Diterpenoids from Euphorbia gedrosiaca as Potential Anti-Proliferative Agents against Breast Cancer Cells

Isolated diterpenes from various species of Euphorbia are important compounds for drug discovery with a broad spectrum of structures and biological effects. In this study, Euphorbia gedrosiaca, one of the endemic species of Iran, was analyzed in terms of the presence and structural determination of diterpenoid compounds. They were extracted with dichloromethane/acetone (2:1) from aerial parts of this plant and purified by chromatographic methods such as MPLC and HPLC. Four premyrsinane compounds and one myrsinane diterpene were isolated from Euphorbia gedrosiaca. They were characterized by extensive 1D and 2D NMR and HRMS analyses. Additionally, their activities were evaluated against two breast cancer cell lines, MDA-MB-231 and MCF-7, by MTT proliferation assay. They exhibited cytotoxic effects in a dose-dependent manner with promising results, which can help to find possible therapeutic application of diterpenoids in breast cancer treatment.


Introduction
As the main genera of the Euphorbiaceae family, Euphorbia has more than 2000 species that grow in all temperate and tropical regions. They cover a wide range of habitats and are distinguished by their particular inflorescences, designated as Cyathia [1,2]. Medicinal species are traditionally used to treat warts, and as a paste to relieve pains [3]. From 2013 to 2019, 455 diterpenoids were isolated from 53 species of Euphorbia [4], most of which are effective in a broad range of biological activities with potential usage in health maintenance [5]. Cyclomyrsinane diterpenoids with 5/7/6/3-tetracyclic carbon framework, myrsinane with 5/7/6 tricyclic ring system ( Figure 1A), and premyrsinane diterpenoids with 5/7/6/3-tetracyclic carbon ( Figure 1B) belong to myrsinane-type diterpenoids [4]. Premyrsinane diterpenoids are obtained by the cyclization at C-12 and C-6 of lathyrane diterpenoids [4] and might be rearranged to myrsinane structures by breaking of C-9 and C-10 bond [4]. They have chemotaxonomic significance and were isolated only from Euphorbia species, including E. prolifera [6,7], E. sogdiana [8], E. dracunculoides [9,10], E. lathyris [11] E. sanctae-catharinae [12], E. macroclada [13], E. falcata [14,15], and E. aleppica [16] with antifungal effects against plant pathogenic fungi, cytotoxic activities, and modulating the drug resistance in cancer cells [7,8,15]  They have chemotaxonomic significance and were isolated only from Euphorbia species, including E. prolifera [6,7], E. sogdiana [8], E. dracunculoides [9,10], E. lathyris [11] E. sanctaecatharinae [12], E. macroclada [13], E. falcata [14,15], and E. aleppica [16] with antifungal effects against plant pathogenic fungi, cytotoxic activities, and modulating the drug resistance in cancer cells [7,8,15] E. gedrosiaca, also identified by the synonym Tithymalus gedrosiacus, is closely related to E. erythradenia. It is a rare localized endemic plant in the Iranian plateau that grows mainly in the center and southeastern parts of Iran [17,18]. In the previous research on this plant, two new myrsinane and two known cyclomyrsinane-type diterpenes with moderate cytotoxicity and proapoptotic properties against B16F10 and A375 melanoma skin tumor cells were reported. In the cycle arrest analysis, a significant increase was reported in the G2/M phases for both myrsinane and cyclomyrsinane compounds [19]. In this paper, our investigation led to the isolation of four premyrsinane diterpenes and one myrsinane diterpene from this plant, along with the determination of their cytotoxic activities against MCF-7 and MDA-MB 231 breast cancer cells by standard MTT assay.

Plant Materials
Aerial parts of E. gedrosiaca Rech.f., Aellen and Esfand., were collected from the South of Nehbandan, South Khorasan province (Iran) in May 2013 in its flowering time. It was identified by Amir Hossein Pahlevan, Department of Botany, Herbaceous Sciences Research Center at the Ferdowsi University of Mashhad, and a voucher specimen (Sam-3638) was kept at the Samsam-Shariat herbarium, Department of Pharmacognosy, Isfahan University of Medical Sciences, Iran.

Extraction Procedure
Plant material was air-dried in the shade (4 kg), and extracted with dichloromethane: acetone (2:1 v/v) to obtain 170 g of deep green crude extract. It was filtered on a C-18 cartridge using MeOH: H2O (6:4) to get a brown color extract free from unwanted chlorophylls, and fatty contents. The fraction eluted by MeOH: H 2 O (6:4 v/v, 57.1 g) was concentrated and resubmitted on MPLC silica gel (25-

MTT Viability Assay
MDA MB-231 and MCF-7 (ATCCR HTB-26 and ATCCR HTB-22) breast cancer cells, originated from Pasteur Institute (IPI, Tehran, I.R. Iran), were cultured in RPMI1640 with streptomycin, penicillin, and fetal bovine serum (FBS) (100 µg/mL, 100 units/mL, 10%) with criteria of 5% carbon dioxide, 37 • C, and 95% humidity for a few days. Mouse normal fibroblast cell line (NCTC clone 929 from the same institute) was cultured in DMEM+10% FBS+ 1% penicillin/streptomycin in the same conditions. After incubation, cells were seeded at 7 × 10 3 cells in each well of ninety-six well plates for 12 h. Then, the media were changed, and sample compounds in concentrations of 1, 10, and 100 µM were added. For L929 normal cell line, samples were added in the concentrations of 1, 10, 100, 250, and 500 µM. Placebo with the same amount of DMSO in each concentration used for compounds and Taxol in concentrations of 0.001, 0.01, 0.1, 1, and 10 µM were added as the negative and positive controls. After 24 h incubation time, the reduction agent (MTT, 5 mg/mL, 20 µL) was added to each well and incubated for 4 h to let the MTT be reduced into formazan derivative by the mitochondria of survived cells. The media was then changed with DMSO (200 µL) to solubilize the insoluble formazan crystals, and the absorbance was read by a Synergy ELISA reader (BioTek Instruments, Winooski, VT, USA) at 570 nm [20].

