The cytotoxic activities of cardiac glycosides from Streptocaulon juventas and the structure–activity relationships

Fitoterapia 98 (2014) 228–233

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The cytotoxic activities of cardiac glycosides from Streptocaulon juventas and the structure–activity relationships Rui Xue a, Na Han a, Chun Ye a, Lihui Wang b, Jingyu Yang b, Yu Wang c, Jun Yin a,⁎ a Development and Utilization Key Laboratory of Northeast Plant Materials, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China c The People's Liberation Army 463 Hospital, Shenyang 110042, China

a r t i c l e

i n f o

Article history: Received 16 May 2014 Accepted in revised form 3 August 2014 Available online 13 August 2014 Keywords: Cardiac glycosides Streptocaulon juventas Anti-tumor activity A549 NCI-H460 MRC-5

a b s t r a c t A series of cardiac glycosides were isolated and identified from the anti-tumor fraction of the root of Streptocaulon juventas in previous studies. In the present research, the cytotoxic activities of the 43 cardiac glycosides on three cell lines, human lung A549 adenocarcinoma cell, large cell lung cancer NCI-H460 cell and normal human fetal lung fibroblast MRC-5 cell, were evaluated in vitro. Most of the tested compounds showed potent inhibitory activities toward the three cell lines. Then, the structure–activity relationships were discussed in detail. It was indicated that hydroxyl and acetyl groups at C-16 increased the activity, whereas hydroxyl group at C-1 and C-5 can both increase and decrease the activity. Two glucosyl groups which were connected by C1′ → C6′ showed better inhibitory activity against cancer cell lines, while the C1′ → C4′ connection showed stronger inhibitory activity against the normal cell line. Also, this is the first report that the activities of these compounds exhibited different variation trends between A549 and NCI-H460 cell lines, which indicated that these compounds could selectively inhibit the cell growth. The results would lay a foundation for further research on new anti-tumor drug development. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Streptocaulon juventas (Asclepiadaceae) is a liana widely distributed in Southeast Asia, especially as a native plant mainly in the southwest of China [1]. It is medicinally used in Yunnan and Guangxi provinces as a tonic for rheumatism, neurasthenia, dyspepsia and so on. In our previous studies, 75% ethanol extract of S. juventas which showed strong inhibitory effect on the growth of human lung A549 adenocarcinoma cells, was subjected to bioassay-guided fractionation. Then, the most effective fraction was evaluated for anti-tumor activity on lung cancer in athymic nude mice with A549 tumors. Notably, tumor proliferation was inhibited significantly during the treatment

⁎ Corresponding author at: School of Traditional Chinese Materia Medica 48#, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China. Tel./fax: +86 24 23986491. E-mail address: [email protected] (J. Yin).

http://dx.doi.org/10.1016/j.fitote.2014.08.008 0367-326X/© 2014 Elsevier B.V. All rights reserved.

without physical side effects [2]. Next, a systematic phytochemical study of S. juventas led to obtain 43 cardiac glycosides [3,4]. In addition, it was confirmed that the cardiac glycosides were the main active constituents in the anti-tumor fraction by bioassay-guided isolation, which was also in agreement with the reports of references [5–8]. Cardiac glycosides (CGs) are a large family of natural products distributed in several plants and animal species [9]. They were used for medicinal purpose more than 1500 years ago as arrow poisons, diuretics, heart tonics and emetics [10]. Actually, plant extracts containing CGs were used for treating malignant conditions from an early age already [11–14]. However, studies on applications of CGs to the anticancer field only intensified in the 1960s [15]. Over the years, the use of CGs was abandoned as a result of the well-known cardiac toxicity. It was only recently reported that digitalis could induce apoptosis in human tumor cells at concentrations without toxicity in human [5,16,17]. The promising result triggered plenty of studies on the effects of CGs

