Annotation of the results obtained in 2020
Investigation of the anticancer activity of the sea sponges secondary metabolites. The change in the activity of protein kinases under the action of guanidine alkaloid monanchoxymycalin (MomC) was studied using functional kinomics using PamTechnology® technology, which allows the identification of kinases that are specifically activated or inhibited by a particular drug. As a result, there was an overall increased STK activity in the samples treated with MomC compared to the control group. It should be noted that no significant inhibition of MomC-induced phosphorylation was observed for any of the peptides tested. Also, in the course of the kinome study, the activation of PRKY kinase was predicted. The analysis predicted that kinases p38, JNKs, and ERKs are predominantly activated by MomC in cancer cells. To confirm these results, Western blot analysis of the expression of the active forms of these kinases after different MomC treatment times was performed. It was shown that the dose- and time-dependent activation of JNK1/2 was observed as early as 6 hours after the start of cell treatment. In contrast, no activation of caspase-3 was detected after this treatment period. It should be noted that no changes in the ratio of active to inactive forms of ERK1/2 or p38 kinases were found even after 48 h of treatment. Inhibition of both phosphorylated and total kinase p38 was associated with cytotoxic processes that begin to manifest themselves 12 h after the onset of MomC exposure to cells. To determine the specific effects on prostate cancer cells, MomC and SP600125, a known specific kinase inhibitor JNK1/2, were co-treated. To study this process, the Chow-Talalay method was used. A map of the cytotoxic activity of MomC and SP600125 was obtained individually, as well as in combination with each other. Further analysis of the data revealed the antagonistic effect of SP600125 on the cytotoxic activity of MomC in 22Rv1 cells over the entire concentration range of the studied marine alkaloid. Thus, it was found that JNK1/2 activation was identified as a pro-cytotoxic stimulus important for the implementation of the substance-induced cytotoxic program. Since JNK kinase activation may result from oxidative stress, ROS levels in MomC-treated 22Rv1 cells were examined. Indeed, an increased regulation of ROS was found 6 h after the treatment of cells with the substance. Moreover, pretreatment of cells with the well-known antioxidant N-acetyl-L-cysteine (NaC) significantly suppressed the cytotoxic effect of MomC. Thus, ROS activation makes a significant contribution to the antitumor activity of the studied marine alkaloid. In addition, the release of a number of cytotoxic mitochondrial proteins into the cellular cytoplasm was also found. Such proteins were, for example, cytochrome c and apoptosis induction factor. This fact indicates an increase in the permeability of mitochondrial membranes under the action of MomC, which can be both the cause and the result of increased ROS production. The effect of the PARP inhibitor olaparib on MomC activity was investigated. However, the expected synergistic effect of olaparib was not observed, but on the contrary, a pronounced antagonistic effect was found. In the experiments performed, an increased regulation of cytotoxic ROS and damage to mitochondria under the influence of MomC were observed. In addition, active PARP was required for MomC-induced cytotoxic activity. Thus, the increased ROS production caused not the classical apoptotic cell death mediated by the induction of single-stranded DNA breaks, but rather a specific JNK activation leading to further PARP activation. The results show that MomC achieves its cytotoxic effect in this way. Study of starfish secondary metabolites. Six individual triterpene glycosides were isolated from the starfish Solaster pacificus by chromatography on Polychrome 1 and silica gel, as well as HPLC on reverse phase columns: new pacificusosides A-C and known cucumariosides C1, C2, and A10. The cytotoxic activity of the isolated compounds was investigated in normal embryonic kidney cells HEK 293, colorectal carcinoma cells HT-29, human melanoma cells RPMI-7951 and breast cells MDA-MB-231 using the MTS method. Cucumariosides C1, C2 and A10 have high cytotoxic activity against the tested cell lines. Pacificusoside C also has high cytotoxic activity against HEK 293, HT-29 and MDA-MB-231 cells and moderate against RPMI-7951 cells. Pacificusoside