Natural Trienoic Acids as Anticancer Agents: First Stereoselective Synthesis, Cell Cycle Analysis, Induction of Apoptosis, Cell Signaling and Mitochondrial Targeting Studies

Simple Summary Currently, the whole world is acutely concerned with the selection of effective treatment regimens for oncological diseases. This problem is becoming more and more catastrophic every year due to the phenomenon of multidrug resistance, the consequence of which is the loss of the effectiveness of drugs against tumor cells. One of the solutions to the problem described above is the synthesis of new low molecular weight compounds that can effectively affect molecular cellular targets, for example, enzymes of the cell cycle, and, as a consequence, interrupt DNA synthesis, contributing to tumor death. Within the framework of this article, we carried out the Z-stereoselective synthesis of natural unsaturated acids containing a 1Z,5Z,9Z-triene moiety, for which it was shown that they are effective inhibitors of human topoisomerase I, and also affect mitochondria. At the same time, using multiplex analysis, the activation of signaling pathways was studied and a probable mechanism of the antitumor action of the synthesized trienoic acids was proposed. Abstract The first Z-stereoselective method was developed for the synthesis of unsaturated acids containing a 1Z,5Z,9Z-triene moiety in 61–64% yields using the new Ti-catalyzed cross-coupling of oxygen-containing and aliphatic 1,2-dienes as the key synthetic step. It was shown for the first time that trienoic acids with non-methylene-interrupted Z-double bonds show moderate cytotoxic activities against tumor cell lines (Jurkat, K562, U937, HL60, HeLa), human embryonic kidney cells (Hek293), normal fibroblasts and human topoisomerase I (hTop1) inhibitory activity in vitro. The synthesized acids efficiently initiate apoptosis of Jurkat tumor cells, with the cell death mechanism being activated by the mitochondrial pathway. A probable mechanism of topoisomerase I inhibition was also hypothesized on the basis of in silico studies resorting to docking. The activation and inhibition of the most versatile intracellular signaling pathways (CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3 and STAT5 tyrosine kinases) responsible for cell proliferation and for initiation of apoptosis were studied by multiplex assay technology (Luminex xMAP).


Introduction
According to reports and predictions of the World Health Organization, cancer diseases permanently rank first among human fatal diseases and this sad statistic does not tend to improve in the near future (Causes of Death. Available online: https://ourworldindata. org/causes-of-death, accessed on 8 April 2021). The mortality rate caused by malignant neoplasms is also very high; now, the worldwide average is 150 per 100,000 population. By as soon as 2030, the number of people diagnosed with cancer may increase 1.5-fold. About In continuation of these studies, we assumed that this method of synthesis of unsaturated acids could be extended to unique trienoic acids containing a 1Z,5Z,9Z-triene moiety in the molecule, particularly, natural acids that were found in phospholipids of the sea anemone Stoichactis helianthus [29] (Figure 2). The minor content of these acids in natural products and the difficulty of isolation are the key factors that hamper investigations of their biomedical potential.
Therefore, development of stereoselective methods for the preparative synthesis of natural trienoic acids and studies of the biological activities of the products were among the primary goals.

Chemistry
Our study started with the retrosynthetic analysis of the structure of trienoic acids with non-methylene-interrupted Z-double bonds, which demonstrated that the total synthesis can be implemented through Ti-catalyzed cross-cyclomagnesiation of (6Z)-alka-1,2,6-trienes with tetrahydropyran ethers of allene alcohols (Scheme 1). The final step of the synthesis of the desired acids is the Jones oxidation of the trienol tetrahydropyran ether that is formed upon acid hydrolysis of the reaction mixture (Scheme 1).

Scheme 4.
Putative scheme of the predominant formation of cross-cyclomagnesiation products from aliphatic and oxygen-containing allenes.
Thus, the key point is the original (Z,Z,Z)-stereoselective total synthesis of trienoic acids, which contain the 1Z,5Z,9Z-triene system, developed by us, using a new Ti-catalyzed cross-cyclomagnetation 1,2-dienes with readily available Grignard reagents. This method is an important step for the development of new highly effective anticancer drugs.

