Indole Phytoalexins Research Articles

Anticancer Potential of Indole Phytoalexins and Their Analogues

Indole phytoalexins, found in economically significant Cruciferae family plants, are synthesized in response to pathogen attacks or stress, serving as crucial components of plant defense mechanisms against bacterial and fungal infections. Furthermore, recent research indicates that these compounds hold promise for improving human health, particularly in terms of potential anticancer effects that have been observed in various studies. Since our last comprehensive overview in 2016 focusing on the antiproliferative effects of these substances, brassinin and camalexin have been the most extensively studied. This review analyses the multifaceted pharmacological effects of brassinin and camalexin, highlighting their anticancer potential. In this article, we also provide an overview of the antiproliferative activity of new synthetic analogs of indole phytoalexins, which were synthesized and tested at our university with the aim of enhancing efficacy compared to the parent compound.

Exploring the Antiproliferative and Modulatory Effects of 1-Methoxyisobrassinin on Ovarian Cancer Cells: Insights into Cell Cycle Regulation, Apoptosis, Autophagy, and Its Interactions with NAC.

Ovarian cancer, a highly lethal malignancy among reproductive organ cancers, poses a significant challenge with its high mortality rate, particularly in advanced-stage cases resistant to platinum-based chemotherapy. This study explores the potential therapeutic efficacy of 1-methoxyisobrassinin (MB-591), a derivative of indole phytoalexins found in Cruciferae family plants, on both cisplatin-sensitive (A2780) and cisplatin-resistant ovarian cancer cells (A2780 cis). The findings reveal that MB-591 exhibits an antiproliferative effect on both cell lines, with significantly increased potency against cisplatin-sensitive cells. The substance induces alterations in the distribution of the cell cycle, particularly in the S and G2/M phases, accompanied by changes in key regulatory proteins. Moreover, MB-591 triggers apoptosis in both cell lines, involving caspase-9 cleavage, PARP cleavage induction, and DNA damage, accompanied by the generation of reactive oxygen species (ROS) and mitochondrial dysfunction. Notably, the substance selectively induces autophagy in cisplatin-resistant cells, suggesting potential targeted therapeutic applications. The study further explores the interplay between MB-591 and antioxidant N-acetylcysteine (NAC), in modulating cellular processes. NAC demonstrates a protective effect against MB-591-induced cytotoxicity, affecting cell cycle distribution and apoptosis-related proteins. Additionally, NAC exhibits inhibitory effects on autophagy initiation in cisplatin-resistant cells, suggesting its potential role in overcoming resistance mechanisms.

Indole phytoalexins-derived bis-indoles: Design, synthesis and in vitro antiproliferative evaluation

Natural indole phytoalexins isolated from Brassicaceae plants have been identified as promising anticancer leads. Herein, we designed and synthesized six novel bis-indole series as indole phytoalexins analogues. Our design is based on replacing the SCH3 group in the natural leads by the peripheral indole ring. The key first series — bis indole thioureas, prepared from starting [1-(tert-butoxycarbonyl)indol-3-yl]methyl isothiocyanate and 1-substituted (indol-3-yl)methylamine derivatives, were further transformed by oxidative spirocyclization with chromium oxide, bromospirocyclization protocol or methyl bromoacetate. The in vitro antiproliferation/cytotoxicity assays against human cancer cell lines demonstrated the bis-indole spirocompounds cis-28b and cis-31b were the most active compounds exhibiting a similar extent of effects against HeLa and HCT116 cells (a range of IC50 values between 7.4 and 10.8 μM). At the same time, their activities were higher or comparable to cisplatin. Furthermore, compounds cis-28b and cis-31b displayed good selectivity indexes (SIs) spanning in the range 9.3–13.5. Collective, these results show that reported bis-indoles are promising candidate for cervix and colon cancer treatment and suitable for further investigations.

