Podophyllotoxin derivatives-tubulin complex reveals a potential binding site of tubulin polymerization inhibitors in α-tubulin adjacent to colchicine site.

The colchicine site of ß-tubulin has been proven to be essential binding sites of microtubule polymerization inhibitors. Recent studies implied that GTP pocket of α-tubulin adjacent to colchicine sites is a potential binding site for developing tubulin polymerization inhibitors. However, the structural basis for which type of structural fragments was more beneficial for enhancing the affinity of α-tubulin is still unclear. Here, podophyllotoxin derivatives-tubulin complex crystals indicated that heterocyclic with the highly electronegative and small steric hindrance was conducive to change configuration and enhance the affinity of the residues in GTP pocket of α-tubulin. Triazole with lone-pairs electrons and small steric hindrance exhibited the strongest affinity for enhancing affinity of podophyllotoxin derivatives by forming two hydrogen bonds with αT5 Ser178. Pyrimidine with the secondary strong affinity could bind C:Asn101 to make the αH7 configuration deflection, which reduces the stability of tubulin result in its depolymerization. Conversely, 4ß-quinoline-podophyllotoxin with the weakest affinity did not interact with α-tubulin. The molecular dynamics simulation and protein thermal shift results showed that 4ß-triazole-podophyllotoxin-tubulin was the most stable mainly due to two hydrogen bonds and the higher van der Waals force. This work provided a structural basis of the potential binding sites for extending the α/ß-tubulin dual-binding sites inhibitors design strategy.

Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.

Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.

Reversal of the tamoxifen­resistant breast cancer malignant phenotype by proliferation inhibition with bromosulfonamidine amino­podophyllotoxin.

One of the lignans isolated from plants within the genus Podophyllum is podophyllotoxin (PPT). PPT and its derivatives are pharmacologically active compounds with potential antiproliferative properties in several kinds of tumors. Although these compounds have been used to treat other malignancies, no PPT derivative-based chemotherapeutic agent has been used to cure tamoxifen (TAM)-resistant breast cancer in clinical trials, to the best of our knowledge. Thus, using TAM-resistant breast cancer as a disease model, the present study assessed the effects of a recently synthesized PPT derivative, bromosulfonamidine amino-PPT (BSAPPT), on TAM-resistant breast cancer. Using the tamoxifen-resistant breast cancer cell model (MCF-7/TAMR) in vitro, Cell Counting Kit-8 and colony formation assays were adopted to evaluate the effect of BSAPPT on cell proliferation. Cell apoptosis and cell cycle assays were used to assess the influence of BSAPPT on cell apoptosis and the cell cycle in MCF-7/TAMR. The targets of the potential mechanism of action were analyzed by RT-qPCR and western blotting. The present study demonstrated that BSAPPT suppressed MCF-7/TAMR cell proliferation in a dose-dependent manner. By modulating the level of expression of genes linked to both apoptosis and the cell cycle, BSAPPT triggered MCF-7/TAMR cells to undergo apoptosis and prevented them from entering the cell cycle. Consequently, BSAPPT blocked these cells from proliferating, thereby halting the malignant advancement of TAM-resistant breast cancer. Therefore, these findings indicate that new therapeutic agents involving BSAPPT may be developed to facilitate the treatment of TAM-resistant breast cancer.

GuiErBai: a potent inhibitor, exhibiting broadly antitumor effect against cervical cancer in vitro and in vivo.

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 5 mg/mL and 10 mg/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 (10 mg/mL), and low-dose GEB (5 mg/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 10 mg/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 via SIRT1/PPAR /NF-κB axis induced cardiac injury in rats based on the toxicological evidence chain (TEC) concept.