Cytotioxic Activity In Vitro
Trienoic acids 13a-g, which we synthesized, were evaluated for antitumor activity in vitro using the Jurkat, K562, U937, HL60, HeLa and HEK293 cell lines and normal fibroblasts ( Table 1). The assays included determination of IC 50 by flow cytofluorimetry. The assays showed that trienoic acids 13a-g exhibit a cytotoxic effect against the chosen cell lines, which is comparable with or somewhat higher than the effect of the previously synthesized 5Z,9Z-dienoic acids. Acid 13c had the highest cytotoxicity against all of the cell lines chosen for the assays. The selectivity index (SI) of the therapeutic and cytotoxic action for the Jurkat, K562, U937 and HL60 cell lines was 4-9 times higher than that for normal fibroblasts and conditionally normal human embryonic kidney cells Hek293 (Table 1).

Apoptosis and Cell Cycle Research
In accordance with the results of the cytotoxicity of the studied compounds, we more deeply assessed the ability of compound 13c to induce apoptosis in Jurkat cell culture and to influence the phases of the cell cycle ( Figure 3).  The effect of compound 13c resulted in apoptosis of Jurkat cells and this effect was comparable to that of camptothecin and etoposide, the popular cytostatic agents in medicine. The highest rate of apoptosis in Jurkat cells (71.41%) was the exposure to test compound 13c at a concentration of 0.2 µM and a concentration of 0.025 µM caused the lowest percentage of Jurkat cells in the late apoptosis stage (6.56%) (Figure 3). High activity of compound 13c towards live cells may be attributable to the presence of cis-double bonds in the molecule because, as already known, these compounds show high inhibitory activity against topoisomerase I.
Jurkat cells in the control sample show the balance of all phases of the cell cycle with a predominance of cells in the G0-G1 phase ( Figure 4). The analysis of the cell cycle with the addition of compound 13c to the Jurkat cell culture demonstrates a significant predominance of the population of hypodiploid cells (sub-G0-G1), a dramatic increase in the S-phase and a pronounced decrease in the percentage of G2 + M cells, which corresponds to the standard picture of the effect of classical cytostatics-camptothecin and etoposide. In summary, it can be argued that the compound 13c has antitumor activity against cell T cell leukemia caused by apoptosis-inducing activity.

Topoisomerase I Inhibition Assay and Molecular Docking Studies
The ability of trienoic acids 13a-g ( Table 2) to interact with topoisomerase I in vitro with the formation of relaxation of the supercoiled plasmid pHOT1 was studied ( Figure 5).  The data of this study, showing the dependence of the amount of relaxed DNA on the structure of the obtained trienoic acids, will serve as further chemical modifications that will significantly improve the chemotherapeutic properties of these fatty acids ( Figure 5).
The addition of trienoic acids to the relaxation reaction of supercoiled DNA at a concentration of IC 50 makes it possible to detect a gradual decrease in the formed topoisomers and, as a consequence, to observe an increase in the open circular form of the plasmid, which directly indicates a decrease in the enzyme activity. When no test compound was added, no such effect was observed ( Figure 5, line 6). All seven unsaturated acids we prepared behaved almost identically in the given concentration range; they started to inhibit topoisomerase I when present in concentrations below 0.1-0.8 µM. A high inhibitory activity against human topoisomerase I was found for 5Z,9Z,13Z-docosatrienoic acid 13c in concentrations above 0.1 µM. Thus, we found that even micromolar concentrations of compounds 13a-g are able to suppress the catalytic activity of topoisomerase I.
Molecular dynamics studies of the mechanism of topoisomerase I inhibition by eicosapentaenoic acid (cEPA) showed that the K443, K587 and N722 residues are the preferable binding sites ( Figure 6) [30]. Presumably, the interaction of the carboxylate group of unsaturated carboxylic acids with the above-mentioned sites of Topo I gives a stable complex, which prevents the catalytic center at Y723 from performing a nucleophilic attack on the DNA phosphate. Similar results were obtained by molecular docking for the mechanism of topoisomerase I inhibition by unsaturated cholesterol-containing acids and synthetic analogues of natural 5Z,9Z-dienoic acids [27,31]. It can be assumed that synthetic Z,Z,Ztrienoic acids 13a-g should inhibit human Topo I by similar mechanisms. A truncated hTopI form (70 kDa) complexed with the 22-pair duplex oligonucleotide (PDBID 1A36), from which the duplex oligonucleotide fragment and water molecules were removed, served as the initial model for the molecular docking ( Figure 6). The interaction of synthetic Z,Z,Z-trienoic acids 13a-g with the 1A36 fragment were analyzed using the OPLS3 method (Glide XP). We found that the preferable binding sites for the carboxylate group in the compounds 13a-g are the K443, K587 and N722 residues. The docking score calculated by the OPLS3 (Glide XP) method increases in the following series of compounds: 13b (−7.5) < 13c (−6.8) < 13e (−5.9) < 13g (−5.1) < 13a (−4.6) < 13d (−4.4) < 13f (−2.9), which is generally correlated with the experimentally observed activities of the synthetic Z,Z,Z-trienoic acids.