Design, Synthesis and Antiproliferative Evaluation of Bis-Indole Derivatives with a Phenyl Linker: Focus on Autophagy

This work deals with the study of the synthesis of new bis-indole analogues with a phenyl linker derived from indole phytoalexins. Synthesis of target bis-indole thiourea linked by a phenyl linker was achieved by the reaction of [1-(tert-butoxycarbonyl)indol-3-yl]methyl isothiocyanate with p-phenylenediamine. By replacing the sulfur of the thiocarbonyl group in bis-indole thiourea with oxygen using mesityl nitrile oxide, a bis-indole homodimer with a urea group was obtained. A cyclization protocol utilizing bis-indole thiourea and methyl bromoacetate was applied to synthesize a bis-indole homodimer with a thiazolidin-4-one moiety. Bis-indole homodimers derived from 1-methoxyspirobrassinol methyl ether were prepared by bromospirocyclization methodology. Among the synthesized analogues, compound 49 was selected for further study. To evaluate the mode of the mechanism of action, we used flow cytometry, Western blot, and spectroscopic analyses. Compound 49 significantly inhibited the proliferation of lung cancer cell line A549 with minimal effects on the non-cancer cells. We also demonstrated that compound 49 induced autophagy through the upregulation of Beclin-1, LC3A/B, Atg7 and AMPK and ULK1. Furthermore, chloroquine (CQ; an autophagy inhibitor) in combination with compound 49 decreased cell proliferation and induced G1 cell cycle arrest and apoptosis. Compound 49 also caused GSH depletion and significantly potentiated the antiproliferative effect of cis-platin.

Aminoanalogues of isobrassinin, erucalexin and isocyclobrassinin: Synthesis and evaluation of the antiproliferative and cytotoxic properties

This work deals with a synthetic effort to obtain regioisomeric amino analogues of the natural indole phytoalexins brassinin, 1-methoxyspirobrassinin and cyclobrassinin. In order to improve the antiproliferative effect, our attention was focused on bioisosteric and regioisomeric optimization of the structure of indole phytoalexins. Starting from 1-substituted (indol-2-yl) methylamine derivatives, thioureas were prepared and then transformed by oxidative spirocyclization with chromium dioxide, Hugerschoff ring-closure or methyl bromoacetate. The twenty-three novel compounds were evaluated for in vitro antiproliferative activity against human cancer cell lines. Compounds 20 and 27 were shown to be potent antiproliferative agents with IC50 values in the low micromolar range. Compounds 20 and 27 exhibited better antiproliferative/cytotoxic effect against Jurkat, MCF-7 and HCT116 cells (IC50 < 10 μM) than cisplatin. This work revealed that efficacy against cancer cell lines does not improve depending on the regioisomeric position.

Brassinin Enhances Apoptosis in Hepatic Carcinoma by Inducing Reactive Oxygen Species Production and Suppressing the JAK2/STAT3 Pathway

Plants from the family Brassicaceae produce brassinin (BSN), which is an essential indole phytoalexin. BSN can kill certain types of cancer cells. Using hepatocarcinoma (HCC) cells, we examined the molecular mechanisms of BSN. We found that HCC cell growth was suppressed and apoptosis was induced by BSN via the downregulation of the JAK/STAT3 pathway. The cytoplasmic latent transcription factor STAT3, belonging to the STAT family, acted as both a signal transducer and an activator and was linked to tumor progression and decreased survival. BSN incubation caused HCC cells to produce reactive oxygen species (ROS). By activating caspase-9/-3 and PARP cleavage, Bcl-2 was reduced, and apoptosis was increased. BSN inhibited constitutive STAT3, JAK2, and Src phosphorylation. The JAK/STAT signaling cascade was confirmed by siRNA silencing STAT3 in HCC cells. BSN also suppressed apoptosis by Z-Val-Ala-Asp-Fluoromethylketone (Z-VAD-FMK), an apoptotic inhibitor. N-acetylcysteine (NAC) inhibited the production of ROS and diminished BSN-induced apoptosis. Our findings suggested that BSN has potential as a treatment for cancer.

