Mechanism of Action of Cytotoxic Compounds from the Seeds of Euphorbia lathyris

Abstract

Background

The seeds of Euphorbia lathyris are used in traditional Chinese medicines for the treatment of various medical conditions. E. lathyris contains many natural diterpenes with a lathyrane skeleton.

Purpose and study design

Five lathyrane-type diterpenoids named Euphorbia factors L₁, L₂, L₃, L₈, and L₉ (1–5), were investigated for cytotoxicity against A549, MDA-MB-231, KB, and MCF-7 cancer cell lines and the KB-VIN multidrug resistant (MDR) cancer cell line. Also, a tetraol derivative (6) of Euphorbia factor L₂ (2) was synthesized to assess the effect of hydroxy moieties.

Methods

An ethanolic extract of seeds of Euphorbia lathyris was prepared and separated into petroleum ether, EtOAc, n-butanol, and n-hexane extracts. The natural diterpenes were isolated by using silica gel and Sephadex LH-20 column chromatography as well as preparative thin-layer chromatography. Saponification of 2 gave tetraol derivative 6. Cytotoxic activity was determined by the sulforhodamine B (SRB) colorimetric assay. Mechanism of action studies focused on the impact of compounds on the cell cycle progression as well as cell morphology.

Results

Compound 5 exhibited the strongest cytotoxicity against all cell lines, while compound 2 showed selectivity against KB-VIN. In cells treated with 3 and 5, accumulation of G1 to early S phase cells was obvious, while no effect was seen on G2/M phase.

Conclusion

Analysis of the screening data compared with compound structures suggested that the substitutions at C-3, C-5, C-7, and C-15 are critical for cytotoxicity, as well as cell type-selectivity. Furthermore, results of cytotoxic mechanism analysis demonstrated for the first time that compounds 3 and 5 disrupted normal cell cycle progression, whereas compounds 2–5 induced obvious actin filament aggregation, as well as partial interference of the microtubule network.

Graphical Abstract

Introduction

The seeds of Euphorbia lathyris are used in traditional Chinese medicines for the treatment of hydropsy, ascites, amenorrhea, scabies, terminal schistosomiasis, and snakebite (Shi et al., 2008). E. lathyris contains many natural diterpenes with a lathyrane skeleton, including a series of diterpenoids named Euphorbia factors L₁–L₂₁ (EFL1–21) (Adolf and Hecker, 1971, 1975; Appendino et al., 2003; Lu et al., 2014), as well as lathyranoic acid A (Liao et al., 2005) and lathyranone A (Gao et al., 2007), two rearranged lathyrane-type compounds.

In cancer treatment, multidrug resistance (MDR) has been recognized as a major cause of chemotherapy failure. The development of resistance to anticancer drugs is a major limitation for cancer chemotherapy. One of the most effective mechanisms of MDR involves the transporter P-glycoprotein (P-gp), a 170 KDa phosphorylated and glycosylated membrane protein belonging to the ATP-binding cassette (ABC) superfamily of transporter proteins (Juliano and Ling, 1976).

In the discovery of MDR reversing agents from plants, numerous diterpenes isolated from E. lathyris, E. dendroides (Corea et al., 2003), E. serrulata, E. esula, E. salicifolia, E. peplus (Hohmann et al., 2002), E. characias, E. amygdaloides, E. paralias, and E. helioscopia have exhibited potent P-gp inhibitory effects. With a structurally homogeneous skeleton differing only in the substitution pattern or positions, these compounds can serve as ideal targets for a structure activity relationship (SAR) study among this new class of P-gp inhibitors (Corea et al., 2009).

In prior biological activity studies, lathyrane-type diterpenes from E. lathyris have shown significant cytotoxicity toward cancer cells, as well as powerful P-gp inhibitory (Barile et al., 2008) and MDR modulating effects (Appendino et al., 2003; Jiao et al., 2009; Zhang et al., 2010). We also reported the isolation, identification, and evaluation of P-gp mediated ATP hydrolysis to determine the P-gp substrates among ten compounds from a petroleum ether extract of Euphorbia seed (Duan et al., 2014).

As part of our ongoing work, the present study investigated the in vitro cytotoxicity of compounds 1–5 against A549, MDA-MB-231, KB, P-gp-overexpressing KB subline KB-VIN, and MCF-7 cancer cells. Furthermore, compound 6, a new tetraol derivative of 2, was synthesized and evaluated for cytotoxicity. The detailed cytotoxic mechanism of Euphorbia factors L_1–3, L₈, and L₉ (EFL1–3, EFL8, EFL9) (Fig. 1) were further investigated and are described herein.