B inhibited cell survival of all cell types tested with an IC50 greater than 20 μM. At the same time, pacificusoside A was non-toxic against all cell types at concentrations up to 40 μM. The effect of the isolated triterpene glycosides mixed with cholesterol on cell viability was analyzed. It was found that the cytotoxic activity of the compounds under study in combination with cholesterol decreased markedly in comparison with the activity of individual triterpene glycosides. The data obtained suggested that the mechanism of the cytotoxic action of triterpene glycosides can be explained by the binding of plasma membranes to cholesterol, which leads to membranolysis and cell death. The effect of triterpene glycosides on the growth and formation of colonies of tumor cells at non-toxic concentrations of 0.1, 0.5 and 1 μM was investigated. Pacificusosides A and B, as well as cucumarioside A10 at a concentration of 1 μM, have moderate inhibitory activity. Pacificusoside C and cucumariosides C1 and C2 almost completely suppressed the growth of colonies of cells HT-29, RPMI-7951, and MDA-MB-231 at concentrations of 0.5 μM and 1 μM. To our knowledge, this is the first evidence that triterpene glycosides in non-toxic concentrations significantly inhibited the formation of human tumor cell colonies. Based on the data obtained, it was hypothesized that the activity of triterpene glycosides depends on the side chain in the triterpene aglycone. The hypothesis that triterpene glycosides have a membranolytic effect and exhibit their cytotoxic effect by binding the components of the plasma membrane of target cells was also confirmed once again. We studied low-polarity compounds obtained from the combined chloroform-metal and ethanol extracts of the starfish Ceramaster patagonicus. In particular, using partition chromatography, silica gel chromatography, and HPLC, seventeen subfractions were obtained, which contained mixtures of ceramides. Based on the chemical shifts of carbon atoms of the terminal methyl groups in the 13C NMR spectra of the isolated ceramides, it was concluded that there are normal, iso- and anti-iso types of fatty acids and a sphingosine base. The absolute stereochemistry of the asymmetric center in 2-(R)-2-hydroxy fatty acids was determined based on the specific rotation value. The absolute stereochemistry of the phytosphingosine base was determined in a similar manner. Also, a series of cerebrosides was isolated from the low-polarity portion of the extract of the starfish Ceramaster patagonicus. The 1H and 13C NMR spectra of these compounds showed signals characteristic of sphingosine-type glucocerebroside containing a 2-hydroxy fatty acid residues. The structures of the isolated compounds were determined in a similar way. Results of studying the anticancer activity of the recombinant actinoporin analog of the sea anemone Heteractis crispa. Comparative analysis of amino acid sequences of known actinoporins and Hct-S3 showed that Hct-S3 combines a high degree of identity (87–89%) with giantoxin-4 from Stichodactyla gigantea, as well as RTX-A from H. crispa and StnI from Stichodactyla helianthus, which have anticancer activity. The recombinant peptide was obtained as part of a fusion protein with glutathione-S-transferase, a cleavage site for enteropeptidase, and a polyhistidine sequence as a product of gene expression in the bacterial system. The target actinoporin yield was 1 mg/L of cell culture. The molecular weight of the peptide according to MALDI-TOF MS data was 19393 Da, which corresponds to the calculated one (19390 Da). The cytotoxic activity of actinoporin was investigated on human intestinal cancer cells HT-29, breast cancer MDA-MB-231, melanoma SK-MEL-28, as well as normal mouse epidermal cells JB6 C141 and human embryonic kidney cells HEK 293 by the MTS method. It was shown that actinoporin exhibited cytotoxic activity against all cell lines. Actinoporin at concentrations of 1, 2, and 4 μM inhibited EGF – induced neoplastic transformation of JB6 C141 cells by 10% ± 5.0, 23% ± 2.5, and 34% ± 0.2, respectively. At the same concentrations, recombinant Hct-S3 reduced the number of HT-29 colonies by 25%±1.8, 33%±0.1, and 47%±0.9; MDA-MB-231 - by 17%±2.4, 20%±2.5 and 37%±1.2; SK-MEL-28 - 18%±1.5, 24%±0.5, 34%±3.6, respectively. Hct-S3 at concentrations of 1, 2, and 4 μM inhibited the migration of HT-29 cells by 33% ±10.2, 50%±7.5, and 99%±6.4, compared with the control group. In addition, the antiproliferative activity