Structure Identification and In Vitro Anticancer Activity of Lathyrol-3-phenylacetate-5,15-diacetate

Natural products from the genus Euphorbia show attention-attracting activities, such as anticancer activity. In this article, classical isolation and structure identification were used in a study on Caper Euphorbia Seed. Subsequently, MTT and wound healing assays, flow cytometry, western blotting, Hoechst 33258 staining and fluorescence microscopy examination were applied to investigate the anticancer activity of the obtained compounds. In a result, lathyrol-3-phenyl- acetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) was isolated from Caper Euphorbia Seed. Moreover, the NMR signals were totally assigned. DEFL1 showed potent inhibition against lung cancer A549 cells, with an IC50 value of 17.51 ± 0.85 μM. Furthermore, DEFL1 suppressed wound healing of A549 cells in a concentration-dependent manner. Mechanically, DEFL1 induced apoptosis, with involvement of an increase of reactive oxygen species (ROS), decrease of mitochondrial membrane potential (ΔΨm), release of cytochrome c, activity raise of caspase-9 and 3. Characteristic features of apoptosis were observed by fluorescence microscopy. In summary, DEFL1 inhibited growth and induced apoptosis in lung cancer A549 cells via a mitochondrial pathway.


Introduction
Cancer has become the increasing public health problem and the major threat to human health for its high morbidity and mortality [1][2][3]. Under current understanding, chemotherapy is one of the key treatment methods, which has achieved great therapeutic success for certain malignant tumors [4][5][6]. However, some aspects are limiting factors of chemotherapy, including side effects and resistance, particularly multi-drug resistance (MDR) [7][8][9]. Thus, developing novel drugs with better efficacy to treat cancer is the effective strategy [10][11][12].
Natural resources are still the key provider of novel drugs despite the development of combinatorial chemistry, which can synthesize thousands of compounds quickly [13,14]. Euphorbia is the largest genus of the spurge family, containing more than 2000 species, some of which have been applied as medicinal plants to treat various diseases, including cancer [15]. Caper Euphorbia Seed, used as a Traditional Chinese Medicine is the seeds of Euphorbia lathyris L. [16]. The main constituents of Caper Euphorbia Seed are lathyrane diterpenoids [17], and a series of diterpenes L1-L8 based on the lathyrane skeleton have been isolated from the seeds. The biological activities of these lathyrane-type diterpenes have been studied, showing cytotoxicity to cancer cells and the ability to reverse MDR [18,19].
As part of ongoing research, we have exhaustively investigated Caper Euphorbia Seed, examining the chemical isolation and structure identification of its components and their pharmacological aspects [20][21][22][23]. During these studies, lathyrol-3-phenylacetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) was isolated from Caper Euphorbia Seed. In this article we report its structure identification and total NMR signal assignment. Furthermore, DEFL1 was shown to inhibit the growth of and induce apoptosis in A549 cells via a mitochondrial pathway.

Structure Identification and NMR Signal Assignments
Lathyrol-3-phenylacetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) was obtained as white flaky crystals. Colorless crystals were obtained from dichloromethane-petroleum ether. The molecular formula C 32 H 40 O 7 was determined by high resolution mass spectrometry (HRMS) showing m/z 537.2848 (M + H), which indicates 13 degrees of unsaturation. The 1 H-NMR spectrum displayed five benzene protons with chemical shifts between 7.15 and 7.50. Moreover, 30 carbon signals were visible in the 13 C-NMR data. The information supplied by the molecular formula, 1 H-NMR and 13 C-NMR implied that DEFL1 contains one monosubstituted benzene ring. The 13 C-NMR spectrum also showed four carbonyls at 196.8, 171.3, 170.7 and 169.8 ppm. The DEPT spectrum indicated that DEFL1 has six methyl carbons, five methylene carbons, ten methine carbons, and nine quaternary carbons. The 1 H-, 13 C-NMR and heteronuclear multiple bond correlation (HMBC) data listed in Table 1, together with the data in reference [19] allowed the full assignment of the 1D NMR signals and the determination of the structure as shown in Figure 1A. Seed, used as a Traditional Chinese Medicine is the seeds of Euphorbia lathyris L. [16]. The main constituents of Caper Euphorbia Seed are lathyrane diterpenoids [17], and a series of diterpenes L1-L8 based on the lathyrane skeleton have been isolated from the seeds. The biological activities of these lathyrane-type diterpenes have been studied, showing cytotoxicity to cancer cells and the ability to reverse MDR [18,19]. As part of ongoing research, we have exhaustively investigated Caper Euphorbia Seed, examining the chemical isolation and structure identification of its components and their pharmacological aspects [20][21][22][23]. During these studies, lathyrol-3-phenylacetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) was isolated from Caper Euphorbia Seed. In this article we report its structure identification and total NMR signal assignment. Furthermore, DEFL1 was shown to inhibit the growth of and induce apoptosis in A549 cells via a mitochondrial pathway.