Studying the Effect of Trienoic Acids on Mitochondria
Mitochondria are extremely important as the main organelles that ensure the energy balance of the cell. Moreover, they regulate a number of apoptotic and proapoptotic proteins, are the main source of ATP and interact with reactive oxygen species (ROS). Today mitochondria are key organelles involved in the pathogenesis of many forms of cancer, inflammatory and neurodegenerative processes [32].
The mitochondrial regulation of apoptosis and permeability of mitochondrial membrane upon variation of the potential difference between the inner and outer membrane parts are closely interrelated, which has been demonstrated by a large number of research groups all over the world. According to Mitchell's theory, the proton electrochemical potential ∆Ψ, arising during the transfer of electrons through the inner membrane, determines the interaction of oxidation and phosphorylation processes, the conjugation of which serves as a source of energy for the formation of adenosine triphosphate [33]. Various changes in ∆Ψ detected by the MitoSense Red dye, in combination with the externalization of phosphatidylserine on the cell membrane surface, is a reliable sign of apoptosis due to mitochondrial damage and the formation of ROS ions. The expression of phosphotidylserine on the membrane is assessed by the binding of annexin V and the permeability of the plasma membrane to the 7AAD dye is a clear indicator of apoptosis in the cell. Multivariate analysis carried out using three dyes-annexin V Alexa fluor 488, 7AAD and MitoSense Red-makes it possible to clearly establish the event of apoptosis depending on damage to the mitochondrial membrane and dysfunction of mitochondria.
The change of potential (∆Ψ) in Jurkat cells after treatment with compound 13c was assessed using the fluorescent cationic dye MitoSense Red, which selectively accumulates only in intact mitochondria of living cells and does not penetrate into dead objects.
As a result of the experiment, it was found that in the samples treated with compound 13c, the number of cells with damaged membrane potential (∆Ψ) increased dramatically (84.18%), while when treated with staurosporin, this number of cells was 58.15% (Figure 7, histograms B and C). Out of all trienoic acids, compound 13c induced the most pronounced decrease in the mitochondrial potential; moreover, this effect from compound 13c was dose-dependent and significantly exceeded the values of cells treated with staurosporin, a well-known inhibitor of protein kinases (Figure 7, histogram B). The percentage of apoptotic cells in the samples treated with compound 13c (0.079 µM) was 48.24% (40.00% early apoptosis and 8.24% late apoptosis, respectively) ( Figure 7, histogram C). The results indicate that compound 13c activates the mitochondrial pathway of apoptosis via disjunction of oxidation and phosphorylation pathways in the mitochondria of Jurkat cells.

