Veratrum Californicum Research Articles

Integration of induction, system optimization and genetic transformation in Veratrum californicum var. vitro cultures to enhance the production of cyclopamine and veratramine

Cyclopamine, a compound found in wild Veratrum has shown promising potential as a lead anti-cancer drug by effectively blocking cancer signaling pathways. However, its complex chemical structure poses challenges for artificial synthesis, thus limiting its supply and downstream drug production. This study comprehensively utilizes induction, system optimization, and transgenic technologies to establish an efficient suspension culture system for the high-yield production of cyclopamine and its precursor, veratramine. Experimental results demonstrate that methyl jasmonate (MeJA) effectively promotes the content of veratramine and cyclopamine in Veratrum californicum var. callus tissue, while yeast extract (YE) addition significantly increases cell biomass. The total content of veratramine and cyclopamine reached 0.0638 mg after synergistic treatment of suspension system with these two elicitors. And the content of the two substances was further increased to 0.0827 mg after the optimization by response surface methodology. Subsequently, a genetic transformation system for V. californicum callus was established and a crucial enzyme gene VnOSC1, involved in the steroidal alkaloid biosynthesis pathway, was screened and identified for genetic transformation. Combined suspension culture and synergistic induction system, the total content of the two substances in transgenic suspension system was further increased to 0.1228 mg, representing a 276.69% improvement compared to the initial culture system. This study proposes a complete and effective genetic transformation and cultivation scheme for V. californicum tissue cells, achieving milligram-level production of the anticancer agent cyclopamine and its direct precursor veratramine for the first time. It provides a theoretical basis for the industrial-scale production of these substances.

Efficient production of 22(R)-hydroxycholesterol via combination optimization of Saccharomyces cerevisiae.

22(R)-hydroxycholesterol (22(R)-HCHO) is a crucial precursor of steroids biosynthesis with various biological functions. However, the production of 22(R)-HCHO is expensive and unsustainable due to chemical synthesis and extraction from plants or animals. This study aimed to construct a microbial cell factory to efficiently produce 22(R)-HCHO through systems metabolic engineering. First, we tested 7-dehydrocholesterol reductase (Dhcr7s) and cholesterol C22-hydroxylases from different sources in Saccharomyces cerevisiae, and the titer of 22(R)-HCHO reached 128.30mg L-1 in the engineered strain expressing Dhcr7 from Columba livia (ClDhcr7) and cholesterol 22-hydroxylase from Veratrum californicum (VcCyp90b27). Subsequently, the 22(R)-HCHO titer was significantly increased to 427.78mg L-1 by optimizing the critical genes involved in 22(R)-HCHO biosynthesis. Furthermore, hybrid diploids were constructed to balance cell growth and 22(R)-HCHO production and to improve stress tolerance. Finally, the engineered strain produced 2.03g L-1 of 22(R)-HCHO in a 5-L fermenter, representing the highest 22(R)-HCHO titer reported to date in engineered microbial cell factories. The results of this study provide a foundation for further applications of 22(R)-HCHO in various industrially valuable steroids.

Determination of Veratrum alkaloid contents in three Veratrum species by HPLC-MS/MS.

Veratrum alkaloids have gained attention due to their toxic effects and potential pharmaceutical applications, particularly in cancer and cardiology. Over 200 alkaloids are found in species of the Veratrum genus. The alkaloid composition and concentrations can greatly vary in plants depending on factors like species, plant part, location, season, weather, or nutrients. This study aims an analytical approach to analyze and quantify Veratrum alkaloids in different plant parts of Veratrum species. The purpose is to contribute essential alkaloid concentration data for future research on the pharmacological and toxicological aspects of Veratrum alkaloids. This study focuses on five Veratrum alkaloids (cevadine, jervine, protoveratrine A, veratramine, and veratridine) in three Veratrum species (Veratrum album L., Veratrum californicum Durand, and Veratrum nigrum L.) collected from four German botanical gardens (Dresden, Leipzig, Marburg, and Schellerhau). A liquid-liquid extraction method and a sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method operating in multiple reaction monitoring (MRM) mode were applied for the alkaloid determination. Quantification revealed varying alkaloid concentrations among plant parts and Veratrum species in the μg/g to mg/g range. Protoveratrine A exhibited the highest content, while veratramine concentrations were generally lower. Especially in fruit, roots and rootstock of Veratrum album L. alkaloid concentrations were significant high. The developed HPLC-MS/MS method successfully determined Veratrum alkaloid concentrations in plant samples. The study contributes valuable data on Veratrum alkaloid distribution in different species and plant parts, crucial for understanding their potential medicinal and toxicological significance.