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toward the proliferation of human tumor cells in the past decades, so as to find numerous candidates for anticancer agents. From these studies, we could obtain such conclusions that the 5β-14β-card-20(22)-enolide structure is important for the cell growth inhibitory activity of cardiac glycosides; 8,14-epoxy or 8,14-dihydroxy group led to a decrease in the activity [18]; 17βconfiguration of the lactone is crucial for the cytotoxic effect [19,20]; and a double bond or an α-epoxide ring into the C-16,17 position in the aglycone led to the abrogation of cytotoxicity [18,21,22]. Although some relationships between structure and cytotoxic activity of cardiac glycosides had been summarized, there were still confusing and inconsistent conclusions found in different references, such as the effects of the introduction of hydroxyl or acetyl group at C-16 [23–25] and the oxidation status of the C-19 methyl group [26,27]; the influences of glycosylation at C-3 and the number of sugar residues [19,21,28,29], and so on. This may be because their cytotoxic activities were evaluated against different kinds of carcinoma cell lines. It was supposed that these compounds may exhibit selective anti-proliferative activity toward different carcinoma cell lines. Thus, more SAR studies on cardiac glycosides need to be continued. On the basis of the potent in vivo anti-tumor activity of the active fraction from S. juventas [2], and the abundant CGs we obtained in our previous research [3,4], we performed a relatively systemic SAR study toward different cancer cell lines. Meanwhile whether these CGs possess the same cell-killing activity against normal lung cells was confirmed upon MRC-5 cell, laying the foundation for the development of potential anti-cancer agents. In the present research, the anti-proliferative activities of the 43 cardiac glycosides (Fig. 1 and Table 1) on three cell lines, including human lung A549 adenocarcinoma cell, large cell lung cancer NCI-H460 cell and normal human fetal lung fibroblast MRC-5 cell, were tested in vitro, while the SAR was discussed in detail. 2. Results and discussion 2.1. Anti-proliferative activity of compounds 1–43 on A549 and NCI-H460 cell lines In an initial study, the anti-proliferative activity of compounds 1–43 on A549 and NCI-H460 cell lines at 100 μM was examined by MTT assay, with cisplatin as the positive control (Fig. 2). It was shown that most of the compounds displayed more sensitive anti-proliferative activity on A549 than NCIH460 cell line. The compounds with growth inhibition rate above 50% were chosen for further evaluation against the two kinds of tumor cell lines at more different concentrations to obtain the IC50 values (Table 1). The compounds (2, 3, 5–6, 8, 12, 13, 23, 25–26, 28, 31, 34, 37, 39–41) showed strong antiproliferative activities on both of the human lung cancer cell lines, i.e., A549 (IC50, 0.037–27.295 μM) and NCI-H460 (IC50, 0.063–39.963 μM). Compound 42 showed no activity against neither kind of the cell line, and the other compounds exhibited selective potent activity toward the two kinds of cell lines, such as compound 14 (the growth inhibition rates toward A549 and NCI-H460 at 100 μM were 74.85% and 4.61%, respectively), as well as compound 35 (the growth inhibition rates were 44.66% and 83.26%, respectively). The interesting results implied that

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these compounds may possess different structure–activity relationships against different cell lines. 2.2. Toxicity of compounds 1–43 on MRC-5 cell line In order to confirm the security of these compounds, 36 selected compounds with 50% of the growth inhibition rates toward A549 were selected for further study against the normal human fetal lung fibroblast MRC-5 cell line (Table 1). The results demonstrated that all these 36 compounds showed cytotoxic activity against MRC-5 cell line (IC50, 0.001– 46.090 μM), and most of them also exhibit stronger cytotoxicity than cisplatin (IC50, 29.480 μM). The consequences indicated that the structure–toxicity relationships of these compounds toward MRC-5 may be relatively identical with that of A549 cell line, and these compounds may possess obvious cytotoxicity against human lung cell lines in vitro. 2.3. Discussion on the SAR of these compounds With regard to the SAR of the cardenolides against A549 and MRC-5 cell line, compound 18, the simplest cardenolide with only 3β, 14β-dihydroxyl functionality, was designated as the basic structure, and proved to be important for the activity. The hydroxyl group at C-5 of the aglycon part or double bond between C-16 and 17 decreased their activity (33 b 18; 11 b 12; 8 b 5; 41 b 5; 42 b 33), and the β-hydroxyl or acetyl group at C-16 increased their activity obviously (33 b 10; 18 b 12; 5 b 1; 33 b 11). The hydroxyl group at C-1 decreased the toxicity against MRC-5 cells (1 b 12; 5 b 18; 33 b 8). However, the hydroxyl group at C-1 would both increase and decrease the activity against A549 cells (12 b 1; 5 b 18; 8 b 33), which demonstrated that the influence of the hydroxyl group at C-1 depended on whether there were other substituents at the other positions of the aglycon. With respect to the number of sugars on C-3, monoglycosylation and triglycosylation would intensify their activity, whereas diglycosylation and tetraglycosylation would not increase or even weaken the activity, and most of them showed weaker activity than the mono- and tri-glycosylation (3, 4, 5 b 6, 7; 23, 24, 25 b 19, 20; 37 b 32, 34, 39, 40). When the type of sugar was changed, it was found that the compounds with 2,6-dideoxy sugar moiety (like cymarosyl unit and digitoxosyl unit) displayed strongest cytotoxicity, especially against MRC-5 cell line, yet the compounds with digitalosyl unit showed the weakest activity (31 b 29, 27), and acetylation of the hydroxyl group at C-3′ reduced the activity (28 b 29). Then the difference between these sugar units can be summarized as: digitalose b 2-OAc-digitalose b 3-OAcdigitoxose b glucose b digitoxose b cymarose. On the other side, the SAR of the cardenolides against NCI-H460 cell line, contrary to those against A549 and MRC5 cell, the aglycon displayed weaker activity than the others, except for compounds with double bond between C-16 and C-17 (41 b 5; 42 b 33). Among them, the hydroxyl group at C1, C-5 and C-16 could increase their activity (18 b 5; 33 b 8; 18 b 33; 5 b 8, and 33 b 10), and the hydroxyl group at C-1 was more favorable. Acetylation of the hydroxyl group at C16 could either increase (18 b 12) or decrease the activity (1 b 5; 11 b 33), which meant that the effect of acetyl group at C-16 also depended on whether there were other