Figure 1. Structures of Euphorbia factors L_1, L_2, L_3, L_8, and L₉ (EFL1–3, 8, and 9).

Materials and Methods

General Experimental Procedures

All chemicals and solvents were used as purchased. All melting points were measured on a Fisher-Johns melting point apparatus and reported without correction. ¹H and ¹³C NMR spectra were recorded on a Varian Gemini 2000 (300 MHz) or Varian Inova (400 MHz) NMR spectrometer with TMS as the internal standard. All chemical shifts were reported in ppm. NMR spectra were referenced to the residual solvent peak, chemical shifts δ were in ppm, and apparent scalar coupling constants J were in hertz. Mass spectroscopic data were obtained on a Shimadzu LCMS-IT-TOF instrument. Analytical thin layer chromatography (TLC) was carried out on Merck precoated aluminum silica gel sheets (Kieselgel 60 F-254). Biotage Flash or Isco Companion systems were used for flash chromatography. All target compounds were characterized and determined to be at least >95% pure by ¹H NMR and analytical HPLC.

Plant Material

Caper Euphorbia seed was purchased from Huqiao, Anhui Pharmaceutical Co., Ltd. and identified by Professor Fei Li (School of Chinese Materia Medica, Beijing University of Chinese Medicine).

Extraction and Isolation (Duan et al., 2014)

Powdered Euphorbia lathyris seeds (4 kg) were refluxed with 95% alcohol three times for three hours each time. The ethanolic extracts were combined and concentrated to remove solvent, and then suspended in H₂O and partitioned successively with petroleum ether, EtOAc, n-butanol, and n-hexane to afford corresponding extracts.

The petroleum ether extract was separated by silica gel (petroleum ether/EtOAc, petroleum ether/acetone) and Sephadex LH-20 column chromatography (CHCl₃/MeOH, MeOH) as well as preparative thin-layer chromatography to afford compound 1 (1120 mg), compound 2 (320 mg), compound 3 (1100 mg), and compound 4 (40 mg). Compound 5 was purchased from Wuhan ChemFaces Biochemical (Hubei, China).

General Synthetic Procedure for Compound 6

Compound 6 (7β-hydroxylathyrol): A suspension of 2 (321 mg, 0.5 mmol) in 5% potassium hydroxide in MeOH (50 mL) was stirred at room temperature for 24 h. Further purification was performed by flash column chromatography (petroleum ether/EtOAc = 4:1) to afford 6 (120 mg, 80% yield). HRMS-ESI-TOF m/z calcd for (M + Na⁺): 373.20, found: 373.20. ¹H NMR (400 MHz, CDCl₃) δ 1.08 (3H, d, J = 7 Hz, H-16), 1.14 (s, 1H, H-19), 1.18 (s, 1H, H-18), 1.32 (1H, ddd, J = 12, 8.5, 4 Hz, H-9), 1.47 (dd, 1H, J = 11.5, 8.5 Hz, H-11), 1.63 (dd, 1H, J = 13, 11 Hz, H-1β), 1.67 (br s, 3H, H-20), 1.83 (ddd, 1H, J = 15, 12, 8.5 Hz, H-8β), 1.99 (m, 1H, H-2), 2.16 (ddd, 1H, J = 15, 4, 4 Hz, H-8α), 2.29 (dd, 1H, J = 8.5, 3 Hz, H-4), 3.11 (dd, 1H, J = 14, 10 Hz, H-1α), 4.23 (dd, 2H, J = 8.5, 4 Hz, H-7), 4.41 (t, 1H, J = 3 Hz, H-3), 4.80 (d, 1H, J = 8.5 Hz, H-5), 5.01 (br s, 1H, H-17b), 5.16 (br s, 1H, H-17a), 6.81 (d, 1H, J = 11.4 Hz, H-12). ¹³C NMR (400 MHz, DMSO) δ11.1 (q, C-20), 12.8 (q, C-16), 14.8 (q, C-19), 22.8 (t, C-8), 26.6 (s, C-10), 27.4 (d, C-11), 30.0 (q, C-18), 30.3 (d, C-9), 35.8 (d, C-2), 47.2 (t, C-1), 53.2 d (C-4), 63.4 (d, C-5), 76.2 (d, C-7), 76.9 (d, C-3), 87.2 (s, C-15), 111.3 (t, C-17), 132.2 (s, C-13), 147.8 (s, C-6), 147.9 (d, C-12), 199.8 (s, C-14).