BACKGROUND: The study of cardiotoxicity of drugs has become an important part of clinical safety evaluation of drugs. It is commonly known that podophyllotoxin (PPT) and its many derivatives and congeners are broad-spectrum pharmacologically active substances. Clinical cardiotoxicity of PPT and its derivatives has been raised, basic research on the mechanism of cardiotoxicity remains insufficient. PURPOSE: In present study, our group's innovative concept of toxicological evidence chain (TEC) was applied to reveal the cardiac toxicity mechanism of PPT by targeted metabolomics, TMT-based quantitative proteomics and western blot. METHODS: The injury phenotype evidence (IPE) acquired from the toxicity manifestations, such as weight and behavior observation of Sprague-Dawley rat. The damage to rat hearts were assessed through histopathological examination and myocardial enzymes levels, which were defined as Adverse Outcomes Evidence (AOE). The damage to rat hearts was assessed through histopathological examination and myocardial enzyme levels, which were defined as evidence of adverse outcomes.Overall measurements of targeted metabolomics based on energy metabolism and TMT-based quantitative proteomics were obtained after exposure to PPT to acquire the Toxic Event Evidence (TEE). The mechanism of cardiac toxicity was speculated based on the integrated analysis of targeted metabolomics and TMT-based quantitative proteomics, which was verified by western blot. RESULTS: The results indicated that exposure to PPT could result in significant elevation of myocardial enzymes and pathological alterations in rat hearts. In addition, we found that PPT caused disorders in cardiac energy metabolism, characterized by a decrease in energy metabolism fuels. TMT-based quantitative proteomics revealed that the PPAR (Peroxisome proliferators-activated receptor) signaling pathway needs further study. It is worth noting that PPT may suppress the expression of SIRT1, subsequently inhibiting AMPK, decreasing the expression of PGC-1α, PPARα and PPARγ. This results in disorders of glucose oxidation, glycolysis and ketone body metabolism. Additionally, the increase in the expression of p-IKK and p-IκBα, leads to the nuclear translocation of NF-κB p65 from the cytosol, thus triggering inflammation. CONCLUSION: This study comprehensively evaluated cardiac toxicity of PPT and initially revealed the mechanism of cardiotoxicity,suggesting that PPT induced disorders of energy metabolism and inflammation via SIRT1/PPAR/NF-κB axis, potentially contributing to cardiac injury.

Exploring the mechanism of enterotoxicity mediated by the microbiome-butyrate-PPAR axis in podophyllotoxin through the toxicological evidence chain (TEC) concept.

Podophyllotoxin (PPT) is a lignan derived from the roots and stems of the Podophyllum plant. However, its enterotoxicity restricts its clinical application. The underlying mechanisms by which PPT exerts its action remain largely elusive. This study aimed to evaluate the molecular mechanisms underlying PPT-induced enterotoxicity utilizing the concept of toxicological evidence chain. Changes in body weight, behavior, and histopathological and biochemical markers in rats were observed. Additionally, microbiome, metabolome, and transcriptome analyses were integrated to identify potential microorganisms, metabolic markers, and major pathways using a co-occurrence network. Our findings suggested that PPT induced pathological changes in rats, including weight loss, diarrhea, and inflammation accompanied by increased levels of IFN-γ, IL-5, IL-6, GRO/KC, and IL-12p70. The decrease in butyrate levels in the PPT group may be related to the enrichment of Firmicutes. The reduction of butyrate levels may impair the expression of PPARγ, subsequently promoting Escherichia-Shigella proliferation. Additionally, the suppression of PPARs pathway may result in the increased production of inflammatory factors, contributing to enterotoxicity. This study offers a novel understanding of the molecular mechanisms underlying PPT-induced enterotoxicity, making a significant contribution to developing strategies to mitigate PPT toxicity and prevent associated diseases.

Podophyllic Aldehyde, a Podophyllotoxin Derivate, Elicits Different Cell Cycle Profiles Depending on the Tumor Cell Line: A Systematic Proteomic Analysis.