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Fig. 1. The general structures of compounds 1–43.

substituents at the other positions of the aglycone. Referred to the substituents in sugar moieties, the free form of the hydroxyl group at C-2′ raised the activity (7 b 6; 29 b 31), and methylation of the hydroxyl group at C-3′ reduced the activity (27 b 29). Acetylation of the hydroxyl group at C-2′ decreased the activity (17 b 31) while that at C-3′ increased the activity (21 b 26; 29 b 28). Then the activity and regularity of sugar moiety against NCI-H460 cells can be concluded as follows: 2-OAc-digitalose b cymarose b digitoxose b digitalose, glucose b 3-OAc-digitoxose. In addition, tetrasaccharide at C-3 obviously weakens the activity (30 b 18). On the comparison among the effects of compounds on the three cell lines, it could be found that digitalose raised the activity toward NCI-H460 cell line (29, 27 b 31; 33, 34, 39 b 35), and the opposite happened when they were against A549 and MRC-5 cell line. Furthermore, glycosylation at C-6′ obviously improved the activity toward two kinds of cancer cell line (24, 25 b 23), which happened at C-4′ which significantly increased the toxicity against the normal cell line (25 b 23 b 24: the growth inhibition rates at 5 μM, 50.09%, 61.13% and 76.15%, respectively). Thus, the anti-proliferative activity of these cardiac glycosides would depend on not only the substituent groups on the aglycon, but also the sugar moieties. However, the different combinations would bring about significant unexpected changes. Take the situations on A549 cells for example, generally, triglycosylation exhibited stronger activity than diglycosylation (16 b 17; 26 b 28), while the opposite happened between 21 and 29. 6-Deoxy and 2,6-dideoxy sugar moieties are common and characteristic in cardiac glycosides. Involved in binding of glycoside to Na+, K+-ATPase, it was found that 6-deoxy sugars exhibited the most strong binding activity, compared with the other hexose units [30,31]. Also, inotropic potency was enhanced by the presence of a 4′ equatorial oxygen function [32]. In this study, referring to the monoglycosides, it was found that deoxy sugar units showed the potent, non-selective cytotoxic activity against either carcinoma cells or normal cells (compounds 2, 7, 20, 39), which was consistent with the reports that toxicity is a major concern whenever cardiac glycosides are considered for therapy, such as digitoxin, a cardiac glycoside with three 2,6-dideoxy sugar units [33].