Cytochrome C Release from Mitochondria
An important role of mitochondria in apoptosis is associated with the release of the transmembrane protein cytochrome C, which is an activator of procaspase-9 [34]. Cytochrome c is a soluble heme protein that carries electrons in oxidative phosphorylation during respiration and transfers electrons from the cytochrome bc1 complex to cytochrome oxidase on the surface of the inner mitochondrial membrane [35]. Dissipative changes in the transmembrane potential of mitochondria (∆Ψm) indicate the appearance of signs of apoptosis in the cell [36]. Flow cytometry using detection of mitochondrial potential dissipation and cytochrome release into the cytoplasm is the method of choice for this analysis and proves the mitochondrial pathway of apoptosis in cells [37].
A quantitative assessment of the release of cytochrome c from the mitochondria of cells with apoptosis was used to detect the mitochondria-dependent pathway of cell death using flow cytometry. We used labeled antibodies against cytochrome c FITC, control of the isotype anti-IgG1-FITC, as well as optimized fixation, permeabilization and blocking buffer, allowing the detection of cytochrome c by flow cytometry. In the work, buffers were used to achieve selective mitochondrial permeability, while simultaneously leaving the mitochondrial membrane intact. Viable or living cells show higher levels of cytochrome c fluorescence, while apoptotic cells that have released their cytochrome c from the mitochondria into the cytoplasm will show a reduced staining intensity when probed with an anti-cytochrome c antibody FITC.
The detection of cytochrome c-negative cells in samples incubated with the test compound directly attests to the loss of cytochrome c with mitochondria and, as a consequence, to the intrinsic pathway of apoptosis. The histograms depicted in Figure 8

Major Kinases and Their Phosphorylation Status of Nine Signaling Pathways for Cell Growth and Proliferation
Nine major kinases of signaling pathways responsible for cell growth and differentiation were studied in Jurkat tumor cells incubated with novel trienoic acid 13c. The study was carried out using multiplex analysis using Luminex xMAP technology. This technology makes it possible to simultaneously detect in the same experiment a number of protein analytes per unit of time, which significantly reduces the probability of errors associated with sample preparation. This method is based on the use of special polystyrene spheres coated with antibodies with special fluorophores, followed by laser detection and digital processing. In our experiment, the following kinases were analyzed: CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3 and STAT5, more precisely, their phosphorylated and non-phosphorylated forms in cells. Often, in many tumors, it is the activation of these kinases, all at once or only some, that gives the cell immortality, completely excluding the Hayflick limit.
We performed a pairwise comparison of all nine kinases CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3 and STAT5 in an inactive and phosphorylated state in three samples of Jurkat cell lysate (samples containing 0.04 µM and 0.08 µM test compound and control). All of these samples have been tested using Luminex Assay (MILLIPLEX ® MAP 9-Plex Multi-Pathway 9-plex Magnetic Bead Kit) ( Figure 9).
Pairwise comparison of the active and inactive forms of the protein shows that the most significant changes are detected for two signaling pathways, namely Akt and p38 ( Figure 9). Kinases ERK/MAP and Akt are two key families of serine-threonine kinases Ser that are activated by RTK signals and, in turn, activate the kinases p70S6, Msk1, STAT3 (Ser727) and CREB. In addition, the simultaneous activation of FAS receptors or other stressful stimuli generated in response to intracellular signaling can cause the activation of p38, JNK and NF-kB. Figure 9 shows that compound 13c significantly reduces all types of kinase proteins at a concentration of 0.08 µM compared to the control sample. Obviously, a slight increase in the phosphorylated form of p38 is a response to stress exposure of the test compound. Akt kinase is a key enzyme of the PI3K/Akt signaling pathway involved in cell growth and differentiation [38]. In recent years, this kinase has been attributed an important role as a pro-oncogene and a participant in oncogenic cell transformation [39]. In Figure 9, it is plainly seen that the phosphorylated Akt kinase fraction considerably decreases, with this decrease being dose-dependent, in comparison with the concentration of this kinase in the control sample. Akt is an important component of phosphatidylinositol-3-kinase (PI3K) signaling pathway; it has numerous substrates and promotes signal transduction along this pathway. The PI3K and Akt kinases are important targets for the therapeutic action [40]. P38 is a MAPK kinase, that is, a serine threonine protein kinase activated by numerous external stimuli towards signal transduction from the membrane surface to the nucleus. The MAPK signaling pathway kinases are the central components of the Ras/ERK/MAPK cascade responsible for differentiation and cell growth as well. The Ras and Raf proteins are important prognostic markers of tumor diseases and promising targets for the therapy [41].