When new antitumor therapy drugs are discovered, it is essential to address new target molecules from the point of view of chemical structure and to carry out efficient and systematic evaluation. In the case of natural products and derived compounds, it is of special importance to investigate chemomodulation to further explore antitumoral pharmacological activities. In this work, the compound podophyllic aldehyde, a cyclolignan derived from the chemomodulation of the natural product podophyllotoxin, has been evaluated for its viability, influence on the cell cycle, and effects on intracellular signaling. We used functional proteomics characterization for the evaluation. Compared with the FDA-approved drug etoposide (another podophyllotoxin derivative), we found interesting results regarding the cytotoxicity of podophyllic aldehyde. In addition, we were able to observe the effect of mitotic arrest in the treated cells. The use of podophyllic aldehyde resulted in increased cytotoxicity in solid tumor cell lines, compared to etoposide, and blocked the cycle more successfully than etoposide. High-throughput analysis of the deregulated proteins revealed a selective antimitotic mechanism of action of podophyllic aldehyde in the HT-29 cell line, in contrast with other solid and hematological tumor lines. Also, the apoptotic profile of podophyllic aldehyde was deciphered. The cell death mechanism is activated independently of the cell cycle profile. The results of these targeted analyses have also shown a significant response to the signaling of kinases, key proteins involved in signaling cascades for cell proliferation or metastasis. Thanks to this comprehensive analysis of podophyllic aldehyde, remarkable cytotoxic, antimitotic, and other antitumoral features have been discovered that will repurpose this compound for further chemical transformations and antitumoral analysis.

Cytotoxic Cyclolignans Obtained by the Enlargement of the Cyclolignan Skeleton of Podophyllic Aldehyde, a Selective Podophyllotoxin-Derived Cyclolignan.

Podophyllotoxin, a cyclolignan natural product, has been the object of extensive chemomodulation to obtain better chemotherapeutic agents. Among the obtained podophyllotoxin derivatives, podophyllic aldehyde showed very interesting potency and selectivity against several tumoral cell lines, so it became our lead compound for further modifications, as described in this work, oriented toward the enlargement of the cyclolignan skeleton. Thus, modifications performed at the aldehyde function included nucleophilic addition reactions and the incorporation of the aldehyde carbon into several five-membered rings, such as thiazolidinones and benzo-fused azoles. The synthesized derivatives were evaluated against several types of cancer cells, and although some compounds were cytotoxic at the nanomolar range, most of them were less potent and less selective than the parent compound podophyllic aldehyde, with the most potent being those having the lactone ring of podophyllotoxin. In silico ADME evaluation predicted good druggability for most of them. The results indicate that the γ-lactone ring is important for potency, while the α,ß-unsaturated aldehyde is necessary to induce selectivity in these cyclolignans.

Synthesis of Novel 4-Acyloxy-2'-Bromo-6'-Chloropodophyllotoxin Derivatives Displaying Significant Insecticidal Activity Against Mythimna Separata.

In order to explore novel natural product-based insecticidal agent, two important intermediates (2 and 3) and 4-acyloxy-2'-bromo-6'-chloropodophyllotoxin derivatives (4 a-f and 5 a-f) were designed and prepared, and their structures were confirmed by 1H-NMR, 13C NMR, HRMS, ESI-MS, optical rotation and melting point (mp). The stereochemical configuration of compound 4 b was unambiguously confirmed by single-crystal X-ray diffraction. Moreover, we evaluated the insecticidal activity of target compounds 4 a-f and 5 a-f against a serious agricultural pest of Mythimna separata by using the leaf-dipping method. Among all tested compounds, compounds 4 d, 5 d and 5 f exhibited stronger insecticidal activity with a final mortality rate exceeding 60 %. Especially compound 5 d exhibited the best insecticidal activity, with a final mortality rate of 74.1 %. It has been proven that introducing bromine or chlorine atoms at the C-2', C-2' and C-6' positions of the E ring of podophyllotoxin can produce more potent compounds. In addition, the configuration of the C-4 position is important for insecticidal activity, and 4ß-configuration is optimal. This will pave the way for further design, structural modification, and development of derivatives of podophyllotoxin as insecticidal agents.

Antileishmanial effect of podophyllotoxin and podophyllin on Leishmania major in vitro and in vivo.