Cytotoxicity Assays

Cytotoxic activity was determined by the sulforhodamine B (SRB) colorimetric assay as previously described (Nakagawa-Goto et al., 2015) In brief, human tumor cell lines were cultured in RPMI-1640 medium (Corning) supplemented with 2 mM L-glutamine and 25 mM HEPES (Gibco), supplemented with 10% fetal bovine serum (Serum Source), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 0.25 μg/mL amphotericin B (Corning). MDR stock cells (KB-VIN) were maintained in the presence of 100 nM vincristine (VIN) (Sigma-Aldrich).

Freshly trypsinized cell suspensions were seeded in 96-well microtiter plates at densities of 4,000–11,000 cells per well (based on the cell lines) with compounds. After 72 h in culture with test compounds, cells were fixed with 10% trichloroacetic acid and then stained with 0.04% sulforhodamine B (Sigma-Aldrich). The bound SRB was solubilized in 10 mM Tris-base and the absorbance at 515 nm was measured using a Microplate Reader (ELx800, Bio-Tek Instruments, Winooski, VT) operated by Gen5 software (BioTek) after solubilizing the protein-bound dye with 10 mM Tris base. The mean IC₅₀ is the concentration of agent that reduced cell growth by 50% compared with vehicle (DMSO) control under the experimental conditions and is the average from at least three independent experiments with duplicate samples.

The following human tumor cell lines were used in the assay: A549 (lung carcinoma), KB (originally isolated from epidermoid carcinoma of the nasopharynx), KB-VIN (VIN-resistant KB subline showing MDR phenotype by overexpressing P-gp), MCF-7 (estrogen receptor (ER)-positive, HER2-negative breast cancer), MDA-MB-231 (triple-negative breast cancer). All cell lines were obtained from the Lineberger Comprehensive Cancer Center (UNC-CH) or from ATCC (Manassas, VA), except KB-VIN, which was a generous gift of Professor Y.-C. Cheng (Yale University). Paclitaxel was purchased from Sigma-Aldrich.

Cell Cycle Analysis

KB-VIN cells were seeded in 12-well plates at a density of 1 × 10⁵ per well and incubated overnight. After 24 h of treatment with tested compounds at a concentration of one- or three-fold IC₅₀, the cells were harvested and fixed in 70% EtOH at −20 °C overnight followed by staining with propidium iodide (PI) containing RNase (BD Pharmingen) for 30 min at 37 °C. The DNA contents of stained cells were analyzed by flow cytometer (FACSCalibur, BD Biosciences) controlled by FlowJo (FlowJo LLC) with 488 nm excitation laser and FL2 yellow fluorescence channel. All obtained data were analyzed by Flowing Software version 2.5.1. Paclitaxel (PXL) was used at 3 or 6 μM. Combretastatin A4 (CA-4) was purchased from Sigma-Aldrich.

Immunocytochemistry Analysis

KB-VIN cells were seeded in an 8-well chamber slide (Lab-Tech) at a density of 1 × 10⁵ per mL and incubated overnight. After 24 h of treatment with tested compounds, the cells were fixed with 4% paraformaldehyde in PBS for 10 min followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min at room temperature. Cells were then blocked with 5% normal goat serum in 1% BSA in washing buffer (0.01% Tween 20 in PBS) for 30 min. Cells were labeled with monoclonal antibody to α-tubulin (T5168, clone B-5–1-2, Sigma-Aldrich) for 2 h followed by fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG secondary antibody (F5262, Sigma-Aldrich) for 1 h. F-actin was stained with tetramethylrhodamine (TRITC)-labeled phalloidin (P1951, Sigma-Aldrich) for 30 min while DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich) for 5 min. Stained cells were mounted with Vectashield (H-1000, Vector Laboratories). The images were captured by confocal fluorescence microscope (Zeiss LSM700) (Laser lines: 405 nm for DAPI, 488 nm for FITC, and 555 nm for TRITC) operated by ZEN software (Zeiss). Final images were analyzed by ZEN light and processed with Photoshop 7 (Adobe).