In the present research, most of the cardiac glycosides exhibited potent anti-proliferative activities against the two kinds of human lung cancer cell and were significantly superior to cisplatin, the positive control. It illustrated that cardiac glycosides would be a series of promising candidate compounds in anti-cancer research. Especially, it also showed remarkable selectivity between A549 and NCI-H460 cell lines, which indicated that these compounds may possess excellent effect on human lung cancer, particularly for lung adenocarcinoma. It's worthwhile to note that these compounds also showed obvious cytotoxic activity toward normal human lung cell, which would be a serious problem and limited the further development. However, according to our previous study, the in vivo effect on lung cancer of the most active fraction of S. juventas, which was rich in cardiac glycosides, was investigated in athymic nude mice with A549 cells. The promising result illustrated that it remarkably inhibits the tumor growth without any side effects in tumor-bearing mice, of course, no significant differences were found in the pathological sections of nude mice lung when compared with those of the control group [2]. For this reason, the obvious difference may exist between in vitro and in vivo studies. Whether these compounds possessed real toxicity during the therapeutic process should be further confirmed in next study. 3. Conclusion As conclusion, the anti-proliferative activities of 43 compounds were evaluated against two kinds of human lung cancer cell lines and one normal human lung cell line, consequently the structure–activity relationships of CGs concerning antitumor especially lung cancer were summarized. These findings should be of guiding significance for the development of new anti-cancer drugs. 4. Materials and methods 4.1. Chemicals 16-O-Acetyl-hydroxyacovenosigenin (1), 16-O-acetylhydroxyperiplogenin 3-O-β-D-digitoxopyranoside (2), 1α,

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Table 1 In vitro cytotoxicities of compounds 1–43 against A549, NCI-H460 and MRC-5 cell lines. Compounds

R1

R2

R3

R4

IC50 (μM) A549

NCI-H460

MRC-5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Cisplatin

β-OH H α-OH β-OH β-OH β-OH β-OH β-OH β-OH H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H β-OH H H –

H Dig Dtl2-Glc Dtl4-Glc H Glc Dig H Glc H H H Dig4-Glc Cym4-Glc6-Glc Dig4-Glc (2-O-Ac-Dtl)4-Glc (2-O-Ac-Dtl)4-Glc6-Glc H Glc Cym Dig4-Glc Cym4-Glc Glc6-Glc Glc4-Glc Glc2-Glc (3-O-Ac-Dig)4-Glc Cym4-Glc6-Glc (3-O-Ac-Dig)4-Glc6-Glc Dig4-Glc6-Glc Cym4-Dtl4-Glc6-Glc Dtl4-Glc6-Glc Cym4-Glc4-Glc H Glc Dtl Dig Dtl4-Glc Dig4-Glc Cym Cym4-Glc6-Glc H H Glc –

H OH H H H H H OH OH OH OH H H H H H H H H H H H H H H H H H H H H OH OH OH OH OH OH OH OH OH H OH H –

OAc OAc H H H H H H H OH OAc OAc OAc OAc OH H H H H H H H H H H H H H H H H H H H H H H H H H Δ16,17 Δ16,17 Δ16,17 –

0.021 0.343 1.021 63.815 3.323 NA 0.153 27.295 0.492 1.845 0.534 0.219 0.037 0.357 0.734 1.496 0.868 0.783 0.010 0.008 0.020 0.063 0.664 6.876 5.614 NA 0.041 0.262 0.188 0.437 9.048 0.161 17.483 0.065 – NA 1.843 NA 0.047 0.282 26.046 – – 22.980

– 0.075 6.137 – 18.441 0.292 – 7.057 – – – 0.535 0.063 – – – – – – – – – 1.168 – 5.603 8.206 – 0.343 – – 0.873 – – 0.220 0.296 NA 4.042 NA 0.177 0.251 39.963 – 16.227 2.564

0.127 0.032 2.830 1.910 0.860 0.710 0.036 7.460 0.510 NA 5.782 0.008 0.005 0.039 0.200 NA 35.250 0.088 0.008 0.001 0.006 0.003 0.843 0.052 3.710 25.240 0.009 0.009 0.003 1.190 0.400 0.260 2.230 0.320 NA NA 8.372 NA 0.004 0.010 46.090 – NA 29.480

Cisplatin was used as a positive control. NA: represented compounds of which amounts were not enough for rescreening. “–” represented compounds which possessed below 50% growth inhibition rates at 100 μM and were not subjected to IC50 evaluation.