Pharmacology of Veratrum californicum Alkaloids as Hedgehog Pathway Antagonists.

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each.

Hydroxylated cyclopamine analogues from Veratrum californicum and their hedgehog pathway inhibiting activity

Cyclopamine (1), the teratogenic steroidal alkaloid isolated from corn lily (Veratrum californicum), has recently gained renewed interest due to its anticancer potential, that has been translated into the FDA approval of three Hedgehog (Hh) pathway inhibiting antitumor drugs. A chemical analysis of mother liquors obtained from crystallization of cyclopamine, extracted from roots and rhizomes of V. californicum, resulted in the isolation of two unprecedented cyclopamine analogues, 18-hydroxycyclopamine (2) and 24R-hydroxycyclopamine (3), the first compounds of this class to show modifications on rings D-F. The stereostructures of these new natural compounds have been established based on a detailed MS and 1D/2D NMR investigation. The isolated compounds were evaluated with the dual-luciferase bioassay for their inhibition of the hedgehog pathway in comparison to cyclopamine, providing new insights into the structure–activity relationships for this class of compounds.

Development of a New Combination Compound (Cyclopamine) Isolated from Veratrum californicum and 5-FU Acting as Anti-invasive and Initiator of Intrinsic Apoptosis Mechanism against Aggressive Type Colorectal Cancer Cells

Characteristics of programmed cell death are generally regarded as defined by cell outgrowth or cellular aging and distinct morphological characteristics featuring nuclear condensation and formation of apoptotic bodies. Apoptosis is considered to have a function at cellular level and is essential to long-lived cell differentiation undergoing senescence and apoptosis. Inappropriate apoptosis is a factor that results in serious conditions, causing multiple disease progression as well as cancer. The ability to modulate the life or death of a cancer cell is recognized for its immense therapeutic potential. Therefore, the research findings that are focused on the elucidation of a newly synthesized drug or drug derived from a natural plant to stop the cell cycle mechanism and signaling pathways that control cell cycle arrest and apoptosis are considered necessary. The field research of apoptosis is moving forward at a rapid rate. The goal of this paper is to investigate a natural active constituent, which is simply identified as “a drug for near future co-chemotherapy”, as well as to explore the ability to activate the apoptotic pathway. Our results demonstrated that cyclopamine provides an excellent anti-tumor activity and changing of the morphology of cancerous cells in the early and late phases of apoptosis, indicating that cyclopamine has the potential to chemosensitize SW-620 cancer cells to 5-FU based chemotherapy, which may be useful for the treatment of colorectal cancer and the fight to overcome drug resistance in advanced colorectal cancer.

Jerveratrum-Type Steroidal Alkaloids Inhibit β-1,6-Glucan Biosynthesis in Fungal Cell Walls.

