
23. 24. 25.
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