Cucurbitacin C

Identification of seven undescribed cucurbitacins in Cucumis sativus (cucumber) and their cytotoxic activity.[Pubmed:35182783]

Phytochemistry. 2022 May;197:113123.

Cucurbitacin C-type cucurbitacins that are only identified in Cucumis sativus (cucumber) are, in part, responsible for the health benefits and bitter flavor. Nevertheless, detailed information about those functional ingredients in cucumber is scarce. In this study, ten Cucurbitacin C analogues including seven undescribed ones have been isolated from the bitter leaves of cucumber, in which six compounds showed growth inhibition capabilities against tumor cell lines HepG2, A549, DU145 and HCT116. Intriguingly, Cucurbitacin C6 and C7 exhibited a significant inhibition effect compared to the positive control taxol (IC50 = 1.86 +/- 0.17 muM) on HepG2 cell line with IC50 values of 10.06 +/- 0.34 muM and 4.16 +/- 0.42 muM, respectively. The mechanism of cucurbitacin-induced apoptosis is likely down-regulating the expression of caspase-related proteins. This work enlarges the knowledge of the cucurbitacins in cucumber and highlights the importance of cucumber as a source of specialized metabolites in the food and medicinal industries.

Transcriptional and metabolite analysis reveal a shift in direct and indirect defences in response to spider-mite infestation in cucumber (Cucumis sativus).[Pubmed:32306368]

Plant Mol Biol. 2020 Jul;103(4-5):489-505.

KEY MESSAGE: Cucumber plants adapt their transcriptome and metabolome as result of spider mite infestation with opposite consequences for direct and indirect defences in two genotypes. Plants respond to arthropod attack with the rearrangement of their transcriptome which lead to subsequent phenotypic changes in the plants' metabolome. Here, we analysed transcriptomic and metabolite responses of two cucumber (Cucumis sativus) genotypes to chelicerate spider mites (Tetranychus urticae) during the first 3 days of infestation. Genes associated with the metabolism of jasmonates, phenylpropanoids, terpenoids and L-phenylalanine were most strongly upregulated. Also, genes involved in the biosynthesis of precursors for indirect defence-related terpenoids were upregulated while those involved in the biosynthesis of direct defence-related Cucurbitacin C were downregulated. Consistent with the observed transcriptional changes, terpenoid emission increased and Cucurbitacin C content decreased during early spider-mite herbivory. To further study the regulatory network that underlies induced defence to spider mites, differentially expressed genes that encode transcription factors (TFs) were analysed. Correlation analysis of the expression of TF genes with metabolism-associated genes resulted in putative identification of regulators of herbivore-induced terpenoid, green-leaf volatiles and cucurbitacin biosynthesis. Our data provide a global image of the transcriptional changes in cucumber leaves in response to spider-mite herbivory and that of metabolites that are potentially involved in the regulation of induced direct and indirect defences against spider-mite herbivory.

In Vitro and In Vivo Antitumor Activity of Cucurbitacin C, a Novel Natural Product From Cucumber.[Pubmed:31780930]

Front Pharmacol. 2019 Nov 8;10:1287.

Cucurbitacin C (CuC), a novel analogue of triterpenoids cucurbitacins, confers a bitter taste in cucumber. Genes and signaling pathways responsive for biosynthesis of CuC have been identified in the recent years. In the present study, we explored the anti-cancer effects of CuC against human cancers in vitro and in vivo. CuC inhibited proliferation and clonogenic potential of multiple cancer cells in a dose-dependent manner. Low-dose CuC treatment induced cell cycle arrest at G1 or G2/M stage in different cancer lines, whereas high-dose treatment of CuC caused apoptosis in cancer cells. PI3K-Akt signaling pathway was found to be one of the major pathways involved in CuC-induced cell growth arrest and apoptosis by RNA-Seq and Western blotting. Mechanistic dissection further confirmed that CuC effectively inhibited the Akt signaling by inhibition of Akt phosphorylation at Ser473. In vivo CuC treatment (0.1 mg/kg body weight) effectively inhibited growth of cancer cell-derived xenograft tumors in athymic nude mice and caused significant apoptosis. Our findings for the first time demonstrated the potential therapeutic significance of CuC against human cancers.

Metabolomic analysis of the occurrence of bitter fruits on grafted oriental melon plants.[Pubmed:31600335]

PLoS One. 2019 Oct 10;14(10):e0223707.