ABSTRACTThe limited number of available effective agents necessitates the development of new antifungals. We report that jervine, a jerveratrum-type steroidal alkaloid isolated from Veratrum californicum, has antifungal activity. Phenotypic comparisons of cell wall mutants, K1 killer toxin susceptibility testing, and quantification of cell wall components revealed that β-1,6-glucan biosynthesis was significantly inhibited by jervine. Temperature-sensitive mutants defective in essential genes involved in β-1,6-glucan biosynthesis, including BIG1, KEG1, KRE5, KRE9, and ROT1, were hypersensitive to jervine. In contrast, point mutations in KRE6 or its paralog SKN1 produced jervine resistance, suggesting that jervine targets Kre6 and Skn1. Jervine exhibited broad-spectrum antifungal activity and was effective against human-pathogenic fungi, including Candida parapsilosis and Candida krusei. It was also effective against phytopathogenic fungi, including Botrytis cinerea and Puccinia recondita. Jervine exerted a synergistic effect with fluconazole. Therefore, jervine, a jerveratrum-type steroidal alkaloid used in pharmaceutical products, represents a new class of antifungals active against mycoses and plant-pathogenic fungi.IMPORTANCE Non-Candida albicans Candida species (NCAC) are on the rise as a cause of mycosis. Many antifungal drugs are less effective against NCAC, limiting the available therapeutic agents. Here, we report that jervine, a jerveratrum-type steroidal alkaloid, is effective against NCAC and phytopathogenic fungi. Jervine acts on Kre6 and Skn1, which are involved in β-1,6-glucan biosynthesis. The skeleton of jerveratrum-type steroidal alkaloids has been well studied, and more recently, their anticancer properties have been investigated. Therefore, jerveratrum-type alkaloids could potentially be applied as treatments for fungal infections and cancer.

Hydroxylated Cyclopamine Analogues from Veratrum Californicum and Their Hedgehog Pathway Inhibiting Activity

Hydroxylated Cyclopamine Analogues from Veratrum Californicum and Their Hedgehog Pathway Inhibiting Activity

Review: Veratrum californicum Alkaloids.

Veratrum spp. grow throughout the world and are especially prevalent in high mountain meadows of North America. All parts of Veratrum plants have been used for the treatment of ailments including injuries, hypertension, and rheumatic pain since as far back as the 1600s. Of the 17–45 Veratrum spp., Veratrum californicum alkaloids have been proven to possess favorable medicinal properties associated with inhibition of hedgehog (Hh) pathway signaling. Aberrant Hh signaling leads to proliferation of over 20 cancers, including basal cell carcinoma, prostate and colon among others. Six of the most well-studied V. californicum alkaloids are cyclopamine (1), veratramine (2), isorubijervine (3), muldamine (4), cycloposine (5), and veratrosine (6). Recent inspection of the ethanolic extract from V. californicum root and rhizome via liquid chromatography–mass spectrometry has detected up to five additional alkaloids that are proposed to be verazine (7), etioline (8), tetrahydrojervine (9), dihydrojervine (10), 22-keto-26-aminocholesterol (11). For each alkaloid identified or proposed in V. californicum, this review surveys literature precedents for extraction methods, isolation, identification, characterization and bioactivity to guide natural product drug discovery associated with this medicinal plant.

Of embryos and tumors: Cyclopia and the relevance of mechanistic teratology.

Embryos and tumors share several characteristics, but embryos differ from tumors in their coordination of cellular- and tissue-level processes, including organized differentiation, remodeling of tissues through apoptosis, and disciplined migrations of cells. Embryonic cellular events are kept on track through orderly cell-cell communication via signal transduction pathways. If the pathways are disrupted, development is perturbed, and malformation may result. Despite profound differences between embryos and tumors, the study of one has benefited our understanding of the other. Using cyclopia as an example, the history of humans' beliefs concerning and reactions to this horrific malformation are explored. During the latter half of the 20th century, interest in cyclopic sheep from high pastures in western Idaho led to the discovery that cyclopia occurred after pregnant ewes foraged in fields containing corn lily (Veratrum californicum). Eventually, the proximate teratogen was identified as cyclopamine (a steroidal alkaloid). The teratogenic mechanism was identified as inhibition of the sonic hedgehog (Shh) signal transduction pathway. Alert cancer researchers noted that a prominent form of medulloblastoma (a devasting childhood brain tumor) overexpressed Shh. Cyclopamine effectively inhibited the tumor in mice and killed human medulloblastoma cells in vitro. Thus, over a 60-year period, a molecule causing hideous malformations and much emotional pain was discovered and then found capable of restraining a destructive tumor, potentially saving children's lives and sparing emotional devastation of their families. The success of identifying cyclopamine as a cause of cyclopia and a potential cure for medulloblastoma emerged from mechanistic research shared by multiple disciplines.