Structure Identification and NMR Signal Assignments
Lathyrol-3-phenylacetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) was obtained as white flaky crystals. Colorless crystals were obtained from dichloromethane-petroleum ether. The molecular formula C32H40O7 was determined by high resolution mass spectrometry (HRMS) showing m/z 537.2848 (M + H), which indicates 13 degrees of unsaturation. The 1 H-NMR spectrum displayed five benzene protons with chemical shifts between 7.15 and 7.50. Moreover, 30 carbon signals were visible in the 13 C-NMR data. The information supplied by the molecular formula, 1 H-NMR and 13 C-NMR implied that DEFL1 contains one monosubstituted benzene ring. The 13 C-NMR spectrum also showed four carbonyls at 196.8, 171.3, 170.7 and 169.8 ppm. The DEPT spectrum indicated that DEFL1 has six methyl carbons, five methylene carbons, ten methine carbons, and nine quaternary carbons. The 1 H-, 13 C-NMR and heteronuclear multiple bond correlation (HMBC) data listed in Table 1, together with the data in reference [19] allowed the full assignment of the 1D NMR signals and the determination of the structure as shown in Figure 1A.

Growth Inhibition in A549, KB and HCT116 Cells
The growth suppression results of DEFL1 were determined by an MTT assay to give IC 50 values of 17.51 ± 0.85, 24.07 ± 1.06, and 27.18 ± 1.21 µM for A549, KB and HCT116 cells, respectively ( Figure 1B). Among these three cancer cell lines, A549 cells showed the most sensitivity (p < 0.05), thus A549 cells were selected for further investigation.

Increase of Intracellular ROS Levels in A549 Cells
According to current knowledge, a raise of intracellular ROS levels causes apoptosis [20]. To verify this, generation of ROS was measured with DCFH-DA, which can be hydrolyzed and oxidized to DCF, a fluorescent dye that can be detected by flow cytometry [21]. After A549 cells were treated with 18.0 M DEFL1 for 12, 24 and 36 h, the intracellular ROS levels were 126.66 ± 4.46%, 151.15 ± 7.09% and 191.04 ± 11.56% of control, respectively ( Figure 3).

Increase of Intracellular ROS Levels in A549 Cells
According to current knowledge, a raise of intracellular ROS levels causes apoptosis [20]. To verify this, generation of ROS was measured with DCFH-DA, which can be hydrolyzed and oxidized to DCF, a fluorescent dye that can be detected by flow cytometry [21]. After A549 cells were treated with 18.0 µM DEFL1 for 12, 24 and 36 h, the intracellular ROS levels were 126.66 ± 4.46%, 151.15 ± 7.09% and 191.04 ± 11.56% of control, respectively ( Figure 3).

Increase of Intracellular ROS Levels in A549 Cells
According to current knowledge, a raise of intracellular ROS levels causes apoptosis [20]. To verify this, generation of ROS was measured with DCFH-DA, which can be hydrolyzed and oxidized to DCF, a fluorescent dye that can be detected by flow cytometry [21]. After A549 cells were treated with 18.0 M DEFL1 for 12, 24 and 36 h, the intracellular ROS levels were 126.66 ± 4.46%, 151.15 ± 7.09% and 191.04 ± 11.56% of control, respectively ( Figure 3).