Results

Chemistry

Five known macrocyclic diterpenes, Euphorbia factors L₁ (1), L₂ (2), L₃ (3), L₈ (4), and L₉ (5), were purified from a petroleum ether extract of E. lathyris seeds using a chromatographic method (Fig. 1). As shown in Scheme 1, compound 2 was also modified by hydrolysis of all four ester groups to give the tetraol derivative (6). With these six compounds in hand, we could assess the effects of substituents, especially ester groups at positions C-3, C-5, C-7, and C-15, based on elements of size, hydrophobicity, and aromaticity.

Scheme 1.

Synthesis of 6

Cytotoxicity of Compounds 2–5 and Newly Synthesized Tetraol Derivative 6

In several studies (Jiao et al., 2015; Zhang et al., 2013; Zhang et al., 2011) using human tumor MDR cells, compounds 1 and 3, as well as analogues of 3, have exhibited MDR reversal ability. Compounds 2 and 3 were suggested to act as modulators of P-gp, as both compounds could stimulate P-gp ATPase activity (Duan et al., 2014). Therefore, we tested the five known Euphorbia factors (1–5) together with the newly synthesized derivative (6) against chemo-sensitive KB (originally isolated from epidermoid carcinoma of the nasopharynx) and P-gp overexpressing multidrug resistant KB subline KB-VIN, as well as three additional human cancer cell lines, including A549 (lung carcinoma), MDA-MB-231 (triple negative breast cancer), and MCF-7 (estrogen receptor-positive breast cancer), using a sulforhodamine B (SRB) assay. The IC₅₀ values are presented in Table 1.

Table 1.

Cytotoxicity of compounds

Compound	IC50 (μM)a / Cell Line
	A549	MDA-MB-231	KB	KB-VIN	MCF-7
1	>40	>40	>40	>40	>40
2	>40	>40	33.2 ± 7.7	7.2 ± 1.5	>40
3	14.6 ± 0.1	31.6 ± 2.3	7.9 ± 0.3	8.0 ± 1.3	25.9 ± 4.3
4	11.8 ± 1.8	24.4 ± 4.0	17.7 ± 1.2	16.9 ± 2.9	23.8 ± 4.7
5	6.7 ± 0.5	21.9 ± 1.3	6.1 ± 0.2	5.7 ± 0.1	8.4 ± 1.4
6	>40	>40	>40	>40	>40
PXL (nM)	7.4 ± 2.0	9.6 ± 3.0	6.7 ± 2.2	1928.8 ± 183.8	14.3 ± 2.9

^aAll values are the mean ± SD of at least three experiments.

Discussion

Biological Activity Comparison

Except for the surprising potency (IC₅₀ 7.2 μM) of 2 against KB-VIN cells, compounds 1, 2, and 6 were essentially inactive. In fact, while compound 3 exhibited moderate broad spectrum cytotoxicity, compound 1 showed no cytotoxicity against any cell line at 40 μM. Structurally, compounds 1 and 3 differ at C-3 (phenylacetate in 1, benzoate in 3) and C-6/C-17 (epoxide in 1, exocyclic methylene in 3). Interestingly, compound 3 showed its greatest potency against KB and the KB-VIN subline (IC₅₀ 7.9 and 8.0 μM, respectively). Notably, all of the active diterpenes did not show much difference in activity between the parent and drug-resistant sublines, as compared with the nearly 300-fold drop in activity with paclitaxel, the positive control.

Overall, compound 5 was the most potent compound tested with IC₅₀ values ranging from 5.7 to 8.4 μM against four cell lines. Although 5 was less potent against MDA-MB-231 (IC₅₀ 21.9 μM), this cell line was also less responsive to all six tested lathyrane-type diterpenes. Among 3–5, the rank order of potency was 5 > 3 and 4. Compound 5 has a nicotinate ester at C-7, while compounds 3 and 4 are unsubstituted at this position, indicating that substitution at C-7 plays a role in the cytotoxicity. The synthetic compound 6, with four hydroxy rather than ester groups at C-3, C-5, C-7, and C-15, was inactive, again emphasizing the importance of appropriate substitution at C-7.

Interestingly, compound 2 displayed five-fold selective cytotoxicity against the MDR subline KB-VIN compared with parent KB cells. As far as we know, this study is the first to report a Euphorbia factor diterpene as a collateral sensitive agent. A prior study had revealed that the substitutions at C-5 and C-15 were related to the chemo-reversal effects of Euphorbia factors (Jiao et al., 2015). In addition, we found that the identity of the aromatic ring system at C-7 was crucial for the selective cytotoxicity against KB-VIN. Compound 5 with a nicotinoyl rather than benzoyl group at C-7 showed significant and broad cytotoxic activity. Taken together, a combination of acetate groups at C-5 and C-15 as well as benzoate groups at C-3 and C-7 were required for the selective cytotoxicity against KB-VIN.