Camalexin accumulation as a component of plant immunity during interactions with pathogens and beneficial microbes.

This review provides an overview on the role of camalexin in plant immunity taking into account various plant-pathogen and beneficial microbe interactions, regulation mechanisms and the contribution in basal and induced plant resistance. In a hostile environment, plants evolve complex and sophisticated defense mechanisms to counteract invading pathogens and herbivores. Several lines of evidence support the assumption that secondary metabolites like phytoalexins which are synthesized de novo, play an important role in plant defenses and contribute to pathogens' resistance in a wide variety of plant species. Phytoalexins are synthesized and accumulated in plants upon pathogen challenge, root colonization by beneficial microbes, following treatment with chemical elicitors or in response to abiotic stresses. Their protective properties against pathogens have been reported in various plant species as well as their contribution to human health. Phytoalexins are synthesized through activation of particular sets of genes encoding specific pathways. Camalexin (3'-thiazol-2'-yl-indole) is the primary phytoalexin produced by Arabidopsis thaliana after microbial infection or abiotic elicitation and an iconic representative of the indole phytoalexin family. The synthesis of camalexin is an integral part of cruciferous plant defense mechanisms. Although the pathway leading to camalexin has been largely elucidated, the regulatory networks that control the induction of its biosynthetic steps by pathogens with different lifestyles or by beneficial microbes remain mostly unknown. This review thus presents current knowledge regarding camalexin biosynthesis induction during plant-pathogen and beneficial microbe interactions as well as in response to microbial compounds and provides an overview on its regulation and interplay with signaling pathways. The contribution of camalexin to basal and induced plant resistance and its detoxification by some pathogens to overcome host resistance are also discussed.

Camalexin, an indole phytoalexin, inhibits cell proliferation, migration, and mammosphere formation in breast cancer cells via the aryl hydrocarbon receptor.

Breast cancer is the most commonly diagnosed cancer among women worldwide. Despite a variety of drugs available for the treatment of patients with breast cancer, drug resistance remains a significant clinical problem. Therefore, there is an urgent need to develop drugs with new mechanisms of action. Camalexin is the main indole phytoalexin in Arabidopsis thaliana and other crucifers. Camalexin inhibits the proliferation of various cancer cells. However, the mechanism by which camalexin inhibits cell proliferation remains unclear. In this study, we found that camalexin inhibited cell proliferation and migration of breast cancer cell lines. Furthermore, camalexin also suppressed breast cancer stem cell-derived mammosphere formation. We previously reported that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) agonist suppresses mammosphere formation. Several compounds with indole structures are known to act as AhR agonists. Therefore, we hypothesized that the inhibition of mammosphere formation by camalexin may involve AhR activation. We found that camalexin increased the nuclear translocation of AhR, AhR-mediated transcriptional activation, and expression of AhR target genes. In addition, camalexin suppressed mammosphere formation in AhR-expressing breast cancer cells more than in the breast cancer cells that lacked AhR expression. Taken together, the data demonstrate that camalexin is a novel AhR agonist and that the inhibition of cell proliferation, migration, and mammosphere formation by camalexin involves the activation of AhR. Our findings suggest that camalexin, an AhR agonist, may be a novel therapeutic agent for breast cancer.

Synthesis and cytotoxicity evaluation of novel 5-fluorinated indoles

We report herein the synthesis of a small 28-membered library of novel 5-fluorinated indole phytoalexins. Target compounds were designed by a structure-based bioisosterism strategy. The newly prepared compounds were screened in vitro for cytotoxic activity against seven human cancer cell lines. The cytotoxic evaluation revealed that the 2’-(3,4-dichlorophenylamino) analogue of 5-fluorospirobrassinin was the most active against cancer cell lines without causing toxicity to HUVEC cells. Overall, 5-fluoro analogues of indole phytoalexins did not show improved anti-cancer activity compared to the lead compounds. The preliminary structure-activity relationship (SAR) study revealed that placing fluoro substituent at C-5 position of the indole ring is not crucial for inducing cytotoxicity against a cancer cell line.