Fig. 2. Growth inhibition rates of A549 and NCI-H460 cells after treating with compounds 1–43 at 100 μM for 48 h by MTT assay (mean ± SE). Cisplatin was used as the positive control. Illustration for each compound (X-axis) analyzed for the differences (Y-axis) between the mean values of the growth inhibition rates of A549 and NCIH460 cell lines.

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14β-dihydroxy-5β-card-20 (22)-enolide 3-O-[O-β-D-glucopyranosyl-(1 → 2)-β-D-digitalopyranoside] (3), acovenosigenin A 3-O-[O-β-D-glucopyranosyl-(1 → 4)-β-D-digitalopyranoside] (4), acovenosigenin A (5), acovenosigenin A β-glucoside (6), acovenosigenin A 3-O-β-digitoxopyranoside (7), evonogenin (8), glucoevonogenin (9), 5β-hydroxygitoxigenin (10), 16-Oacetyl-hydroxyperiplogenin (11), oleandrigenin (12), subapinoside (13), honghelotrioside A (14), glucogitoroside (15), digitoxigenin 3-O-[O-β-D-glucopyranosyl-(1 → 4)-2-O-acetylβ-D-digitalopyranoside] (16), digitoxigenin 3-O-[O-β-D-glucopyranosyl-(1 → 6)-O-β-D-glucopyranosyl-(1 → 4)-2-O-acetylβ-D-digitalopyranoside] (17), digitoxigenin (18), acetodigin (19), hongheloside G (20), glucoevatromonoside (21), echunbioside (22), digitoxigenin gentiobioside (23), digitoxigenin 3-O-[O-β-D-glucopyranosyl-(1 → 4)-β-D-glucoside] (24), digitoxigenin sophoroside (25), digitoxigenin 3-O-[O-βD-glucosyl-(1 → 4)-3-O-acetyl-β-D-digitoxoside] (26), echujin (27), digitoxigenin 3-O-[O-β-glucopyranosyl-(1 → 6)-O-βglucopyranosyl-(1 → 4)-3-O- acetyl-β-digitoxopyranoside] (28), digitoxigenin 3-O-β-gentiobiosyl-(1 → 4)-O-β-D-digitoxopyranoside (29), digitoxigenin 3-O-[O-β-glucopyranosyl(1 → 6)-O-β-glucopyranosyl-(1 → 4)-O-β-digitalopyranosyl(1 → 4)-β-cymaropyranoside] (30), odoroside G (31), periplogenin 3-O-[O-β-D-glucopyranosyl-(1 → 4)-O-β-Dglucopyranosyl-(1 → 4)-β-D-cymaropyranoside] (32), periplogenin (33), periplogenin glucoside (34), emicymarin (35), periplogenin 3-O-β-digitoxoside (36), periplogenin 3-O-β-glucopyranosyl-(1 → 4)-O-β-digitaloxopyranoside (37), corchorusoside C (38), periplocymarin (39), biondianoside A (40), 1β, 3β, 14β-trihydroxy-5β-card-16, 20 (22)-dienolide (41), griffithigenin (42) and Δ(16)-digitoxigenin β-D-glucoside (43) were isolated from the roots of S. juventas (Asclepiadaceae) as described previously [3,4]. The purities of these compounds were all above 98%. Cisplatin [Sigma-Aldrich, USA, 99% (purity)] was used as the positive control. All chemical reagents used in this study were purchased from Laibo Chemical Company, Ltd. 4.2. Cell culture A549, NCI-H460 and MRC-5 cell lines were used in this research. The three cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI-1640 (Gibco, No. NUDO160) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in a 5% CO2 and 95% air atmosphere. The compound stock solutions for anti-proliferative assay were prepared in DMSO at an initial concentration of 50 or 100 mM. 4.3. Anti-proliferative activity Anti-proliferative activity of the test compounds was analyzed by the reduction of 3-(4,5-dimethylibiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) as described previously [34]. Briefly, cells were seeded into 96-well microtiter plates at a density of 8 × 104 cells/mL (A549 and NCI-H460 cells) and 9 × 104 cells/mL (MRC-5 cell), respectively, and then incubated for 24 h. Culture media containing different concentrations of test samples were then added. After incubating for 48 h, 100 μL of MTT was added in each well from a stock solution (0.25 mg/mL), and incubated for an additional 3–4 h at 37 °C. The yield purple formazans were

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