of actinoporin was tested. It was shown that at concentrations of 1, 2, and 4 μM Hct-S3 weakly reduced the proliferation rate of HT-29 cells after 24 and 48 h, whereas after 96 h of treatment with the polypeptide, cell proliferation was reduced by 11%±3.0, 26%±1.2, and 31%±5.0, respectively, which indicates a moderate antiproliferative activity of actinoporin. To identify the putative mechanism of the anti-migration activity of Hct-S3, the effect of the polypeptide on the expression level of matrix metalloproteinases (MMP)-2 and -9 was assessed. Actinoporin at a concentration of 2 μM effectively inhibited the expression of both MMP-2 and MMP-9. Moreover, the effect of Hct-S3 on the activation of caspase-3, poly (ADP-ribose) polymerase (PARP), as well as the expression level of the factors Bcl-2 and Bax was tested. Actinoporin triggered the cleavage of caspase-3 and PARP, which indicates the initiation of the apoptosis mechanism, as well as activation of the expression of the antiapoptotic factor Bax and suppression of the expression of the proapoptotic factor. Thus, recombinant Hct-S3 reduces the migration of HT-29 intestinal cancer cells, probably by inhibiting MMP-2 and MMP-9, and triggers the process of apoptosis by activating caspase-3. Thus, it has been shown that H. crispa actinoporin has antitumor activity against intestinal cancer cells, breast cancer, and melanoma. It was found that Hct-S3 suppresses the migration of intestinal cancer cells by inhibiting the expression of MMP-2 and MMP-9, triggers the apoptosis mechanism through the activation of caspase-3 and PARP, and also regulates the expression of apoptotic proteins Bcl-2 and Bax. These results indicate the high potential of actinoporin as an antitumor compound.

 

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Annotation of the results obtained in 2018
Using TLC, 14 ethanol extracts of sea sponges were analyzed. One of the most interesting specimens of the sea sponge of the genus Topsentia was selected. A series of polysulfated steroid compounds was isolated from a concentrated ethanol extract of sea sponge of the genus Topsentia by chromatography on various sorbents. The structure of isolated compounds was analyzed by 1H and 13C NMR spectroscopy and mass spectrometry. Twelve ethanol extracts of starfishes were analyzed by TLC. Three species of starfishes Anthenea aspera, Ceramaster patagonicus, and Solaster pacificus were chosen for further investigations. As a result of chromatographic separation three new steroidal bi-glycosides were identified, called V-X anthenosides and seven previously known compounds: the anthenosides E, G, S1, S4 and S6 and a mixture of anthenosides epimers J and K. The structures of the new compounds were established by various one- and two-dimensional NMR methods, as well as by mass spectrometry, including high-resolution mass spectrometry. Anthenoside V contains a rare 5α-cholest-8 (14) -en-3α, 7β, 16α-hydroxysteroid core. Anthenosides W and X were isolated as an inseparable mixture of epimers. A mixture of anthenosides J and K have moderate cytotoxic activity against cell lines RPMI-7951, T-47D and HT-29. It was shown that the isolated compounds in a non-cytotoxic concentration of 40 μM inhibit colony formation and growth of RPMI-7951, T-47D and HT-29 cells to a variable degree. Anthenosides J and K had inhibitory activity against all tested cell lines. These glycosides reduced the number of colonies of RPMI-7951, T-47D and HT-29 cancer cells by 64, 55 and 83%, respectively, compared to untreated cells. Anthenosides J and K have been shown to suppress the expression of the anti-apoptotic protein Bcl-XL and increase the expression of the pro-apoptotic proteins Bax and Bak, which lead to the activation of initiating caspase-9. Activated caspase-9, in turn, induced overexpression of effector caspase-3 in a dose-dependent manner. As a result, a mixture of the studied anthenosides J and K at 40 μM causes proteolytic cleavage of caspase-3 and induced apoptosis of colorectal carcinoma cells. Thus, a mixture of antenosides J and K significantly inhibits the formation of colonies of melanoma cells, breast cancer and colorectal carcinoma and induces apoptosis of colorectal carcinoma cells through mitochondrial signaling pathway. The study of two other starfish species has been started: Ceramaster patagonicus and Solaster pacificus. As a result of chromatographic separation, total fractions of ceramides (28.5 g) and cerebrosides (23.0 g) were