Reduced Mitochondrial Membrane Potential (ΔΨm) of A549 Cells
It is well known that an increase of ROS levels can cause the loss of ΔΨm, which plays a crucial role in the mitochondrial pathway [21]. Indeed, decreases of ΔΨm in a time-dependant manner were observed ( Figure 4). After treatment with 18.0 μM DEFL1, the levels of ΔΨm were 81.42 ± 4.98%, 49.03 ± 5.33% and 33.37 ± 2.19% of control after 12, 24 and 36 h, respectively.

Release of Cytochrome C
Functional mitochondria depend on the maintenance of mitochondrial membrane potential (ΔΨm), loss of which results in release of cytochrome c [21]. To confirm this, cytosolic cytochrome c was detected by western blot analysis. After A549 cells were treated with 18.0 μM DEFL1 for different time, the release of cytochrome c was determined ( Figure 5). Relative gray values of cytochrome c were 3.94 ± 2.17%, 20.16 ± 4.47%, 102.10 ± 4.90%, 144.09 ± 4.89%, for control, 12, 24, 36 h, respectively.

Reduced Mitochondrial Membrane Potential (∆Ψm) of A549 Cells
It is well known that an increase of ROS levels can cause the loss of ∆Ψm, which plays a crucial role in the mitochondrial pathway [21]. Indeed, decreases of ∆Ψm in a time-dependant manner were observed ( Figure 4). After treatment with 18.0 µM DEFL1, the levels of ∆Ψm were 81.42 ± 4.98%, 49.03 ± 5.33% and 33.37 ± 2.19% of control after 12, 24 and 36 h, respectively.

Reduced Mitochondrial Membrane Potential (ΔΨm) of A549 Cells
It is well known that an increase of ROS levels can cause the loss of ΔΨm, which plays a crucial role in the mitochondrial pathway [21]. Indeed, decreases of ΔΨm in a time-dependant manner were observed ( Figure 4). After treatment with 18.0 μM DEFL1, the levels of ΔΨm were 81.42 ± 4.98%, 49.03 ± 5.33% and 33.37 ± 2.19% of control after 12, 24 and 36 h, respectively.

Release of Cytochrome C
Functional mitochondria depend on the maintenance of mitochondrial membrane potential (ΔΨm), loss of which results in release of cytochrome c [21]. To confirm this, cytosolic cytochrome c was detected by western blot analysis. After A549 cells were treated with 18.0 μM DEFL1 for different time, the release of cytochrome c was determined ( Figure 5). Relative gray values of cytochrome c were 3.94 ± 2.17%, 20.16 ± 4.47%, 102.10 ± 4.90%, 144.09 ± 4.89%, for control, 12, 24, 36 h, respectively.

Release of Cytochrome C
Functional mitochondria depend on the maintenance of mitochondrial membrane potential (∆Ψm), loss of which results in release of cytochrome c [21]. To confirm this, cytosolic cytochrome c was detected by western blot analysis. After A549 cells were treated with 18.0 µM DEFL1 for different time, the release of cytochrome c was determined ( Figure 5). Relative gray values of cytochrome c were 3.94 ± 2.17%, 20.16 ± 4.47%, 102.10 ± 4.90%, 144.09 ± 4.89%, for control, 12, 24, 36 h, respectively.