Therapeutic research is very important in the prevention and treatment of leishmaniasis due to problems such as drug resistance, scarring and disease recurrence. The aim of this study was to determine how Leishmania major responds to the anti-leishmaniasis properties of podophyllotoxin and podophyllin. Cultured Leishmania promastigotes were exposed to different concentrations of podophyllotoxin and podophyllin for 24 and 48 h. Then, during the animal phase, Balb/c mice were experimentally injected with Leishmania promastigotes. After wounding, the effects of 0.5% podophyllotoxin and 25% podophyllin on reducing wound diameter and the number of amastigotes in the wound were evaluated. Podophyllotoxin and podophyllin were 83% and 59% lethal to Leishmania major promastigotes at the highest concentrations (200 µg/ml) and time (48 h). In the in vivo study, the mean lesion diameter at the end of treatment in the negative control group was 15.10 mm compared to 14.21 mm and 11.55 mm in the 25% podophyllin and 0.5% podophyllotoxin groups, respectively. Although both agents reduced the size of mice wounds and the number of amastigotes in the wounds, podophyllotoxin was more effective in this regard. Based on the results, podophyllotoxin and podophyllin can be used as leishmaniasis drugs after further research.

Polycarbonate-Based Copolymer Micelles as Biodegradable Carriers of Anticancer Podophyllotoxin or Juniper Extracts.

Podophyllotoxin (PPT) is used in the industrial production of efficient anticancer, antiviral and other drugs. Sinopodophyllum hexandrum or Podophyllum peltatum are natural sources of PPT, but at present they are considered as endangered species. Their PPT content is variable, depending on the growing conditions. Searching for new sources of PPT, some representatives of the genus Juniperus were found to exhibit efficient PPT biosynthesis. However, PPT is highly toxic and poorly soluble in water compound, which limits its clinical applications. In this connection, amphiphilic polymer micelles are considered to be suitable PPT carriers, aimed at increase in water solubility and decrease in toxicity. The present research deals with the evaluation of MPEG-polycarbonate block copolymer micelles loaded with PPT or juniper extracts. The active component-loaded polymer nanocarriers were characterized by dynamic and electrophoretic light scattering, as well as by transmission electron microscopy. The active component loading efficiency and loading capacity were also determined. Highly efficient antiproliferative activity of the loaded micelles was determined in a panel of cancer cell lines. The obtained amphiphilic nanocarriers, loaded with PPT-containing bioactive components, have application in future in vivo preclinical trials of their pharmacokinetics and pharmacodynamics as potential therapeutical agents in the prospective nanomedicine.

Pinobanksin from peony seed husk: A flavonoid with the potential to inhibit the proliferation of SH-SY5Y.

Pinobanksin, as one of the flavonoids, has powerful biological activities but has been under-recognized. In this study, we optimized the extraction method of phragmites from peony seed shells by using organic solvent extraction. The yield of PSMS was 10.54 ± 0.13% under the conditions of ethanol volume fraction 70%, extraction temperature 70°C, material-liquid ratio 1:25 g/mL, and extraction time 60 min; the optimized PSMS could be effectively separated in S-8 macroporous resin coupled with C18. The relative content of PSMS was increased from 0.42% in PSMS to 92.53% after C18 purification; the antioxidant activity test revealed that pinobanksin could exert antioxidant ability by binding catalase (CAT) enzyme. Second, it was found that pinobanksin could effectively inhibit the proliferation of SH-SY5Y cells, mainly by binding to BCL2-associated X (BAX), B-cell lymphoma-2 (BCL-2), and cyclin-dependent Kinase 4/6 (CDK4/6) to produce more hydrogen bonds to inhibit their activities. This study confirms the medicinal potential of pinobanksin and provides the basis for the proper understanding of pinobanksin and the development of related products.

Podophyllotoxin: Recent Advances in the Development of Hybridization Strategies to Enhance Its Antitumoral Profile.

