Podophyllotoxin (POD) serves as the primary therapeuticagent forthe external management of genital warts. However, traditional PODtinctures often cause severe systemic adverse reactions, due to thedrug being absorbed through the skin into the bloodstream. Therefore,it is particularly important to develop new topical preparations forPOD to enhance topical administration and reduce systemic absorption.Herein, POD-loaded keratin-functionalized transfersomes (POD-KTFs)were developed and characterized, and the skin permeation and deposition,the intradermal fluorescence distribution, and the influence of POD-KTFson a skin structure were evaluated. The hydrodynamic diameter andsurface potential of POD-KTFs were 183.1 nm and −33.9 mV, respectively.The image of scanning electron microscopy (SEM) indicated that thePOD-KTFs had a spherical appearance. The X-ray diffraction (XRD) patternconfirmed that POD mainly existed in its amorphous form in POD-KTFs.Meaningfully, POD-KTFs increased the deposition of POD in porcineear skin by 2.14 and 1.80 times, respectively, compared to the PODtincture and POD-loaded transfersomes (POD-TFs), while the transdermalpenetration of POD in POD-KTFs was obviously less than that of PODtincture (p < 0.001) and POD-TFs (p < 0.01). Using coumarin-6 as a fluorescent probe, fluorescenceimaging of skin sections revealed that the POD-KTFs were mainly distributedin the epidermis. In addition, POD-TFs could promote the drug to passthrough the stratum corneum (SC) by affecting the structure of theSC on the skin surface. Therefore, this study might provide an efficientway to improve the topical delivery of POD to the epidermis of theskin, thereby enhancing the therapeutic effect of genital warts.

Polyamines are small aliphatic amines whose metabolic reprogramming is involved in the regulation of various plant cellular reactions. Our previous study showed that polyamines increased lignan production in Linum album; however, little is known about the underlying mechanisms. This study aimed to provide more details on how putrescine (Put) regulates lignan biosynthesis in L. album cell culture. Our results showed that Put leads to podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6MPTOX) accumulation by increasing the expression levels of phenylalanine ammonia-lyase (PAL) and pinoresinol-lariciresinol reductase (PLR) genes, encoding lignan biosynthesis regulatory enzymes. Put also increased hydrogen peroxide (H2O2) content, while its level decreased in the presence of aminoguanidine (AG) and imidazole, inhibitors of diamine oxidase (DAO) and NADPH oxidase (NOX), respectively. Elevated levels of nitric oxide (NO) and cytosolic free Ca2+ caused by Put treatment were reduced after using inhibitors of nitrate reductase (NR) and nitric oxide synthesis-like (NOS-like) enzymes, as well as Ca2+ influx. Besides, pre-treatment of cells with AG, imidazole, ethylene glycol-bis (β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid (EGTA) (Ca2+ chelator), Nɷ-nitro-l-arginine methyl ester (L-NAME), and Sodium tungstate (TUN) (NO generation inhibitors) diminished PAL and PLR transcript levels and PTOX and 6MPTOX accumulation, indicating the involvement of H2O2, NO, and Ca2+ in regulating lignan biosynthesis in L. album cells. Put also stimulated salicylic acid (SA) accumulation, being sensitive to all inhibitors used. Overall, this study suggests that Put-induced H2O2 generation in combination with NO and Ca2+ signals can regulate PAL and PLR genes expression and lignan production, likely in a SA-dependent manner.

Sinopodophyllum hexandrum (Royle) Ying, the only species of Sinopodophyllum in Berberidaceae, is an endangered traditional Tibetan medicine. The harsh plateau growth environment makes S. hexandrum tough to breed and meet the global demand for clinical medications such as podophyllotoxin (PTOX) and etoposide. Jasmonic acid (JA) is acknowledged as a key phytohormone that modulates stress responses by activating defense mechanisms and promoting the production of specialized metabolites, which offers valuable insights for developing varieties that are more resilient to stress or yield higher amounts of secondary metabolites. In this study, JA treatment was used as a simulated source of stress to investigate the spatiotemporal changes in phytohormones, such as JA, cis-(+)-12-oxo-10, 15(Z)-phytodienoic acid (cis-(+)-OPDA), and abscisic acid (ABA), and transcriptional regulation following hormonal regulation in intact plants. Some correlations through changes in phytohormone levels and the expression level of related signaling pathway genes were observed to confirm the overall regulatory effect after the JA treatment. Furthermore, the JA treatment caused the differential expression of various genes including transcription factors (TFs), of which the most typical one is myelocytomatosis oncogene like protein 2 (MYC2), ShMYC2_3. Therefore, we proposed that a plant hormone-mediated regulatory network exists endogenously in S. hexandrum, enabling it to respond to JA treatment. This study provides a new direction for the germplasm improvement and the sustainable utilization of S. hexandrum when facing exogenous stimulation.