Discussion
Among different kinds of cancers, lung cancer remains the most common cancer throughout the world, regarding new cases and death for its high rates of morbidity and mortality [24,25]. Furthermore, non-small cell lung cancer (NSCLC) is the most common form of lung cancer [26,27]. Cancer chemotherapy offers effective treatment and developing novel anticancer drugs is a promising strategy to overcome cancer [28,29]. More and more evidence suggests that the success of anti-tumor chemotherapy depends on the discovery and development of novel drugs [23,30].
Despite the advent of combinatorial chemistry which can generate thousands of new chemicals rapidly, natural products remain the key source for developing novel drugs [31][32][33]. The genus Euphorbia provides various types of compounds including sesquiterpenoids, diterpenoids, triterpenoids, steroids, phenolics and flavonoids, among which, diterpenoids are the vital type for the biological activity [15,34]. Considerable attention has been focused on macrocyclic diterpenoids derived from lathyranes with a 5/11/3-membered ring that have exhibited anticancer and MDRreversing activities [35,36].
In this paper we report the isolation, structure identification and apoptosis induction in NSCLC A549 cells of lathyrol-3-phenyl-acetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) showed strong growth inhibition in A549 cells with an IC50 value of 17.51 ± 0.85 μM ( Figure 1B). What's more, DEFL1 suppressed cell growth and migration in experiments of wound healing of A549 cells in a dose-dependent manner (Figure 2). To elucidate the anticancer mechanism, apoptosis induced by DEFL1 was investigated.
Apoptosis is a highly regulated death process deciding homeostasis and development of multicellular organisms [37][38][39]. It is characterized by a series of well-organized processes including activation of the family of cysteinyl aspartate-specific proteinases (caspase), reduction of cell volume, membrane blebbing, internucleosomal DNA cleavage, compaction of cytoplasmic organelles and fragmentation of nuclear chromatin [40][41][42]. What's more, apoptosis is one of the important mechanisms of action of anticancer agents [43,44]. The mitochondrial pathway and death

Discussion
Among different kinds of cancers, lung cancer remains the most common cancer throughout the world, regarding new cases and death for its high rates of morbidity and mortality [24,25]. Furthermore, non-small cell lung cancer (NSCLC) is the most common form of lung cancer [26,27]. Cancer chemotherapy offers effective treatment and developing novel anticancer drugs is a promising strategy to overcome cancer [28,29]. More and more evidence suggests that the success of anti-tumor chemotherapy depends on the discovery and development of novel drugs [23,30].
Despite the advent of combinatorial chemistry which can generate thousands of new chemicals rapidly, natural products remain the key source for developing novel drugs [31][32][33]. The genus Euphorbia provides various types of compounds including sesquiterpenoids, diterpenoids, triterpenoids, steroids, phenolics and flavonoids, among which, diterpenoids are the vital type for the biological activity [15,34]. Considerable attention has been focused on macrocyclic diterpenoids derived from lathyranes with a 5/11/3-membered ring that have exhibited anticancer and MDR-reversing activities [35,36].
In this paper we report the isolation, structure identification and apoptosis induction in NSCLC A549 cells of lathyrol-3-phenyl-acetate-5,15-diacetate (deoxy Euphorbia factor L1, DEFL1) showed strong growth inhibition in A549 cells with an IC 50 value of 17.51 ± 0.85 µM ( Figure 1B). What's more, DEFL1 suppressed cell growth and migration in experiments of wound healing of A549 cells in a dose-dependent manner (Figure 2). To elucidate the anticancer mechanism, apoptosis induced by DEFL1 was investigated.
Apoptosis is a highly regulated death process deciding homeostasis and development of multicellular organisms [37][38][39]. It is characterized by a series of well-organized processes including activation of the family of cysteinyl aspartate-specific proteinases (caspase), reduction of cell volume, membrane blebbing, internucleosomal DNA cleavage, compaction of cytoplasmic organelles and fragmentation of nuclear chromatin [40][41][42]. What's more, apoptosis is one of the important mechanisms of action of anticancer agents [43,44]. The mitochondrial pathway and death receptor pathway are two major apoptosis pathways that depend on different apoptotic stimuli [45,46]. Mitochondria have been suggested to play a critical role in the regulation of apoptosis, which is related to the generation of ROS and the disruption of redox homeostasis [47][48][49]. Indeed, an increase of ROS levels was observed in A549 cells after treatment with DEFL1 ( Figure 3). Mitochondrial dysfunction, including permeability transition, loss of mitochondrial potential (∆Ψm) and release of cytochrome c from mitochondria into cytosol can lead to apoptosis [50,51]. In our research, the loss of ∆Ψm (Figure 4) and release of cytochrome c ( Figure 5) were detected in DEFL1-treated A549 cells.
As far as the mitochondrial pathway is concerned, caspase-9 is activated after cytochrome c is released to cytosol [52,53]. Subsequently, complex formation of cytochrome c, apoptotic protease activation factor 1 (Apaf-1) and procaspase-9 can activate the caspase-9, which brings up the activation of downstream caspases, including casapse-3 [54][55][56]. Consistently, an activity increase of caspase-9 and -3 was recorded ( Figure 6A). Furthermore, Hoechst 33258 staining provided more evidence showing apoptosis of reduction of cell volume and fragmentation of nuclear chromatin ( Figure 6B). Our results implied that DEFL1 induced apoptosis of A549 cells via a mitochondrial pathway and the related mechanisms are summarized in Figure 7.
receptor pathway are two major apoptosis pathways that depend on different apoptotic stimuli [45,46]. Mitochondria have been suggested to play a critical role in the regulation of apoptosis, which is related to the generation of ROS and the disruption of redox homeostasis [47][48][49]. Indeed, an increase of ROS levels was observed in A549 cells after treatment with DEFL1 ( Figure 3). Mitochondrial dysfunction, including permeability transition, loss of mitochondrial potential (ΔΨm) and release of cytochrome c from mitochondria into cytosol can lead to apoptosis [50,51]. In our research, the loss of ΔΨm (Figure 4) and release of cytochrome c ( Figure 5) were detected in DEFL1treated A549 cells.
As far as the mitochondrial pathway is concerned, caspase-9 is activated after cytochrome c is released to cytosol [52,53]. Subsequently, complex formation of cytochrome c, apoptotic protease activation factor 1 (Apaf-1) and procaspase-9 can activate the caspase-9, which brings up the activation of downstream caspases, including casapse-3 [54][55][56]. Consistently, an activity increase of caspase-9 and -3 was recorded ( Figure 6A). Furthermore, Hoechst 33258 staining provided more evidence showing apoptosis of reduction of cell volume and fragmentation of nuclear chromatin ( Figure 6B). Our results implied that DEFL1 induced apoptosis of A549 cells via a mitochondrial pathway and the related mechanisms are summarized in Figure 7.     Table 1.

