YU Kun LI Ying CHEN Hai-Jiao LIU Bo③ YAO Qing-Qiang③

a (School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan 250200, China)

b (Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250062, China)

ABSTRACT We isolated cucurbitacin IIa from the rhizomes of Hemsleya pengxianensis, and tested the cytotoxicity of cucurbitacin IIa against various cancer cell lines by the sulforhodamine B assay (SRB). At the same time, we preliminarily found that cucurbitacin IIa has a certain inhibitory activity on kinase CDK1/cyclin B, and shows potent inhibitory activities against many cancer cell lines. The cucurbitacin IIa was structurally characterized by specific optical rotation measurement, high-resolution mass spectroscopy and NMR spectroscopic analysis. In addition, the molecular structure of cucurbitacin IIa was further determined by X-ray single-crystal crystallography.

Keywords: cucurbitacin IIa, crystal structure, sulforhodamine B assay, CDK1/cyclin B, anti-cancer activity; DOI: 10.14102/j.cnki.0254-5861.2011-2608

1 INTRODUCTION

Cucurbitacin IIa mainly exists in the rhizome of Hemsleya, which is a cucurbitaceous plant. It has the functions of anticancer, bacteriostasis, anti-gastric ulcer, antipyretic, detoxification, pain relief and anti-HIV. It is widely used in the treatment of hepatitis, coronary heart disease and tracheitis[1-3]. Cucurbitacin IIa was investigated for in vitro anti-cancer activities in different cancer cell lines such as HepG2 (hepatic carcinoma), MCF-7 (breast carcinoma), SKOV3 (ovarian carcinoma), HT-29 and LOVO (colon carcinoma) using SRB assay[4].

In these experiments, cisplatin, a known anti-cancer drug, was used as a positive control[5,6]. In this paper, cucurbitacin IIa was evaluated for the inhibition of CDK1/cyclinB, AMPKα2, EGFR, GSK3α, JAK2, MAPK1, mTOR, PKBα, and PI3K p110α(E542K)/p85α via a KinaseProfiler radiometric protein kinase assay, and the structural characteristic and anti-cancer activity of cucurbitacin IIa were introduced in order to provide a reference for further study of this excellent natural medicine.

Fig. 1. Structure of cucurbitacin IIa

2 EXPERIMENTAL

2. 1 Materials

DMSO, methanol, Hexane, dichloromethane, sulforhoda- mine B, fetal bovine serum, sodium bicarbonate and other materials were obtained from commercial sources and used without further purification.

2. 2 Apparatuses

High-resolution mass spectroscopy (HRMS) was per- formed on an AB SCIEX X500R Accurate Mass Q-TOF by using electrospray ionization (ESI). The NMR spectra were recorded on a Bruker AM-600 spectrometer (Billerica, MA).

2. 3 Structure of cucurbitacin IIa

White solid, HRMS (ESI) calcd. for C32H50O8Na (M+Na)+585.3403, found 585.3388. = +53 (c = 0.1, EtOH), m.p.: 226 ℃. IR (KBr, cm-1) v: 3566, 3453, 3408, 2984, 2832, 1692. Spectroscopic data for cucurbitacin IIa were identical to those of the reported sample[7,8].1H NMR (600 MHz, pyridine-d5, ppm): δ 6.44 (d, J = 4.5 Hz, 1H), 5.89 (s, 1H), 5.74 (dt, J = 6.2, 2.1 Hz, 1H), 4.92 (td, J = 8.0, 3.8 Hz, 1H), 4.10 (ddd, J = 11.4, 9.1, 4.1 Hz, 1H), 3.43 (d, J = 9.1 Hz, 1H), 3.37～3.28 (m, 2H), 3.09 (ddd, J = 17.7, 10.9, 5.0 Hz, 1H), 2.96 (d, J = 7.2 Hz, 1H), 2.83 (d, J = 14.5 Hz, 1H), 2.74～2.68 (m, 1H), 2.48～2.40 (m, 2H), 2.34 (ddt, J = 11.2, 8.4, 5.3 Hz, 2H), 1.93 (m, 6H), 1.71 (d, J = 12.9 Hz, 1H), 1.61 (s, 3H), 1.54 (m, 4H), 1.51 (s, 3H), 1.48 (m, 6H), 1.30 (s, 3H), 1.25 (s, 3H), 1.22 (s, 3H).13C NMR (150 MHz, pyridine-d5, ppm): δ 215.1, 213.1, 170.1, 142.4, 118.7, 81.6, 81.4, 80.2, 71.0, 70.4, 59.0, 51.1, 49.2, 48.8, 48.7, 46.4, 43.1, 42.8, 35.4, 34.7, 34.4, 32.2, 26.0, 26.0, 25.5, 25.5, 24.2, 22.4, 22.2, 20.4, 20.4, 19.1.