Understanding the change in plant-associated microbial diversity and secondary metabolite biosynthesis in medicinal plants due to their cultivation in non-natural habitat (NNH) is important to maintain their therapeutic importance. Here, the bacterial endomicrobiome of Podophyllum hexandrum plants of natural habitat (NH; Kardang and Triloknath locations) and NNH (Palampur location) was identified and its association with the biosynthesis of podophyllotoxin (PTOX) was revealed. Rhizomes (source of PTOX) of plants of NH had highest endophytic bacterial diversity compared to NNH-plants. Presence of plant-location and tissue-specific distinct and common taxa were also identified. Acinetobacter, Ralstonia and Pseudomonas were identified as core taxa, present in plants of both NH and NNH. Predictive functional analysis of endophytic communities revealed abundant presence of genes encoding initial enzymes of PTOX biosynthesis and plant growth promotion in the rhizomes and roots of Kardang locations. Higher accumulations of secondary metabolites such as PTOX (2.78 and 2.11 folds in Kardang and Triloknath rhizomes, respectively; 1.48 and 1.71 fold in Kardang and Triloknath roots, respectively), Picropodophyllotoxin (3.08 fold in Kardang rhizomes), Quercetin (1.65 fold in Kardang and 1.32 fold in Triloknath rhizomes; 3.07-fold in Kardang and 1.60 fold in Triloknath roots) and Kaempferol (1.66 and 1.24-fold in Kardang and Triloknath rhizomes, respectively; 2.91 and 1.94-fold in Kardang and Triloknath roots, respectively) were also found in NH compared to NNH. This study provides novel insight into the change in the endomicrobiome of NH and NNH-plants and their correlation to secondary metabolites biosynthesis, and that must be considered for cultivation practices.

Podophyllotoxin (PTOX), produced by Linum album, is a monolignol that participates in plant defense strategies. Our previous study established that methyl jasmonate (MeJA) significantly stimulates PTOX production in L. album cells. However; the mechanisms by which MeJA regulates PTOX biosynthesis are uncovered. In the present study, we demonstrated that MeJA induces a time-dependent hydrogen peroxide (H2O2) and salicylic acid (SA) accumulation but reduces nitric oxide (NO) generation in L. album cells. PTOX biosynthetic genes such as PAL, CCR, CAD, and PLR were upregulated in response to MeJA exposure. Furthermore, the results of RT-qPCR revealed a positive correlation between the expression of PTOX biosynthetic genes and MeJA-induced upregulation of four miRNAs such as miR156, miR159, miR172, and miR396 at 12 h. Generally, this study revealed that MeJA mediates PTOX biosynthesis in L. album cells by inducing H2O2 and SA formation, which can probably upregulate the expression level of some miRNAs and biosynthetic genes in a redox balance-dependent manner.

Pharmacological studies have shown that podophyllotoxin (PTOX) has anti-tumor, antiviral, and anti-inflammatory effects. More and more derivatives of PTOX, such as VM-26, NK-611 and GL-331, have gradually been synthesized and are widely used in clinical practice. Sinopodophyllum hexandrum rhizome is rich in PTOX, which is much higher than other PTOX source plants. At present, research on S. hexandrum mainly focuses on artificial cultivation, chemical composition, pharmaceutical value, and the biosynthesis pathway of PTOX. However, the researches are relatively scattered, and systematic review on PTOX is very limited. Therefore, this study performed a comprehensive investigation of the plant origin source, content profile, and biological activities to provide an integrated reference for in-depth research and clinical application of PTOX in the fields of chemistry and pharmacy, which can promote the innovative use of S. hexandrum resources and research and development of new anticancer drugs.