Cell Lines and Culture
A549, KB and HCT116 cells provided by Prof. Li-Wu Fu (Cancer Center, Sun Yat-sen University) were cultivated in RPMI 1640 medium containing 100 U/mL penicillin, 100 µg/mL streptomycin and 10% FBS in an incubator with a humidified atmosphere of 5% CO 2 at 37 • C [57]. Mycoplasma contamination was determined every two months regularly.

Cell Viability Assay
A549 cells were plated in 96-well plates and allowed to adhere for 24 h. Cells were then incubated with varying concentrations of indicated compounds or fractions. After 68 h, MTT was added to each well and plates were then incubated for another 4 h. After that, formazan crystals were dissolved with 200 µL DMSO and the absorbance at 540 nm was measured by a microplate reader with 655 nm as reference wavelength. Finally, cell survival was calculated as: survival (%) = (mean experimental absorbance/mean control absorbance) × 100% [58].

Wound Healing Assay
To perform the wound healing experiments, A549 cells were seeded in the six-well plates and cells were cultured to reach confluence overnight. Then, the 200 µL pipette tip was applied to scratch the monolayer cell to build the wound healing model. After that, the wounded cell layer was washed to discard loose cells. Medium containing different concentrations of DEFL1 was added to the plates. After indicated time of culture, images were captured. Results of cell growth and motility were examined based on the percentage of the healing area [4].

Measurement of ROS Generation
Briefly, DCF fluorescence intensity is directly related to the amount of ROS produced by the cells. After A549 cells were treated with DEFL1 for 24 h, 5 × 10 5 cells were harvested, washed with ice-cold PBS and incubated with DCFH-DA (50 µM of the final concentration) in the dark at 37 • C for 20 min. Subsequently, cells were washed twice with ice-cold PBS and resuspended in 1 mL PBS. ROS generation quantity was examined based on 10,000 cells each sample by a FACS Caliber flow cytometer (Beckman Coulter, Brea, CA, USA) at 488 nm of the excitation wavelength and 530 nm of the emission wavelength. The data were analyzed by CellQuest software (BD Biosciences Inc., Franklin Lakes, NJ, USA) and expressed as MFI [59].