Grafting has been widely applied to melon (Cucumis melo L.) production to alleviate obstacles of continuous cropping and control soil-borne diseases. However, grafting often leads to a decline of fruit quality. For example, sometimes bitter fruits are produced on grafted plants. However, the underlying physiological mechanism still remains unclear. This study investigated the effects of different rootstocks on the taste of fruits of the Balengcui, an oriental melon cultivar, during summer production. The results showed that all grafted plants with Cucurbita maxima Duch. rootstocks produced bitter fruits, while non-grafted plants and plants grafted onto muskmelon rootstocks produced no bitter fruits. Liquid chromatography-mass spectrometry and metabonomic analysis were performed to investigate the mechanism underlying the occurrence of bitter fruits. Metabolite comparisons of fruits from plants grafted onto Ribenxuesong rootstocks both with non-grafted plants and plants grafted onto muskmelon rootstocks showed that 17 metabolites including phospholipids, cucurbitacins and flavonoids, exhibited changes. The three Cucurbitacins, Cucurbitacin O, Cucurbitacin C, and Cucurbitacin S, increased dramatically. The 10 phospholipids PS(18:1(9Z)/18:2(9Z,12Z)), PS(P-18:0/15:0), PA(18:1(11Z)/18:1(11Z)), PE(16:0/18:0), PS(O-16:0/17:2(9Z,12Z)), PI(16:0/18:2(9Z,12Z)), PA(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PS(P-16:0/17:2(9Z,12Z)), PS(22:0/22:1(11Z)), and PA(17:1(9Z)/0:0)) were significantly decreased, while two PA (16:0/18:2 (9Z, 12Z) and 16:0/18:1 (11Z)), two flavonoids (pelargonidin 3-(6''-malonylglucoside)-5-glucoside and malvidin 3-rutinoside) significantly increased in fruits of plants grafted onto Cucurbita maxima Duch. rootstocks. These metabolites were involved in the glycerophospholipid metabolic pathway, the mevalonate pathway, and the phenylpropanoid pathway. In summary, these results showed that the bitter fruits of grafted Balengcui were caused by Cucurbita maxima Duch. rootstocks. Phospholipids, cucurbitacins, and flavonoids were the key contributors for the occurrence of bitter fruits in Balengcui melon after grafting onto Cucurbita maxima Duch. rootstocks.

A structural and data-driven approach to engineering a plant cytochrome P450 enzyme.[Pubmed:31119558]

Sci China Life Sci. 2019 Jul;62(7):873-882.

Functional manipulation of biosynthetic enzymes such as cytochrome P450s (or P450s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450s. However, the functional redesign of P450s through directed evolution (DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450 (CYP87D20) from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87D20, which is involved in the Cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450s.

The role of H2S in low temperature-induced cucurbitacin C increases in cucumber.[Pubmed:30707394]

Plant Mol Biol. 2019 Apr;99(6):535-544.

KEY MESSAGE: In this study, we first linked the signal molecule H2S with Cucurbitacin C, which can cause the bitter taste of cucumber leaves and fruit, and specifically discuss its molecular mechanism. Cucurbitacin C (CuC), a triterpenoid secondary metabolite, enhances the resistance of cucumber plants to pathogenic bacteria and insect herbivores, but results in bitter-tasting fruits. CuC can be induced in some varieties of cucumber on exposure to plant stressors. The gasotransmitter hydrogen sulfide (H2S) participates in multiple physiological processes relating to plant stress resistance. This study focused on the effect of H2S on low temperature-induced CuC synthesis in cucumber. The results showed that treatment of cucumber leaves at 4 degrees C for 12 h enhanced the content and production rate of H2S and increased the expression of genes encoding enzymes involved in H2S generation, Csa2G034800.1 (CsaLCD), Csa1G574800.1 (CsaDES1), and Csa1G574810.1 (CsaDES2). In addition, treatment at 4 degrees C or with exogenous H2S upregulated the expression of CuC synthetase-encoding genes and the resulting CuC content in cucumber leaves, whereas pretreatment with hypotaurine (HT, a H2S scavenger) before treatment at 4 degrees C offset these effects. In vitro, H2S could increase the S-sulfhydration level of His-Csa5G156220 and His-Csa5G157230 (both bHLH transcription factors), as well as their binding activity to the promoter of Csa6G088690, which encodes the key synthetase for CuC generation. H2S pretreatment enhanced the cucumber leaves resistance to the Phytophthora melonis. Together, these results demonstrated that H2S acts as a positive regulator of CuC synthesis as a result of the modification of proteins by S-sulfhydration, also providing indirect evidence for the role of H2S in improving the resistance of plants to abiotic stresses and biotic stresses by regulating the synthesis of secondary metabolites.

Conspecific and Heterospecific Aboveground Herbivory Both Reduce Preference by a Belowground Herbivore.[Pubmed:26313185]

Environ Entomol. 2015 Apr;44(2):317-24.