Steroidal alkaloid variation in Veratrum californicum as determined by modern methods of analytical analysis

Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant in over twenty types of cancer. These alkaloids first became known in the 1950's due to their teratogenic properties, which resulted in newborn and fetal lambs developing cyclopia as a result of pregnant ewes consuming Veratrum californicum. It was discovered that the alkaloids in V. californicum were concentrated in the root and rhizome of the plant with much lower amounts of the most active alkaloid, cyclopamine, present in the aerial plant, especially in the late growth season. Inspired by the limitations in analytical instrumentation and methods available to researchers at the time of the original investigation, we have used state-of-the-art instrumentation and modern analytical methods to quantitate four steroidal alkaloids based on study parameters including plant part, harvest location, and growth stage. The results of the current inquiry detail differences in alkaloid composition based on the study parameters, provide a detailed assessment for alkaloids that have been characterized previously (cyclopamine, veratramine, muldamine and isorubijervine), and identify at least six alkaloids that have not been previously characterized. This study provides insight into optimal harvest time, plant growth stage, harvest location, and plant part required to isolate, yet to be characterized, alkaloids of interest for exploration as Hh pathway antagonists with desirable medicinal properties.

Teratogenic jervine increases the activity of doxorubicin in MCF-7/ADR cells by inhibiting ABCB1

Jervine is a natural teratogenic compound isolated from Veratrum californicum. In this study, for the first time, we revealed a novel activity of jervine in sensitizing the anti-proliferation effect of doxorubicin (DOX). We demonstrated that the synergistic mechanism was related to the intracellular accumulation of DOX via modulating ABCB1 transportation. Jervine did not affect the expression of ABCB1 in mRNA nor protein levels. However, jervine increased the ATPase activity of ABCB1 and possibly served as a substrate of ABCB1. The molecular docking results indicated that jervine was bound to a closed ABCB1 conformation and blocked drug entrance to the central binding site at the transmembrane domain. The present study identifies jervine acts as a substrate of ABCB1, and has potential to be developed as a novel and potent chemotherapy sensitizer used for patients developing multidrug resistance.

Native V. californicum Alkaloid Combinations Induce Differential Inhibition of Sonic Hedgehog Signaling.

Veratrum californicum is a rich source of steroidal alkaloids such as cyclopamine, a known inhibitor of the Hedgehog (Hh) signaling pathway. Here we provide a detailed analysis of the alkaloid composition of V. californicum by plant part through quantitative analysis of cyclopamine, veratramine, muldamine and isorubijervine in the leaf, stem and root/rhizome of the plant. To determine whether additional alkaloids in the extracts contribute to Hh signaling inhibition, the concentrations of these four alkaloids present in extracts were replicated using commercially available standards, followed by comparison of extracts to alkaloid standard mixtures for inhibition of Hh signaling using Shh-Light II cells. Alkaloid combinations enhanced Hh signaling pathway antagonism compared to cyclopamine alone, and significant differences were observed in the Hh pathway inhibition between the stem and root/rhizome extracts and their corresponding alkaloid standard mixtures, indicating that additional alkaloids present in these extracts are capable of inhibiting Hh signaling.

Biosynthesis of Veratrum californicum specialty chemicals in Camelina sativa seed

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.