obtained. Chromatographic separation of glycoside fractions from the starfish Solaster pacificus has also been begun. Recombinant analogues of actinoporins Hct-A2 and Hct-S3 of anemones Heteractis crispa were obtained using the methods of molecular cloning and metal-affinity chromatography. Expression constructs containing a nucleotide sequence encoding actinoporin were created on the basis of pET-41a (+), intended for the expression of fusion proteins with a carrier protein, glutathione-S-transferase (GST) in a bacterial system. The target genes were inserted into the vector by the restriction endonuclease sites PshAI and HindIII. Recombinant plasmids pET41a / hct-a2 and pET41a / hct-s2 were isolated, sequenced and used to transform E. coli Rosetta (DE3) strain cells by electroporation. Next, an expression procedure was carried out that allowed the preparation of recombinant actinoporins in the form of chimeric proteins, including GST, a polyhistidine site and an enteropeptidase cleavage site (DDDDK). During the expression, optimal conditions were selected to increase the yield of chimeric protein. The molecular mass of the hybrid protein is ~ 50 kDa, which corresponds to the calculated data (~ 52 kDa). The selection of recombinant actinoporins, rHct-A2 and rHct-S3, is carried out by metal-affinity chromatography on Ni-NTA-agarose under native conditions. The overall yield of rHct-A2 and rHct-S3 was 2.3 mg and 3.0 mg from 1 liter of cell culture, respectively. As a result, 6 mg of rHct-A2 and 8 mg of rHct-S3 were obtained. The molecular masses of actinoporins, by the MALDI-VP MS method, are 19172 Yes for rHct-A2 and 19573 Yes for rHct-S3. The hemolytic activity of rHct-A2 is 2.3 × 103 GU / mg. The cytotoxic activity of recombinant actinoporin, rHct-A2, was studied. It has been shown that the inhibiting concentrations (IC50) of rHct-A2 which caused death of 50% of colon cancer cells (HT-29), breast cancer (MDA-MB-231) and melanoma (SK-MEL-28) were 34, 46 and 63 μM, respectively. Recombinant actinoporin, rHct-A2 inhibited colony formation of HT-29 cells by 47% at concentration of 20 µM

 

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Annotation of the results obtained in 2019
Seven new unusual polysulfated steroids were isolated from the sea sponge Halichondria vansoesti. A scheme of hypothetical biosynthesis pathways of unusual side chains of trisulfated steroids has been proposed. Some of the metabolites studied showed the ability to inhibit the expression of the prostatic specific antigen (PSA) in drug-resistant 22Rv1 cells in low micromolar concentrations. Thus, a decrease in the expression level may lead to suppression of the androgen receptor signal. Also, a number of isolated steroids showed the ability to inhibit glucose uptake by tumor cells. This study is the first report on the ability of marine steroid compounds to inhibit PSA/androgen receptor signaling, as well as glucose uptake by human tumor cells. The mechanism of the anticancer effect of the new guanidine alkaloid urupocidin C (Ur-C), previously isolated from the deep sea sponge Monanchora pulchra, was investigated. We have shown that in tumor cells of the prostate cell, Ur-C is capable of causing cell cycle arrest in the G1 and S phases, as well as inducing caspase-3-mediated cell death. Initially, mitochondrial targeting was defined as the central mechanism of anticancer actions for these molecules. Thus, treatment with isolated alkaloids leads to permeabilization of mitochondrial membranes, resulting in the release of cytotoxic mitochondrial proteins into the cell cytoplasm, activation of reactive oxygen species (ROS), subsequent activation of caspase-9 and -3, PARP cleavage, DNA synthesis and apoptosis. In addition, with the combined use of Ur-C with a clinically approved PARP-olaparib inhibitor, synergistic effects were observed. Finally, this alkaloid exhibits an additive effect in combination with docetaxel and enzalutamide (an androgen receptor inhibitor used in chemotherapy for prostate cancer). In addition, the anticancer activity and mechanism of action of the new marine pentacyclic guanidine alkaloid monanoxymicalaline C isolated from the same sea sponge species have also been studied in human prostate cancer cells 22Rv1. As a result, it was shown that treatment of cells with monanoxyomicalin leads to the externalization of phosphatidylserine, activation of caspase-3, as well as cleavage of PARP. The expression of the anti-apoptotic survivin protein