2. 4 X-ray crystallographic studies

A single crystal of cucurbitacin IIa suitable for X-ray diffraction analysis was grown by slow evaporation of CH2Cl2/hexane solutions of cucurbitacin IIa at 4 ℃. X-ray diffraction was performed on a Bruker D8 Venture diffrac- tometer[9]. Data were collected at 173 K by using a graphite monochromator with CuKα radiation (1.54178 ?) in the ω-? scanning mode. Using ShelXL-2014/7, the structure was solved with the ShelXS17 structure solution program by direct methods and refined with the ShelXL-2014/7 refinement package using the least-squares minimization. Hydrogen and oxygen atoms were located using the geometric method. Details of crystal data, data collections, and structure refinement are summarized in Table 1.

Fig. 2. X-ray crystal structure of cucurbitacin IIa

2. 5 Cell culture

LOVO (colon carcinoma), SKOV3 (ovarian carcinoma), HT-29 (colon carcinoma), MCF-7 (breast carcinoma), and HepG2 (hepatic carcinoma) cell lines (purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in minimum essential medium (modified) with 1.5 mM L-glutamine adjusted to contain 2.2 g/L sodium bicarbonate (90%) and fetal bovine serum (10%). All cells were cultured in a humidified atmosphere containing 5% CO2at 37 ℃.

2. 6 Antiproliferative activities against tumor cell lines

The antiproliferative activities of cucurbitacin IIa against LOVO, SKOV3, HT-29, MCF-7 and HepG2 cell lines were measured using the sulforhodamine B (SRB) assay with cisplatin as reference. Cells were seeded in 96-well plates and then treated with different drug concentrations. After incubation for 72 h, cells were fixed with 10% trichloroacetic acid for 1 h at 4 ℃, washed five times with tap water, and air-dried. Cells that survived were stained with 0.4% (w/v) sulforhodamine B (SRB) for 20 min at room temperature and washed five times with 1% acetic acid. Bound SRB was solubilized with 10 mM Tris and absorbance was measured at 540 nm[4].

2. 7 Kinase inhibitory activities

Kinases CDK1/cyclinB, AMPKα2, EGFR, GSK3α, JAK2, and PKBα were diluted with a buffer comprising 20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% glycerol, 0.1% β-mercaptoethanol, and 1 mg/mL BSA before adding to the reaction mixture. Kinase mTOR was diluted with a buffer comprising 500 mM HEPES, 10 mM EGTA, and 0.1% Tween 20 before adding to the reaction mixture. Kinase MAPK1 was diluted with a buffer comprising 50 mM TRIS, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% β-mercaptoethanol, and 1 mg/mL BSA before adding to the reaction mixture. CDK1/cyclinB (h) was incubated with 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg/mL histone H1, 10 mM magnesium acetate, and [γ-33P]-ATP (specific activity and concentration as required). The reaction was initiated by adding the Mg/ATP mix. After incubating for 40 min at room tempera- ture, the reaction was stopped by adding phosphoric acid to a concentration of 0.5%. A 10 μL sample of the reaction was then spotted onto a P30 filtermat and washed with 0.425% phosphoric acid four times for 4 min, and once in methanol, prior to drying and scintillation counting.