Cytotoxic Mechanisms of Action of 2–5 in KB-VIN Cells

We also performed mechanism of action studies focusing on the impact on the cell cycle progression as well as cell morphology. KB-VIN cells were treated with compounds 2–5 at concentrations of IC₅₀ (1× IC₅₀) or three-fold IC₅₀ (3× IC₅₀), and the cell cycle was analyzed by flow cytometer (Fig. 2). Compounds 2 and 4 showed no significant effect on cell cycle progression at the higher dose (3× IC₅₀). In contrast, both 3 and 5 disrupted normal cell cycle progression. In fact, accumulation of G1 to early S phase cells was obvious, while no effect was seen on G2/M phase, as compared with the mitotic inhibitors combretastatin A-4 (CA-4) and paclitaxel (PXL). These observations suggested that 3 and 5 may target the same protein, which is required for the G1 to S transition.

Figure 2. Effects of compounds 2–5 on cell cycle progression.

KB-VIN cells were treated with vehicle (CTRL) and compounds 2–5 at the concentrations of one (1 × IC₅₀) or three fold IC₅₀ (3 × IC₅₀) for 24 h. Harvested cells were stained with propidium iodide (PI) subjected to flow cytometry. Cell cycle phases are indicated as sub-G1, G1, S, and G2/M based on the DNA contents. CA-4 and paclitaxel (PXL) were used as control for mitotic inhibitors.

A cell’s structure is maintained by its cytoskeleton, which mainly consists of microfilaments and microtubules. These cytoskeletons are also important in controlling the entire dynamics of cell life and chromosome separation in mitosis. We investigated the effects of compounds on cellular cytoskeletons by staining KB-VIN cells with antibody to α-tubulin and phalloidin to visualize the microtubules and F-actin, respectively (Fig. 3). KB-VIN cells were treated for 24 h with compounds at a concentration of 3× IC₅₀. Although control cells showed clear microtubule networks and actin filaments, cells treated with compounds exhibited slight changes in microtubules with obvious aggregations of actin filaments. These observations suggested that all tested compounds partially disrupted microtubules and actin filaments. Unfortunately, no significantly different effects on cytoskeletons were observed between compounds, suggesting that the core lathyrol diterpene structure in 2–5 partially disrupts microtubules and actin filaments resulting in F-actin aggregation. However, these effects were not closely related to the inhibitory effects on G1/S transition. Further detailed analysis of mechanism of action is merited.

Figure 3. Effects of compounds 2–5 on cytoskeletons.

KB-VIN cells were treated with 2–5 for 24 h at a concentration of 3 × IC₅₀. Cytoskeletons were visualized by monoclonal antibody to α-tubulin for microtubules (green), phalloidin-TRITC for F-actin (red), and DAPI for DNA (blue). Labeled cells were observed by confocal fluorescence microscope, and confocal images were reconstructed by stacking. F-actin aggregations are shown by arrows. Scale bar, 20 μm. Additional images are available in Supplementary Information Figure 1.

Conclusion

Five lathyrane-type diterpenoids, named Euphorbia factors L₁, L₂, L₃, L₈, L₉ (1–5) isolated from seeds of Euphorbia lathyris, together with a newly synthesized tetraol derivative (6) of 2 were investigated for cytotoxicity against human tumor cell lines, including the MDR subline KB-VIN. Compound 5 exhibited the strongest cytotoxicity against all cell lines, while compound 2 showed selectivity against KB-VIN cells. SAR analysis suggested that the substitutions at C-3, C-5, C-7, and C-15 are critical for cytotoxicity or selectivity against KB-VIN. Furthermore, results of cytotoxic mechanism of action analysis revealed for the first time that compounds 3 and 5 disrupted normal cell cycle progression, whereas compounds 2–5 exhibited obvious actin filament aggregation as well as partial disruption of microtubule networks.

List of Abbreviations

EFL1–21

Euphorbia factors L₁–L₂₁

MDR

multidrug resistance

P-gp

P-glycoprotein

ABC

ATP-binding cassette

SAR

structure activity relationship