Cyclopamine bioactivity by extraction method from Veratrum californicum

Veratrum californicum, commonly referred to as corn lily or Californian false hellebore, grows in high mountain meadows and produces the steroidal alkaloid cyclopamine, a potent inhibitor of the Hedgehog (Hh) signaling pathway. The Hh pathway is a crucial regulator of many fundamental processes during vertebrate embryonic development. However, constitutive activation of the Hh pathway contributes to the progression of various cancers. In the present study, a direct correlation was made between the extraction efficiency for cyclopamine from root and rhizome by eight methods, and the associated biological activity in Shh-Light II cells using the Dual-Glo® Luciferase Assay System. Alkaloid recovery ranged from 0.39 to 8.03mg/g, with ethanol soak being determined to be the superior method for obtaining biologically active cyclopamine. Acidic ethanol and supercritical extractions yielded degraded or contaminated cyclopamine with lower antagonistic activity towards Hh signaling.

Micropropagation, Acclimatization, and Greenhouse Culture of Veratrum californicum.

Micropropagation and production of Veratrum californicum is most successful when using a premixed Murishage and Skoog basal medium with vitamins and a 5-week subculture cycle at 16 °C for multiplication. These culture conditions provide the best percent survival after acclimatization in the greenhouse. However, clone response to temperature and light quality within culture conditions varies. Micropropagated plants have mass and morphology similar to 2- or 3-year-old seedlings. Acclimatized plantlets can then be grown in the greenhouse using sub-irrigation (ebb and flood) to maintain substrate volumetric water content > 44 %. Growth cycle in the greenhouse must be about 100 days, followed by dormancy for 5 months at 5 °C.

Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells.

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V.californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

Erratum to: Establishing clones of Veratrum californicum, a native medicinal species, for micropropagation

Erratum to: Establishing clones of Veratrum californicum, a native medicinal species, for micropropagation

Habitat relationships of great gray owl prey in meadows of the Sierra Nevada Mountains

ABSTRACTAnnual productivity of great gray owls (Strix nebulosa) in California, USA, is thought to be largely dependent on vole (Microtus spp.) and pocket gopher (Thomomys spp.; hereafter, gopher) abundance, yet factors influencing these prey populations have not been thoroughly investigated. The abundance of voles and gophers has been influenced by vegetation and cattle grazing in other regions; and many meadows are grazed within the breeding range of great gray owls in the Sierra Nevada Mountains of California. We evaluated the influence of vegetation and cattle grazing on great gray owl foraging habitat by quantifying vole abundance, gopher abundance, and vegetation features in grazed and ungrazed meadows in 2010 and 2011. We found that vole presence was positively associated with sward height and corn lily (Veratrum californicum) dominance; vole abundance was weakly negatively associated with cattle grazing. Gopher abundance was negatively associated with site wetness, and weakly positively associated with stem density, the frequency of forb presence, cattle grazing, and the dominance of corn lily. To efficiently manage vegetation and cattle grazing to benefit great gray owl prey, we suggest prioritizing habitat for voles in wet meadows because gophers were not likely to be abundant in wet sites. Particularly in areas with moist soils, we recommend maintaining sward height commensurate with the habitat relationships of voles found in this study; >290 mm where corn lily is not dominant and 125 mm where it is. Sustaining sward height for voles should benefit great gray owls and other meadow or forest edge carnivores. © 2014 The Wildlife Society.

Cyclopamine: from cyclops lambs to cancer treatment.

In the late 1960s, the steroidal alkaloid cyclopamine was isolated from the plant Veratrum californicum and identified as the teratogen responsible for craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in the western United States. Cyclopamine was found to inhibit the hedgehog (Hh) signaling pathway, which plays a critical role in embryonic development. More recently, aberrant Hh signaling has been implicated in several types of cancer. Thus, inhibitors of the Hh signaling pathway, including cyclopamine derivatives, have been targeted as potential treatments for certain cancers and other diseases associated with the Hh signaling pathway. A brief history of cyclopamine and cyclopamine derivatives investigated for the treatment of cancer is presented.