was reduced. Further experiments on the inhibition of caspase showed that, under the influence of substances in the cells, non-apoptotic cell flexibility occurs, which is also independent of caspase. Screening for kinase activity has revealed that activation of the mitogen-activated protein kinase (MAPK) of the c-Jun N-terminal protein kinase (JNK1/2) is the primary molecular effect of monanhoxymicalin C in prostate cancer cells. Four new conjugates, long chain fatty acid esters of polyhydroxysteroids, were isolated from the deep-sea Far Eastern starfish Ceramaster patagonicus. The structures of the isolated compounds were established by IR, 1D and 2D NMR spectroscopy and mass spectrometry, as well as by chemical transformations. The cytotoxic and antiproliferative activities of the isolated compounds were studied on normal JB6 Cl41 epidermal mouse cells, MDA-MB-231 human breast cancer cells and HCT 116 colorectal carcinoma. It was found that the test compounds were non-toxic to test cells at concentrations up to 100 μM. In addition, we determined the cytostatic (antiproliferative) effect of the isolated compounds on JB6 Cl41, MDA-MB-231 and HCT 116 cells in non-toxic concentrations. It was shown that treatment of all tested cell lines with these compounds at a concentration of 20 μM led to a slight inhibition of cell proliferation even after 72 hours of treatment. In the present study, it was found that the isolated compounds inhibited the formation of colonies of MDA-MB-231 tumor cells and HCT 116. In addition, we examined the ability of these compounds to inhibit the migration of MDA-MB-231 breast cancer cells and HCT 116 human colorectal carcinoma cells. with high invasive potential. It was demonstrated that two compounds (at a concentration of 20 μM) are able to prevent the migration of MDA-MB-231 cells by 42% and 50%, respectively, compared with the control after 48 hours of incubation. The other two compounds had moderate antimetastatic activity against MDA-MB-231 cells. On the other hand, one of the compounds almost completely prevented the migration of HCT 116 cells. In addition, we studied other low-polar compounds obtained from the starfish Ceramaster patagonicus. As a result of chromatographic separation, individual ceramides and cerebrosides were obtained. Eight new compounds, pacificusosides A-H, were isolated from the starfish Solaster pacificus, and one compound was identified as the previously known triterpene glycoside cucumarioside D. The structures of the new compounds were determined using two-dimensional NMR spectroscopy as well as high resolution ESI mass spectrometry and ESI MS/MS. We investigated the cytotoxic and cancer-preventive activities of the isolated compounds with respect to normal epidermal mouse cells JB6 Cl41. It was shown that the studied substances suppress cell viability in low micromolar concentrations. It was found that a number of compounds at a non-toxic concentration of 0.5 μM inhibited TPA- and EGF-induced neoplastic transformation of JB6 Cl41 cells. It should be noted that the data on biological activity are in good agreement with the structural features of the isolated compounds. Thus, the most active substances contain the same pentasaccharide carbohydrate chain with terminal 3-O-Me glucose and a similar triterpene aglycon, differing from each other only in the configuration of the double bond in the side chain of the aglycon. At the same time, the compounds that showed the least activity also have a common structural feature - a shortened tetrasaccharide carbohydrate chain in which the terminal monosaccharide residue does not have a C-3 methyl group. In all likelihood, the presence of such a labeled carbohydrate fragment, as well as the length of the carbohydrate chain, play an important role in the manifestation of the antitumor properties of the isolated compounds. Recombinant analogues of actinoporins Hct-A2 and Hct-S3 of anemone Heteractis crispa were obtained according to the previously described scheme. The hemolytic activity of rHct-S3 on human erythrocytes was 3.58∙103 GE/mg, which is comparable with the activity of rHct-A2. rHct-S3 was investigated in an in vitro model of EGF-induced neoplastic transformation of mouse JB6 Cl41 epidermal cells in soft agar. It was found that rHct-S3 at a concentration of 20 μM inhibited the neoplastic transformation of JB6 Cl41 cells induced by EGF by 25% compared with transformed cells.

 

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