Insect herbivores damage plants both above- and belowground, and interactions in each realm can influence the other via shared hosts. While effects of leaf damage on aboveground interactions have been well-documented, studies examining leaf damage effects on belowground interactions are limited, and mechanisms for these indirect interactions are poorly understood. We examined how leaf herbivory affects preference of root-feeding larvae [Acalymma vittatum F. (Coleoptera: Chrysomelidae)] in cucumber (Cucumis sativus L.). We manipulated leaf herbivory using conspecific adult A. vittatum and heterospecific larval Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) herbivores in the greenhouse and the conspecific only in the field, allowing larvae to choose between roots of damaged and undamaged plants. We also examined whether leaf herbivory induced changes in defensive Cucurbitacin C in leaves and roots. We hypothesized that induced changes in roots would deter larvae, and that effects would be stronger for damage by conspecifics than the unrelated caterpillar because the aboveground damage could be a cue to plants indicating future root damage by the same species. In both the greenhouse and field, plants with damaged leaves recruited significantly fewer larvae to their roots than undamaged plants. Effects of conspecific and heterospecific damage did not differ. Leaf damage did not induce changes in leaf or root Cucurbitacin C, but did reduce root biomass. While past work has suggested that systemic induction by aboveground herbivory increases resistance in roots, our results suggest that decreased preference by belowground herbivores in this system may be because of reduced root growth.

Plant science. Biosynthesis, regulation, and domestication of bitterness in cucumber.[Pubmed:25430763]

Science. 2014 Nov 28;346(6213):1084-8.

Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of Cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors Bl (Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors.

23,24-Dihydrocucurbitacin C: a new compound regarded as the next metabolite of cucurbitacin C.[Pubmed:24896808]

Nat Prod Res. 2014;28(15):1165-70.

Cucurbitacin C, a bitter substance in Cucumis sativus L., was isolated from green leaves by using phytochemical methods. An analytical method using high-performance liquid chromatography (HPLC) was established for the quantification of Cucurbitacin C in different parts of the cucumber plant at different growth periods. Cucurbitacin C was detected in the leaves and stems but not in the female flowers, fruits, roots and leafstalks. The level of Cucurbitacin C decreased significantly with the process of young leaves turning old. A new compound named 23,24-dihydroCucurbitacin C, regarded as the next metabolite of Cucurbitacin C, was determined unambiguously by HPLC-quadrupole-time-of-flight mass spectrometry and nuclear magnetic resonance.

Linking agricultural practices, mycorrhizal fungi, and traits mediating plant-insect interactions.[Pubmed:24261037]

Ecol Appl. 2013 Oct;23(7):1519-30.

Agricultural management has profound effects on soil communities. Activities such as fertilizer inputs can modify the composition of arbuscular mycorrhizal fungi (AMF) communities, which form important symbioses with the roots of most crop plants. Intensive conventional agricultural management may select for less mutualistic AMF with reduced benefits to host plants compared to organic management, but these differences are poorly understood. AMF are generally evaluated based on their direct growth effects on plants. However, mycorrhizal colonization also may alter plant traits such as tissue nutrients, defensive chemistry, or floral traits, which mediate important plant-insect interactions like herbivory and pollination. To determine the effect of AMF from different farming practices on plant performance and traits that putatively mediate species interactions, we performed a greenhouse study by inoculating Cucumis sativus (cucumber, Cucurbitaceae) with AMF from conventional farms, organic farms, and a commercial AMF inoculum. We measured growth and a suite of plant traits hypothesized to be important predictors of herbivore resistance and pollinator attraction. Several leaf and root traits and flower production were significantly affected by AMF inoculum. Both conventional and organic AMF reduced leaf P content but increased Na content compared to control and commercial AMF. Leaf defenses were unaffected by AMF treatments, but conventional AMF increased root Cucurbitacin C, the primary defensive chemical of C. sativus, compared to organic AMF. These effects may have important consequences for herbivore preference and population dynamics. AMF from both organic and conventional farms decreased flower production relative to commercial and control treatments, which may reduce pollinator attraction and plant reproduction. AMF from both farm types also reduced seed germination, but effects on plant growth were limited. Our results suggest that studies only considering AMF effects on growth may overlook changes in plant traits that have the potential to influence interactions, and hence yield, on farms. Given the effects of AMF on plant traits documented here, and the great importance of both herbivores and pollinators to wild and cultivated plants, we advocate for comprehensive assessments of mycorrhizal effects in complex community contexts, with the aim of incorporating multispecies interactions both above and below the soil surface.