Accumulation of podophyllotoxin in the root culture of Hyptis suaveolens (L.) Poit: A potential natural lignan for clinically useful anticancer drugs

Previous studies have reported the functional role, biochemical features and synthesis pathway of podophyllotoxin (PTOX) in plants. In this study, we employed combined morphological and molecular techniques to identify an endophytic fungus and extract PTOX derivatives. Based on the analysis of ITS sequences and the phylogenetic tree, the isolate was classified as Penicillium herquei HGN12.1C, with a sequence identity of 98.58%. Morphologically, the HGN12.1C strain exhibits white colonies, short‐branched mycelia and densely packed hyphae. Using PacBio sequencing at an average read depth of 195×, we obtained a high‐quality genome for the HGN12.1C strain, which is 34.9 Mb in size, containing eight chromosomes, one mitochondrial genome and a GC content of 46.5%. Genome analysis revealed 10 genes potentially involved in PTOX biosynthesis. These genes include VdtD, Pinoresinollariciresinol reductase (PLR), Secoisolariciresinol dehydrogenase (SDH), CYP719A23, CYP71BE54, O‐methyltransferase 1 (OMT1), O‐methyltransferase 3 (OMT3), 2‐ODD, CYP71CU and CYP82D61. Notably, the VdtD gene in fungi shares functional similarities with the DIR gene found in plants. Additionally, we identified peltatin, a PTOX derivative, in the HGN12.1C extract. Docking analysis suggests a potential role for the 2‐ODD enzyme in converting yatein to deoxypodophyllotoxin. These findings offer invaluable insights into the synthesis mechanism of PTOX in fungi, shedding light on the relationship between host plants and endophytes.

Supramolecular self-sensitized dual-drug nanoassemblies potentiating chemo-photodynamic therapy for effective cancer treatment

Introduction: Cervical cancer (CC) ranks as the fourth most prevalent malignant tumor among women worldwide, and is the fourth leading cause of cancer-related mortality. GuiErBai (GEB), a compound preparation developed by our research team, is derived from the ancient Chinese medicine of the Miao nationality and is comprised of podophyllotoxin (PTOX), imperatorin, isoimperatorin, and A. dahurica alkaloids. These individual components have demonstrated notable efficacy in tumor treatment. However, the specific anti-tumor effect of the compound Chinese medicine GEB in the context of CC has yet to be validated. Methods: HeLa and SiHa cell lines were utilized for in vitro experiments and treated with 5mg/mL and 10mg/mL GEB concentrations, respectively. The cell cycle changes after GEB treatment were assessed using flow cytometry. Transmission electron microscopy was employed to observe autophagic bodies and apoptotic bodies, while MDC staining evaluated the occurrence of autophagy. CCK-8 was used to observe the effect of GEB on cell proliferation, and Transwell assays assessed cell migration and invasion. Western blotting detected cell cycle and apoptosis-related protein expression, along with the expression level of autophagy-related protein LC3I/II. Changes in ROS and mitochondrial membrane potential in cervical cancer cells following GEB treatment were determined using ROS detection and mitochondrial membrane potential detection kits. For the in vivo experiment, a nude mouse model of cervical cancer transplantation based on HeLa cells was established. Experimental animals were divided into negative control, positive control, high-dose GEB (10mg/mL), and low-dose GEB (5mg/mL) groups. Results: In HeLa and SiHa cell lines, the G0/G1 phase of tumor cells significantly decreased (p < 0.001), while the G2/M phase increased notably (p < 0.001) following various GEB treatments. Electron microscopy showed GEB promoted apoptotic body and autophagosome formation in both cell lines. Compared to untreated HeLa and SiHa cells, GEB-treated cells exhibited significantly reduced caspase3 protein expression, and substantially increased autophagy-related protein LC3I/II expression. GEB treatment significantly reduced migration and invasion capabilities in both cell lines (p < 0.001), while ROS content and mitochondrial membrane potential were significantly elevated (p < 0.001). GEB effectively inhibited cervical cancer cell proliferation, with the optimal concentration being 10mg/mL. A successful nude mouse model of cervical cancer transplantation was established using HeLa cells. Post-GEB treatment, the tumor volume and weight in nude mice significantly decreased (p < 0.001), with diminished expression of CD34, VEGF, and caspase3 proteins in tumor tissues. Discussion: GEB exhibits a robust antitumor effect against cervical cancer, both in vitro and in vivo, in a concentration-dependent manner, by regulating autophagy and apoptosis of tumor cells.