Responses of the Necrotrophic Fungus Alternaria brassisicola to the Indolic Phytoalexin Brassinin.

Alternaria brassicicola causes black spot disease in Brassicaceae. During host infection, this necrotrophic fungus is exposed to various antimicrobial compounds, such as the phytoalexin brassinin which is produced by many cultivated Brassica species. To investigate the cellular mechanisms by which this compound causes toxicity and the corresponding fungal adaptive strategies, we first analyzed fungal transcriptional responses to short-term exposure to brassinin and then used additional functional approaches. This study supports the hypothesis that indolic phytoalexin primarily targets mitochondrial functions in fungal cells. Indeed, we notably observed that phytoalexin treatment of A. brassicicola disrupted the mitochondrial membrane potential and resulted in a significant and rapid decrease in the oxygen consumption rates. Secondary effects, such as Reactive oxygen species production, changes in lipid and endoplasmic reticulum homeostasis were then found to be induced. Consequently, the fungus has to adapt its metabolism to protect itself against the toxic effects of these molecules, especially via the activation of high osmolarity glycerol and cell wall integrity signaling pathways and by induction of the unfolded protein response.

Design, synthesis, and biological evaluation of novel 5-bromo derivatives of indole phytoalexins

The increasing diversity of small molecule libraries is a major source for the discovery of new drug candidates. In terms of this trend, we report the synthesis of five series 5-bromosubstituted derivatives of indole phytoalexins Types A–E using a straightforward synthetic approach. Novel compounds were screened in vitro for antiproliferative/cytotoxic activity against seven human cancer cell lines by MTT assay. Evaluation of their antiproliferative potency showed that the activity of some analogues was better or comparable to that of cisplatin and at the same time the toxicity of these compounds on 3T3 cells was lower than that of cisplatin. We found that all 5-bromosubstituted analogues of indole phytoalexins Types A–E exhibited lower or approximately the same activities as previously studied corrensponding non-brominated compounds.

Brassinin, a brassica-derived phytochemical, regulates monocyte-to-macrophage differentiation and inflammatory responses in human monocytes and murine macrophages.

The effects and molecular mechanisms of brassinin (BR), an indole phytoalexin from cruciferous vegetables, on monocyte-to-macrophage differentiation and inflammatory responses were investigated in this study. Inflammatory responses from RAW264.7 cells and THP-1 were stimulated by lipopolysaccharide (1µg/ml), and monocyte-to-macrophage differentiation of THP-1 was induced by phorbol myristate acetate (50ng/ml). The production of inflammatory mediators was determined by ELISA, Western blot or real-time PCR. Reactive oxygen species were examined by DCFH-DA assay. Brassinin at 50µm suppressed lipopolysaccharide-induced production of nitric oxide synthase, cyclooxygenase-2, prostaglandin E2 and reactive oxygen species by 90%, 69%, 52% and 41%, respectively, in RAW264.7 cells. In THP-1 cells, BR inhibited phorbol myristate acetate-induced monocyte-to-macrophage differentiation by suppressing cluster of differentiation molecule β and CD36. In addition, BR suppressed translocation of nuclear factor 'kappa-light-chain-enhancer' of activated B cells (NF-κB) into the nucleus. However, BR activated the nuclear factor erythroid-derived 2-like 2 (Nrf2) and its target molecules hemoxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), with an increase in nuclear translocation of Nrf2. Brassinin suppressed monocyte-to-macrophage differentiation and inflammatory responses by differentially regulating Nrf2 and NF-κB signallings.

Reactive oxygen species dosage in Arabidopsis chloroplasts can improve resistance towards Colletotrichum higginsianum by the induction of WRKY33.