Chemistry
IR spectra were recorded on Bruker VERTEX 70V using KBr discs over the range of 400-4000 cm −1 . 1 H and 13 C NMR spectra were obtained using a Bruker Ascend 500 spectrometer in CDCl 3 operating at 500 MHz for 1 H and 125 MHz for 13 C and Bruker AVANCE 400 spectrometer in CDCl 3 operating at 400 MHz for 1 H and 100 MHz for 13 C. High resolution mass spectra (HRMS) were measured on a Bruker maXis instrument using electrospray ionization (ESI). In experiments on selective collisional activation (CAD) activation energy was set at maximum abundance of fragment peaks (see figures legend). A syringe injection was used for solutions in MeCN-H 2 O, 50/50 vol.% (flow rate 3 mL/min). Nitrogen was applied as a dry gas; interface temperature was set at 180 • C. Elemental analyses were measured on a 1106 Carlo Erba apparatus. The purity of the synthesized compounds was controlled using TLC on Sorbfil plates; anisic aldehyde in AcOH was used for color development. Column chromatography was carried out on Acrus silica gel (0.060-0.200 mm). All solvents were dried (1,4-dioxane, THF, Et 2 O over Na) and freshly distilled before use.

Cell Culturing
Cell culturing were carried out following the known procedure [42]. Cells (Jurkat, K562, U937, HL60, HeLa, HEK293 and normal fibroblasts) were purchased from HPA Culture Collections (Salisbury, UK) and cultured according to standard mammalian tissue culture protocols and sterile technique.

DNA Topoisomerase I Assay
Inhibition of DNA topoisomerase I tests were carried out following the known procedure [25]. Human topoisomerase I inhibition studies were carried out using commercially available Topoisomerase I Drug Screening Kit (TG-1018-2, Topogen, CO, USA).

Cytotoxicity Assay
Cytotoxicity tests were carried out following the known procedure [42]. Viability (live/dead) assessment was performed by staining cells with 7-AAD (7-Aminoactinomycin D) (Biolegend). After treatment cells were harvested, washed 1-2 times with phosphatebuffered saline (PBS) and centrifuged at 400 g for 5 min. Cell pellets were resuspended in 200 mL of flow cytometry staining buffer (PBS without Ca 2+ and Mg 2+ , 2.5% FBS) and stained with 5 µL of 7-AAD staining solution for 15 min at room temperature in the dark. Samples were acquired on NovoCyteTM 2000 FlowCytometry System (ACEA) equipped with 488 nm argon laser. Detection of 7-AAD emission was collected through a 675/30 nm filter in FL4 channel.

Viability and Apoptosis
Induction of apoptosis tests were carried out following the known procedure [42]. Apoptosis was determined by flow cytometric analysis of Annexin V and 7-aminoactinomycin D staining. After treatment cells during 24 h were harvested, washed 1-2 times with phosphate-buffered saline (PBS) and centrifuged at 400 g for 5 min. Cell pellets were resuspended in 200 µL of flow cytometry staining buffer (PBS without Ca 2+ and Mg 2+ , 2.5% FBS). Then, 200 µL of Guava Nexin reagent (Millipore, Bedford, MA, USA) was added to 5 × 10 5 cells in 200 µL and the cells were incubated with the reagent for 20 min at room temperature in the dark. At the end of incubation, the cells were analyzed on NovoCyteTM2000 FlowCytometry System (ACEA).

Cell Cycle Analysis
Cell cycle analysis was carried out following the known procedure [42]. Guava Cell Cycle Reagent (Millipore) was used. Samples were analyzed on NovoCyteTM 2000Flow-Cytometry System (ACEA).

Mitochondrial Damage
Mitochondrial damage tests were carried out following the known procedure [42]. Cytometric assay (Millipore's FlowCellect™MitoDamage Kit) which allowed multiparametric evaluation of three cell health markers: change in mitochondrial potential (early apoptosis and cellular stress), phosphatidylserine expression on the cell surface (late apoptosis) and membrane permeabilization (cell death) was used.