Podophyllotoxin (PTOX) is naturally produced by the plant Podophyllum species. Some of its derivatives are anticancer drugs, which are produced mainly by using chemical semi-synthesis methods. Recombinant bacteria have great potential in large-scale production of the derivatives of PTOX. In addition to introducing the correct enzymes, the transportation of PTOX into the cells is an important factor, which limits its modification in the bacteria. Here, we improved the cellular uptake of PTOX into Escherichia coli with the help of the zero-valent sulfur transporter YedE1E2 in the presence of cetyltrimethylammonium bromide (CTAB). CTAB promoted the uptake of PTOX, but induced the production of reactive oxygen species. A protein complex (YedE1E2) of YedE1 and YedE2 enabled E. coli cells to resist CTAB by reducing reactive oxygen species, and YedE1E2 was a hypothetical transporter. Further investigation showed that YedE1E2 facilitated the uptake of extracellular zero-valent sulfur across the cytoplasmic membrane and the formation of glutathione persulfide (GSSH) inside the cells. The increased GSSH minimized oxidative stress. Our results indicate that YedE1E2 is a zero-valent sulfur transporter and it also facilitates CTAB-assisted uptake of PTOX by recombinant bacteria.

The purpose of this study was to evaluate L-cysteine-modified transfersomes as the topical carrier for enhanced epidermal delivery of podophyllotoxin (POD). L-cysteine-deoxycholic acid (LC-DCA) conjugate was synthesized via an amidation reaction. POD-loaded L-cysteine-modified transfersomes (POD-LCTs) were prepared via a thin membrane dispersion method and characterized for their particle size, zeta potential, morphology, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and in vitro release. Subsequently, in vitro skin permeation and retention, fluorescence distribution in the skin, hematoxylin-eosin staining and in vivo skin irritation were studied. The POD-LCTs formed spherical shapes with a particle size of 172.5 ± 67.2 nm and a zeta potential of -31.3 ± 6.7 mV. Compared with the POD-Ts, the POD-LCTs provided significantly lower drug penetration through the porcine ear skin and significantly increased the skin retention (p < 0.05). Meaningfully, unlike the extensive distribution of the POD-loaded transfersomes (POD-Ts) throughout the skin tissue, the POD-LCTs were mainly located in the epidermis. Moreover, the POD-LCTs did not induce skin irritation. Therefore, the POD-LCTs provided an enhanced epidermal delivery and might be a promising carrier for the topical delivery of POD.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168h of culture, PTOX content in the PDB with plant extract reached the peak with 314µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

Linum album is a well-known rich source of anticancer compounds, i.e., podophyllotoxin (PTOX) and other lignans. These compounds play an important role in the plant’s defensive system. The RNA-Seq data of flax (L. usitatissimum) were analyzed under various biotic and abiotic stresses to comprehend better the importance of lignans in plant defense responses. Then, the association between the lignan contents and some related gene expressions was experimented with HPLC and qRT-PCR, respectively. Transcriptomic profiling showed a specific expression pattern in different organs, and just the commonly regulated gene EP3 was detected with a significant increase under all stresses. The in silico analysis of the PTOX biosynthesis pathway identified a list of genes, including laccase (LAC11), lactoperoxidase (POD), 4-coumarate-CoA ligase (4CL), and secoisolariciresinol dehydrogenase (SDH). These genes increased significantly under individual stresses. The HPLC analysis showed that the measured lignan contents generally increased under stress. In contrast, a quantitative expression of the genes involved in this pathway using qRT-PCR showed a different pattern that seems to contribute to regulating PTOX content in response to stress. Identified modifications of critical genes related to PTOX biosynthesis in response to multiple stresses can provide a baseline for improving PTOX content in L. album.