Arabidopsis plants overexpressing glycolate oxidase in chloroplasts (GO5) and loss-of-function mutants of the major peroxisomal catalase isoform, cat2-2, produce increased hydrogen peroxide (H2 O2 ) amounts from the respective organelles when subjected to photorespiratory conditions like increased light intensity. Here, we have investigated if and how the signaling processes triggered by H2 O2 production in response to shifts in environmental conditions and the concomitant induction of indole phytoalexin biosynthesis in GO5 affect susceptibility towards the hemibiotrophic fungus Colletotrichum higginsianum. Combining histological, biochemical, and molecular assays, we found that the accumulation of the phytoalexin camalexin was comparable between GO genotypes and cat2-2 in the absence of pathogen. Compared with wild-type, GO5 showed improved resistance after light-shift-mediated production of H2 O2 , whereas cat2-2 became more susceptible and allowed significantly more pathogen entry. Unlike GO5, cat2-2 suffered from severe oxidative stress after light shifts, as indicated by glutathione pool size and oxidation state. We discuss a connection between elevated oxidative stress and dampened induction of salicylic acid mediated defense in cat2-2. Genetic analyses demonstrated that induced resistance of GO5 is dependent on WRKY33, but not on camalexin production. We propose that indole carbonyl nitriles might play a role in defense against C.higginsianum.

Mechanochemical Synthesis and Isomerization of N-Substituted Indole-3-carboxaldehyde Oximes †.

Performing solution-phase oximation reactions with hydroxylamine hydrochloride (NH2OH·HCl) carries significant risk, especially in aqueous solutions. In the present study, four N-substituted indole-3-carboxaldehyde oximes were prepared from the corresponding aldehydes by solvent-free reaction with NH2OH·HCl and a base (NaOH or Na2CO3) using a mechanochemical approach, thus minimizing the possible risk. In all cases, the conversion to oximes was almost complete. The focus of this work is on 1-methoxyindole-3-carboxaldehyde oxime, a key intermediate in the production of indole phytoalexins with useful antimicrobial properties. Under optimized conditions, it was possible to reach almost 95% yield after 20 min of milling. Moreover, for the products containing electron-donating substituents (-CH3, -OCH3), the isomerization from the oxime anti to syn isomer under acidic conditions was discovered. For the 1-methoxy analog, the acidic isomerization of pure isomers in solution resulted in the formation of anti isomer, whereas the prevalence of syn isomer was observed in solid state. From NMR data the syn and anti structures of produced oximes were elucidated. This work shows an interesting and possibly scalable alternative to classical synthesis and underlines environmentally friendly and sustainable character of mechanochemistry.

Liquid chromatographic chiral recognition of phytoalexins on immobilized polysaccharides chiral stationary phases. Unusual temperature behavior

Three immobilized polysaccharide chiral stationary phases, Chiralpak IA, Chiralpak IB and Chiralpak IC, were used for the study of enantioseparation of 36 derivatives of natural indole phytoalexins, in most cases bioactive, including racemic spirobrassinin, 1-methoxyspirobrassinin and 1-methoxyspirobrassinol methyl ether. Almost all analytes were baseline resolved at least on two different polysaccharide columns in normal phase mode. The effects of mobile phase composition, the analyte structure and the column temperature on the retention and enantioseparation were investigated. Evaluation of the corresponding thermodynamic parameters using van´t Hoff plots (ln k versus 1/T) in the temperature range -15 to 50 °C indicated that separations were enthalpy controlled in most cases, but some entropy controlled separations were also observed. Moreover, unusual phenomenon, an increase retention with increasing temperature accompanied with increased resolution was observed on the Chiralpak IC column. The elution order of enantiomers was determined in some cases and reversed elution order was also observed.