Histone H2A.X Analysis
Histone H2A.X analysis was carried out following the known procedure [42]. Cell preparation for phosphorylation of Histone H2A.X in Jurkat cells analysis were grown in culture flask at 48 h. The medium was changed at 24 h before drug treatment. Phosphorylation of histone H2A.X was measured with the FlowCellect™ DNA Damage Histone H2A.X Dual Detection Kit (FCCS025153, Millipore, MA, USA).

Cytochrome C Release Analysis
The commercially available Millipore FlowCellect ™ Cytochrome c kit measures the loss of mitochondrial cytochrome c in cells in which apoptosis has been induced. The kit includes a directly labeled anti-cytochrome cFITC antibody, an anti-IgG1-FITC isotype control, as well as buffers for fixation and permeabilization of cells, allowing the detection of cytochrome by flow cytometry. Viable or living cells show higher levels of fluorescence of cytochrome c and the mitochondria themselves, while apoptotic cells that have released their cytochrome c from the mitochondria into the cytoplasm will show a reduced intensity of staining when probed with an anti-cytochrome c FITC antibody. The incubation time of the cells with the test substances was 4 h.

Procedure for Preparation of Alka-3-in-1-ols (4a-e)
To a solution of alk-1-yne (80 mmol) in THF (50 mL) was slowly added ethyl magnesium bromide (3.0 M in THF, 32.0 mL, 96.0 mmol). The mixture was heated under reflux for a further 4 h then cooled over ice. Ethylene oxide (5.85 g, 130 mmol) was decanted into ice cold dry THF (30 mL) and slowly added to the cooled Grignard reagent. A slight exotherm was observed on this addition. The mixture was allowed to warm to room temperature then stirred overnight prior to cooling over ice then hydrolyzed by careful addition of HCl (50 mL 5%). The mixture was extracted with EtOAc (2 × 100 mL), dried over MgSO 4 and evaporated to give 4a-e as a colorless oil.

Synthesis of (Z)-alk-3-en-1-ols (5)
To a stirred mixture of Ni(OAc) 2 × 4H 2 O (10.7 g, 42.7 mmol) in ethanol (86 mL) was added NaBH 4 (1.2 g, 31 mmol) in ethanol (43 mL) at room temperature under hydrogen. The mixture was stirred for 60 min and ethylenediamine (10.32 g, 171.5 mmol) and alkynol 4a-e (43 mmol) were added sequentially. After stirring for 12 h at the same temperature under hydrogen, the reaction mixture was concentrated and diluted with ethyl acetate. The resulting mixture was filtered through a celite pad and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (n-hexane/ethyl acetate 5:1) to furnish 5a-e as a colorless oil.

Conclusions
Compound 13c exhibits a pronounced antitumor effect due to the multi-target effect on the main intracellular targets. The levels of all main kinases responsible for cell growth and proliferation markedly decrease under the action of the synthesized trienoic acid 13c; simultaneously, acid 13c actively inhibits topoisomerase I and induces apoptosis of tumor cells according to the intrinsic mitochondrial pathway. Using molecular docking, we found that the preferable binding sites for the carboxylate group in the acids 13a-g are the K443, K587 and N722 residues. Among the activated signaling pathways, the phosphorylation levels of ERK1/2, Akt, JNK and p38 were characterized by different basic kinetics in response to the action of the test compound, but differed in lower values than in the control sample. Interestingly, STAT-3 and STAT-5 phosphorylation, characterized by a progressive increase over time in untreated cultures, was inhibited in samples containing the test compound. In general, these results are consistent with the results of our earlier studies of higher fatty acids with diene and triene groups, showing the ability to suppress these signaling pathways in various tumor cultures. The synthesized trienoic acids can claim the role of multitarget compounds that are able to simultaneously penetrate through various biological membranes, including mitochondrial, uncouple oxidation and phosphorylation, thereby reducing mitochondrial potential, as well as inhibiting topoisomerase I and affecting the main signaling pathways of cell proliferation.

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