Determination of Mitochondrial Potential (∆Ψm)
A549 cells at density of 5 × 10 5 cells/mL were treated with 18.0 µM DEFL1 for indicated time to determine ∆Ψm. Thereafter, the cells were collected, centrifuged at 600 g for 5 min and then washed with ice-cold PBS. Subsequently, cells were incubated with 40 nM DiOC6 (3) for 20 min in the dark at 37 • C. After that, stained cells were washed twice with ice-cold PBS once before being resuspened in 1 mL PBS. Finally, the quantity of DiOC6 (3) maintained 10,000 cells each sample was determined by a FACS Caliber flow cytometer (Beckman Coulter, Brea, CA, USA) at 484 nm of excitation wavelength and 501 nm of emission wavelength. The recorded data were analyzed by CellQuest software (BD Biosciences Inc., Franklin Lakes, NJ, USA) and expressed as a mean fluorescence intensity (MFI) [60].

Subcellular Isolation for Western Blot Analysis of Cytosolic Cytochrome c
The indicated extract buffer contained 250 mM sucrose, 20 mM Hepes-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 0.1 mM PMSF and 0.02 mM aprotinin. After treatment with DEFL1, A549 cells were harvested and resuspended in 5-fold volume of ice-cold extract buffer for 40 min at 4 • C. Subsequently, A549 cells were centrifuged at 1200× g for 10 min at 4 • C and the supernatant was then centrifuged at 12,000× g for 15 min at 4 • C to give the final supernatant as cytosolic fraction. Subsequently, the final supernatant after reduplicative centrifugation was collected and dissolved in 5 × loading buffer (250 mM Tris-Cl of pH 6.8, 50% glycerol, 10% sodium dodecyl sulphate, 1.25‰ bromphenol blue and 0.5 M dithiothreitol). After that the samples were heated for 15 min at 100 • C. Then the samples were subjected to western blotting analysis and cytochrome c protein was examined by anti-cytochrome c antibody in the ratio of 1:1000 [59].

Measurement of Caspase-9 and -3 Activities
The activities of caspase-9 and -3 were determined by a caspase colorimetric assay kit following the manufacturer's protocol. To be brief, 1 × 10 6 A549 cells were exposed to 18.0 µM DEFL1 for 12, 24, 36 and 48 h, respectively. Then, cells were collected, washed twice with ice-cold PBS and lysed with the buffer. After that, the lysates were detected for protease activity by the caspase-specific peptide conjugated with color reporter molecule p-nitroanaline. Generally, the caspase enzymatic activities in the given cell lysates were proportional to the color reaction. Finally, the chromophore p-nitroanaline cleaved by caspases was measured by spectrophotometry at 405 nm [20].

Hoechst 33258 Staining
After exposure to DEFL1, both floating and trypsinized adherent A549 cells were collected, washed once with ice-cold PBS and fixed with 1 mL 4% paraformaldehyde for 20 min. Subsequently, cells were washed once with ice-cold PBS. Then the cells were incubated in 1 mL PBS containing 10 µM Hoechst 33258 for 30 min at 37 • C, washed twice. Finally, the results were observed under a fluorescence microscope equipped with standard excitation filters (Leica Dmirb, Wetzlar, Germany) in random microscopic fields at 400 × magnification [54].

Statistical Analysis
Results were analyzed with t-test or one-way ANOVA with the SPSS software (13.0, SPSS Inc., Chicago, IL, USA). Data were expressed as means ± SD of at least triplicate determinations. * p < 0.05 and ** p < 0.01 were indicative of significance.

Conclusions
In summary, DEFL1 inhibits growth and induces apoptosis in A549 cells with involvement of the mitochondrial pathway (Figure 7), characterized by an increase of ROS levels, decrease of mitochondrial potential (∆Ψm), release of cytochrome c, activation of caspase-9 and -3 and observation of typical apoptosis features after Hoechst 33258 staining.