Methyl jasmonate redirects the dynamics of carbohydrates and amino acids toward the lignans accumulation in Linum album cells

Sinopodophyllum hexandrum (Royle) T. S. Ying, an important source of podophyllotoxin (PTOX), has become a rare and endangered plant because of over-harvesting. Somatic embryogenesis (SE) is the main way of seedling rapid propagation and germplasm enhancement, but the regeneration of S. hexandrum has not been well established, and the PTOX biosynthesis abilities at different SE stages remain unclear. Therefore, it is extremely important to elucidate the SE mechanism of S. hexandrum and clarify the biosynthesis variation of PTOX. In this study, the transcriptomes of S. hexandrum at different SE stages were sequenced, the contents of PTOX and 4'-demethylepipodophyllotoxin were assayed, and the transcript expression patterns were validated by qRT-PCR. The results revealed that plant hormone (such as auxins, abscisic acid, zeatin, and gibberellins) related pathways were significantly enriched among different SE stages, indicating these plant hormones play important roles in SE of S. hexandrum; the expression levels of a series of PTOX biosynthesis related genes as well as PTOX and 4'-demethylepipodophyllotoxin contents were much higher in embryogenic callus stage than in the other stages, suggesting embryogenic callus stage has the best PTOX biosynthesis ability among different SE stages. This study will contribute to germplasm conservation and fast propagation of S. hexandrum, and facilitate the production of PTOX.

Influence of light quality and some growth regulators in inducing the production of Podophyllotoxin, a bioactive compound against cancer, in adventitious roots formed in the leaves of Hyptis suaveolens

In vitro propagation from seeds and enhanced synthesis of podophyllotoxin from root callus of SinoPodophyllum hexandrum Royle T.S. Ying (Himalayan Mayapple) − An endangered medicinal plant

Multi modular toxicity assessment of nephrotoxicity in podophyllotoxin exposure rats on account of toxicological evidence chain (TEC) concept

Sinopodophyllum hexandrum is an endangered medicinal herb known for its bioactive lignan podophyllotoxin (PTOX), which is used for the preparation of anticancer drugs. In its natural habitat, S. hexandrum is exposed to a multitude of adversities, such as fluctuating temperatures, water deficit, and high UV radiations. Transcriptional regulation of genes, which is regulated by the condition-specific binding of transcriptional factors to precise motifs in the promoter region, underlines responses to an environmental cue. Therefore, analysis of promoter sequences could ascertain the spatio-temporal expression of genes and overall stress responses. Unavailability of genomic information does not permit such analysis in S. hexandrum, especially on regulation of PTOX pathway. Accordingly, this study describes isolation and in silico analysis of 5′-upstream regions of ShPLR (PINORESINOL-LARICIRESINOL REDUCTASE) and ShSLD (SECOISOLARICIRESINOL DEHYDROGENASE), the two key genes of the PTOX biosynthetic pathway. Data showed a range of motifs related to basal transcription, stress-responsive elements, such as those for drought, low temperature, and light, suggesting that the expression of these genes and resulting PTOX accumulation would be affected by, at least, these environmental cues. While the impact of temperature and light on PTOX accumulation is well studied, the effect of water deficit on the physiology of S. hexandrum and PTOX accumulation remains obscure. Given the presence of drought-responsive elements in the promoters of the key genes, the impact of water deficit on growth and development and PTOX accumulation was studied. The results showed decline in relative water content and net photosynthetic rate, and increase in relative electrolyte leakage with stress progression. Plants under stress exhibited a reduction in transpiration rate and chlorophyll content, with a gradual increase in osmoprotectant content. Besides, stressed plants showed an increase in the expression of genes involved in the phenylpropanoid pathway and PTOX biosynthesis, and an increase in PTOX accumulation. Upon re-watering, non-irrigated plants showed a significant improvement in biochemical and physiological parameters. Summarily, our results demonstrated the importance of osmoprotectants during water deficit and the revival capacity of the species from water deficit, wherein PTOX synthesis was also modulated. Moreover, isolated promoter sequences could be employed in genetic transformation to mediate the expression of stress-induced genes in other plant systems.