Podophyllotoxin is a naturally occurring cyclolignan isolated from rhizomes of Podophyllum sp. In the clinic, it is used mainly as an antiviral; however, its antitumor activity is even more interesting. While podophyllotoxin possesses severe side effects that limit its development as an anticancer agent, nevertheless, it has become a good lead compound for the synthesis of derivatives with fewer side effects and better selectivity. Several examples, such as etoposide, highlight the potential of this natural product for chemomodulation in the search for new antitumor agents. This review focuses on the recent chemical modifications (2017-mid-2023) of the podophyllotoxin skeleton performed mainly at the C-ring (but also at the lactone D-ring and at the trimethoxyphenyl E-ring) together with their biological properties. Special emphasis is placed on hybrids or conjugates with other natural products (either primary or secondary metabolites) and other molecules (heterocycles, benzoheterocycles, synthetic drugs, and other moieties) that contribute to improved podophyllotoxin bioactivity. In fact, hybridization has been a good strategy to design podophyllotoxin derivatives with enhanced bioactivity. The way in which the two components are joined (directly or through spacers) was also considered for the organization of this review. This comprehensive perspective is presented with the aim of guiding the medicinal chemistry community in the design of new podophyllotoxin-based drugs with improved anticancer properties.

Anthriscus sylvestris Deoxypodophyllotoxin Synthase Involved in the Podophyllotoxin Biosynthesis.

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum, the isolation of the DPS enzyme gene and the type of the enzyme, e.g. whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism.

Development of hydroxypropyl cellulose and graphene oxide modified molecularly imprinted polymers for separation and enrichment of podophyllotoxin.

A novel hydroxylpropyl cellulose (HPC) modified graphene oxide (GO)-based molecularly imprinted polymers (HPC-GO-MIP) have been developed as a solid phase extraction (SPE) material for the selective separation and extraction of podophyllotoxin. In this strategy, the cellulose with rich hydroxyl groups was introduced to form bi-functional monomers with methacrylic acid to provide more recognition sites for the improving of extraction efficiency, then GO was added as a two-dimensional substrate for MIP to improve the material morphology and surface area. The extraction performances of obtained HPC-GO-MIP material were tested, and the results prove its high efficiency and selectivity for podophyllotoxin extraction. The saturated adsorption capacity reached 23.1 µg/mg, and high enrichment efiiciency of 463.8 folds was realized under the premise of ensuring the recovery rate. The selective imprinting factor was much higher than those of kaempferol and quercetin, which were the main compounds in podophyllum fruit. Under the optimized SPE conditions, the HPC-GO-MIP based SPE-HPLC method showed the detection limit of 14.2 ng/mL for podophyllotoxin assay. When applied to podophyllum fruit samples, the material showed excellent ability of selective separation and enrichment of podophyllotoxin, and the relative standard deviations (RSD) of intra and inter batches were less than 8.1 % and 5.7 % in real samples detection. The HPC-GO-MIP SPE method broadened the application for high multiple extraction in trace analyte samples and provided a valuable solution to improve the selective separation and detection.

Podophyllotoxin-loaded PEGylated E-selectin peptide conjugate targeted cancer site to enhance tumor inhibition and reduce side effect.

E-selectin, which is highly expressed in vascular endothelial cells near tumor and get involved in the all tumor growth steps: occurrence, proliferation and metastasis, is considered as a promise targeted protein for antitumor drug discovery. Herein, we would like to report the design, preparation and the anticancer evaluation of the peptide-PEG-podophyllotoxin conjugate(PEG-Pep-PODO), in which the short peptide (CIELLQAR) was used as the E-selectin ligand for the targeting purpose and the PEG portion the molecule got the conjugate self-assembled to form a water soluble nanoparticle. In vitro release study showed that the conjugated and entrapped PODO could be released simultaneously in the presence of GSH (highly expressed in tumor environmental conditions) and the GSH would catalyze the break of the disufur bond which linked of the PODO and the peptide-PEG portion of the conjugate. Cell adhesion test of the PEG-Pep-PODO indicated that E-selectin ligand peptide CIELLQAR could get specifically and efficiently binding to the E-selectin expressing human umbilical vein endothelial cells (HUVEC). In vitro cytotoxicity assay further revealed that PEG-Pep-PODO significantly improved the selectivity of PEG-Pep-PODO for killing the tumor cells and normal cells compared with PODO solution formulation. More importantly, the in vivo experiment demonstrated that the conjugate would accumulate of the PODO payload in tumor through targeting endothelial cells in the tumor microenvironment, which resulted in the much improved in vivo inhibition of tumor growth, intratumoral microvessel density, and decreased systemic toxicity of this nanoparticle over the free PODO. Furthermore, this water soluble conjugate greatly improved the pharmacokinetic properties of the mother molecule.