Fast enantioseparation of indole phytoalexins in additive free supercritical fluid chromatography

A series of chiral indole phytoalexins with potential anticancer and antimicrobial activity were enantioseparated in supercritical fluid chromatography. Two polysaccharide-based chiral stationary phases composed of tris-(3,5-dimethylphenylcarbamate) derivatives of amylose or cellulose coated on 2.5 μm silica particles were successfully used. The influences of the polysaccharide backbone, co-solvent type and co-solvent content in the mobile phase on retention, enantioselectivity and enantioresolution of indole phytoalexins were investigated. Fast baseline separations were achieved for 26 from 27 tested compounds. Amylose‐based chiral stationary phase provided higher number of baseline resolutions of the indole phytoalexins than the cellulose-based one. However, certain complementary enantioresolution results towards the studied compounds were observed between the investigated columns. The relationship between structure of the indole phytoalexins and their chromatographic behavior in supercritical fluid chromatography was discussed.

The combined treatment of brassinin and imatinib synergistically downregulated the expression of MMP-9 in SW480 colon cancer cells.

In cancer treatment, which is a major cause of mortality today, combination studies with clinically used chemotherapeutics are becoming increasingly important as much as investigating the effects of novel natural compounds. In this context, phytoalexins constitute an important group due to their unique structure. Brassinin is an essential indole phytoalexin and is a biosynthetic precursor for other phytoalexins. The purpose of this study was to evaluate the anticancer effects of brassinin in combination with imatinib in SW480 cells. In the study, it was observed that brassinin-imatinib combination significantly increased cytotoxicity compared with the single treatment of both compounds and inhibited cell cycle at G0/G1 phase. Annexin V binding and fluorescence imaging assays showed that the combination of brasinin-imatinib induces apoptosis in a dose-dependent manner. Furthermore, the effect of brassinin on the activity of MMP-9 in SW480 cells was evaluated for the first time, and it was detected that MMP-9 activity was significantly reduced. The combination of brassinin-imatinib was found to inhibit MMP-9 activity as well as relative MMP-9 gene expression on a higher level compared with control and compounds alone. Our findings have revealed that the combination of brassinin-imatinib synergistically induces cytotoxicity and apoptosis in SW480 cells. The findings on MMP-9 downregulation have also revealed the anti-metastatic potential of treatment.

Indole phytoalexin derivatives induce apoptosis in human colorectal carcinoma cells

Indole phytoalexin derivatives induce apoptosis in human colorectal carcinoma cells

Spirocyclization reactions and antiproliferative activity of indole phytoalexins 1-methoxybrassinin and its 1-substituted derivatives

Spirocyclization reactions and antiproliferative activity of indole phytoalexins 1-methoxybrassinin and its 1-substituted derivatives

Indole phytoalexin derivatives induce mitochondrial-mediated apoptosis in human colorectal carcinoma cells.

AIMTo investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines.METHODSChanges in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and BrdU assay, allowing us to choose the most potent substance. Cell cycle alterations were analyzed using flow cytometric analysis. The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry, as well as acridine orange/propidium iodide (AO/PI) staining. The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis, such as poly (ADP-ribose) polymerase (PARP), caspase-3 and -9, cytochrome c, Bcl-2 family proteins, and also the integrity of the mitochondrial membrane. To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry.RESULTSAmong the ten tested compounds, compound K-453 {(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol} exhibited the most potent activity with IC50 = 32.22 ± 1.14 μmol/L in human colorectal HCT116 cells and was thus selected for further studies. Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G1 DNA content, which is considered as a marker of apoptotic cell death. The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining. The apoptosis was associated with the loss of mitochondrial membrane potential, PARP cleavage, caspase-3 and caspase-9 activation, release of cytochrome c, as well as changes in the levels of Bcl-2 family members. Moreover, flow cytometry showed that compound K-453 stimulates phosphorylation of p38 MAPK but decreases phosphorylation of Akt and Erk 1/2. Activation of p38 MAPK was also confirmed using Western blot analysis. This analysis also revealed down-regulation of NF-κB1 (p50) and RelA (p65) proteins and the loss of their anti-apoptotic activity.CONCLUSIONIn our study compound K-453 exhibited an antiproliferative effect by induction of intrinsic apoptosis as well as modulation of several signaling pathways.