3 RESULTS DISCUSSION

3. 1 Characterization

Cucurbitacin IIa was characterized by IR and NMR spectroscopy. For instance, the IR spectra showed three absorption bands in the range of 3566～3408 cm-1for hydroxyl groups and the 1691 cm-1peak indicates that the compound has carbonyl group. The13C-NMR spectra exhi- bited characteristic peaks at δ 215.1 and 213.1 ppm for carbonyls[10], and at δ 142.4 and 118.7 ppm for the double bond of cucurbitacin IIa.

3. 2 X-ray crystal structure

The single-crystal X-ray diffraction study of cucurbitacin IIa was performed to confirm the structure of the obtained compound. The crystallographic data with refinement parameters, selected bond lengths and bond angles are given in the Supporting Information. The X-ray crystal structure of cucurbitacin IIa is shown in Fig. 2. The selected bond lengths and bond angles are given in Table 1, and the C–C bond angles of the six-membered ring including C(1) to C(5) and C(8) fall in the 108.7～115.7° range, which is consistent with chair conformation[11]. The bond angles of O(1)–C(3)–C(2) and O(2)–C(4)–C(5) are 113.8° and 108.8°, indicating these two hydroxyl groups are in para to each other. The bond angle 111.3° of O(4)–C(23)–C(20) proves the chirality of branched chains. The bond lengths of C(31)–O(7) and C(14)–O(3) of two carbonyl functionalities in the compound are 1.214 and 1.210 ?. The bond length of C(8)–C(9) is 1.325 ?, which proves the existence of double bonds in the ring of cucurbitacin IIa[12].

Table 1. Selected Bond Lengths (?) and Bond Angles (°)

3. 3 Cell proliferation assay

The in vitro cell proliferation assays were performed as previously described. Briefly, the antiproliferative effects of cucurbitacin IIa against HepG2 (hepatic carcinoma), MCF-7 (breast carcinoma), SKOV3 (ovarian carcinoma), HT-29 and LOVO (colon carcinoma) cell lines were measured by the SRB assay, and cucurbitacin IIa has better activity than cisplatin against cancer cell lines.

Table 2. IC50 (μM) Values of Cucurbitacin IIa and Cisplatin against Cancer Cell Lines

3. 4 Determination of kinase inhibitory activities

To explore the mechanism of cucurbitacin IIa, we tested the biochemical activities of cucurbitacin IIa against CDK1/cyclinB, AMPKα2, EGFR, GSK3α, JAK2, MAPK1, mTOR, PKBα, and PI3K p110α(E542K)/p85α via a KinaseProfiler radiometric protein kinase assay[13-15]. As shown in Tables 3, cucurbitacin IIa has a high selectivity because it has a good inhibitory activity aganist the kinase CDK1/cyclin B, but no inhibitory effects against other kinases.

Table 3. Inhibition of Kinases for Cucurbitacin IIa (1 μM)

4 CONCLUSION

In summary, the structure of cucurbitacin IIa from the rhizomes of Hemsleya pengxianensis was characterized by IR, HRMS, NMR spectroscopy, and its structure was confirmed by X-ray crystallography. The antiproliferative effects of cucurbitacin IIa against HepG2, MCF-7, SKOV3, HT-29 and LOVO cell lines were better than the cisplatin against cancer cell lines. Cucurbitacin IIa was evaluated for inhibition of CDK1/cyclinB, AMPKα2, EGFR, GSK3α, JAK2, MAPK1, mTOR, PKBα, and PI3K p110α(E542K)/p85α via a Kinase- Profiler radiometric protein kinase assay. It was found that cucurbitacin IIa has high inhibitory activity and good selec- tivity to CDK1/cyclinB. Structural characterization and acti- vity determination of cucurbitacin IIa provide meaningful references for the future pharmacological applications and structural modification.