Podophyllotoxin reduces the aggressiveness of human oral squamous cell carcinoma through myeloid cell leukemia­1.

Podophyllotoxin (PPT), which is derived from the podophyllum plant, exhibits marked cytotoxic effects against cancer cells; however, the precise molecular mechanism underlying its activity against human oral squamous cell carcinoma (OSCC) has not been elucidated. In the present study, the mechanism by which PPT induced cytotoxicity in two OSCC cell lines, HSC3 and HSC4, was determined. The effects of PPT on cytotoxicity in HSC3 and HSC4 cells were analyzed using Annexin V/PI double staining, Sub­G1 analysis, soft agar assays, western blotting, and quantitative PCR. The changes in the mitochondrial membrane potential were assessed using a JC­1 assay and cytosolic and mitochondrial fractionation. A myeloid cell leukemia­1 (Mcl­1) overexpression cell lines were also established to study the role of Mcl­1 on apoptosis. The results showed that PPT inhibited the growth of the two human OSCC cell lines and induced apoptosis, which was accompanied by mitochondrial membrane depolarization. Compared with the control, PPT reduced the expression of Mcl­1 in both cell lines through a proteasome­dependent protein degradation process. Overall, these results suggested that targeting of Mcl­1 protein by PPT induced apoptosis, providing a foundation for further pre­clinical and clinical study of its value in the management of OSCC.

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor.

(R)-ß-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6 Šfrom substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-ß-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.

Nephrotoxicity assessment of podophyllotoxin-induced rats by regulating PI3K/Akt/mTOR-Nrf2/HO1 pathway in view of toxicological evidence chain (TEC) concept.

Adverse reactions to traditional Chinese medicine have hindered the healthy development and internationalization process of the traditional Chinese medicine industry. The critical issue that needs to be solved urgently is to evaluate the safety of traditional Chinese medicine systematically and effectively. Podophyllotoxin (PPT) is a highly active compound extracted from plants of the genus Podophyllum such as Dysosma versipellis (DV). However, its high toxicity and toxicity to multiple target organs affect the clinical application, such as the liver and kidney. Based on the concurrent effects of PPT's medicinal activity and toxicity, it would be a good example to conduct a systematic review of its safety. Therefore, this study revolves around the Toxicological Evidence Chain (TEC) concept. Based on PPT as the main toxic constituent in DV, observe the objective toxicity impairment phenotype of animals. Evaluate the serum biochemical indicators and pathological tissue sections for substantial toxic damage results. Using metabolomics, lipidomics, and network toxicology to evaluate the nephrotoxicity of PPT from multiple perspectives systematically. The results showed that PPT-induced nephrotoxicity manifested as renal tubular damage, mainly affecting metabolic pathways such as glycerophospholipid metabolism and sphingolipid metabolism. PPT inhibits the autophagy process of kidney cells through the PI3K/Akt/mTOR and Nrf2/HO1 pathways and induces the activation of oxidative stress in the body, thereby causing nephrotoxic injury. This study fully verified the feasibility of the TEC concept for the safety and toxicity evaluation of traditional Chinese medicine. Provide a research template for systematically evaluating the safety of traditional Chinese medicine.

L-Cysteine-Modified Transfersomes for Enhanced Epidermal Delivery of Podophyllotoxin.

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.