Determination of Cytotoxic Activity
Compounds 1-5 were tested by MTT assay to evaluate their cytotoxic activity ( Table 3)

Discussion
All of the diterpenoids presented in this study are isolated and introduced for the first time from Euphorbia gedrosiaca, and despite their rare occurrence, compounds 1, 3, and 4 were isolated during a study on an endemic species of Euphorbiaceae in Egypt called Euphorbia sanctae-catharinae and they showed moderate cytotoxicity against the proliferation of human lung (A549) and colon (Caco-2) tumor cells in different degrees [21]. Compounds 3 and 4 were also elucidated from Euphorbia pithyusa subsp. cupanii previously [28]. Compound 2 was elucidated from Euphorbia dracunculoides [9], and Euphorbia pithyusa [30] until now. Formerly, this compound was isolated from the roots of Euphorbia prolifera and showed acceptable lipid-lowering activities and TG inhibitory effects during an investigation on this plant [31]. Compound 5 has been reported in previous studies from Euphorbia species like Euphorbia prolifera [32], Euphorbia dracunculoides Lam [33], and Euphorbia nematocypha Hand.-Mazz [34].
Regarding the anti-proliferative activity of the premyrsinane diterpenes (1)(2)(3)(4) in the current study, results from the MTT viability assay showed that amongst all compounds, 1 is the most effective against both cell lines, especially MDA-MB-231 with an IC 50 value of 10.8 µM which is considerable. In general, all four diterpenes showed significant apoptotic effects against MDA-MB-231 based on the cell viability in 100 µM concentration of these bioactive agents (p < 0.0001). The evaluated IC 50 from compounds 2, 3, and 4, respectively, as 22.2, 24.5, and 27.3 µM, exhibits that they are similarly effective against MDA-MB-231. Compound 2 is the second most effective one based on our findings, which had quite the same efficacy on both cell lines. Although the effectiveness of 1 against MCF-7 did not differ significantly from 2, both were reasonably potent in 100 µM concentration with P values less than 0.0001. Compounds 3 and 4 demonstrated nearly similar MTT test results with almost three times higher cytotoxicity against the MDA-MB-231 cell line compared to MCF-7, which can be considered a moderate activity and not as potent as other responses in this study. This comparison shows that this similarity is due to their chemical structural analogy, which only differs at C-17 by replacing an acetyl ester group with a nicotinoyl group. Compound 5 was also tested for its cytotoxicity against these two cell lines but showed less potency than the other four compounds. However, the efficacy is acceptable against the MDA-MB-231 with an IC 50 value of 33.7 µM, and can be worthy of consideration, but the IC 50 value of 125.6 µM against MCF-7 shows low activity. The comparison of MTT assay data also reveals that the myrsinane diterpenes have less efficacy as anti-proliferative agents against these two cancer cell lines. New findings about the structure-activity relationship for cytotoxic efficacy can be helpful to approach the best chemical structure in therapeutic applications.
All of the isolated compounds were also tested on mouse normal fibroblast cell lines to prove their safety, and the results were promising, while none of them demonstrated toxicity at concentrations up to 250 µM towards L929 normal cell line.
Former studies concerning the evaluation of cytotoxicity and possible therapeutic application of diterpenoids in breast cancer treatment have also revealed valuable results as well. In an identical study on Euphorbia aleppica, two new cytotoxic premyrsinane diterpenes were introduced and assessed for their anti-cancer effects on MCF-7 and MDA-MB-231 cell lines which determined their probable potency [16]. Similarly, another investigation on cyclomyrsinane diterpenes from Euphorbia species proved the potential activity of compounds that had structural similarities to the ones elucidated in the present research against the same breast tumor cell lines [35].

Conclusions
Five diterpenoids were determined from E.gedrosiaca and evaluated for their antiproliferative properties against MCF-7 and MDA-MB231 cell lines. The MTT viability assay proves that compound 1 is the most effective against both cell lines, especially MDA-MB-231. We can conclude that these diterpenes show better apoptotic effects against MDA-MB-231 than MCF-7, and it is clear that the premyrsinane skeletal structure improves the cytotoxic activity of compounds against breast cancer cell lines. On the other hand, the results displayed the safety of the isolated compounds against normal cells, which can be beneficial in drug development.
Considering all of the above mentioned, in order to achieve valuable and novel discoveries as practical anti-breast cancer treatments, it is worthwhile to survey and precisely determine the most effective bioactive chemical structure of this type of diterpenoids and detect the most potent apoptotic agent by comparing the replacement of different chemical groups and assess their effects.

Data Availability Statement:
The data presented in this study are available in the article.

Conflicts of Interest:
The authors declare no conflict of interest.