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.

PROTAC EZH2 degrader-1 overcomes the resistance of podophyllotoxin derivatives in refractory small cell lung cancer with leptomeningeal metastasis.

BACKGROUND: Leptomeningeal metastasis (LM) of small cell lung cancer (SCLC) is a highly detrimental occurrence associated with severe neurological disorders, lacking effective treatment currently. Proteolysis-targeting chimeric molecules (PROTACs) may provide new therapeutic avenues for treatment of podophyllotoxin derivatives-resistant SCLC with LM, warranting further exploration. METHODS: The SCLC cell line H128 expressing luciferase were mutated by MNNG to generate H128-Mut cell line. After subcutaneous inoculation of H128-Mut into nude mice, H128-LM and H128-BPM (brain parenchymal metastasis) cell lines were primarily cultured from LM and BPM tissues individually, and employed to in vitro drug testing. The SCLC-LM mouse model was established by inoculating H128-LM into nude mice via carotid artery and subjected to in vivo drug testing. RNA-seq and immunoblotting were conducted to uncover the molecular targets for LM. RESULTS: The SCLC-LM mouse model was successfully established, confirmed by in vivo live imaging and histological examination. The upregulated genes included EZH2, SLC44A4, VEGFA, etc. in both BPM and LM cells, while SLC44A4 was particularly upregulated in LM cells. When combined with PROTAC EZH2 degrader-1, the drug sensitivity of cisplatin, etoposide (VP16), and teniposide (VM26) for H128-LM was significantly increased in vitro. The in vivo drug trials with SCLC-LM mouse model demonstrated that PROTAC EZH2 degrader-1 plus VM26 or cisplatin/ VP16 inhibited H128-LM tumour significantly compared to VM26 or cisplatin/ VP16 alone (P < 0.01). CONCLUSION: The SCLC-LM model effectively simulates the pathophysiological process of SCLC metastasis to the leptomeninges. PROTAC EZH2 degrader-1 overcomes chemoresistance in SCLC, suggesting its potential therapeutic value for SCLC LM.

Computational Study Into the Oxidative Ring-Closure Mechanism During the Biosynthesis of Deoxypodophyllotoxin.

The nonheme iron dioxygenase deoxypodophyllotoxin synthase performs an oxidative ring-closure reaction as part of natural product synthesis in plants. How the enzyme enables the oxidative ring-closure reaction of (-)-yatein and avoids substrate hydroxylation remains unknown. To gain insight into the reaction mechanism and understand the details of the pathways leading to products and by-products we performed a comprehensive computational study. The work shows that substrate is bound tightly into the substrate binding pocket with the C7'-H bond closest to the iron(IV)-oxo species. The reaction proceeds through a radical mechanism starting with hydrogen atom abstraction from the C7'-H position followed by ring-closure and a final hydrogen transfer to form iron(II)-water and deoxypodophyllotoxin. Alternative mechanisms including substrate hydroxylation and an electron transfer pathway were explored but found to be higher in energy. The mechanism is guided by electrostatic perturbations of charged residues in the second-coordination sphere that prevent alternative pathways.

Deoxypodophyllotoxin Induces ROS-Mediated Apoptosis by Modulating the PI3K/AKT and p38 MAPK-Dependent Signaling in Oral Squamous Cell Carcinoma.

Deoxypodophyllotoxin (DPT), a naturally occurring flavonolignan, possesses several pharmacological properties, including anticancer property. However, the mechanisms underlying DPT mode of action in oral squamous cell carcinoma (OSCC) remain unknown. This study aimed to investigate the anticancer effects of DPT on OSCC and the underlying mechanisms. Results of the MTT assay revealed that DPT significantly reduced the cell viability in a time- and dose-dependent manner. Flow cytometry analysis revealed that DPT induces apoptosis in OSCC cells in a dose-dependent manner. Moreover, DPT enhanced the production of mitochondrial reactive oxygen species (ROS) in OSCC cells. Mechanistically, DPT induced apoptosis in OSCC cells by suppressing the PI3K/AKT signaling pathway while activating the p38 MAPK signaling to regulate the expression of apoptotic proteins. Treatment with SC79, an AKT activator, reversed the effects of DPT on AKT signaling in OSCC cells. Taken together, these results provide the basis for the use of DPT in combination with conventional chemotherapy for the treatment of oral cancer.

Picropodophyllin Inhibits the Proliferation of Human Prostate Cancer DU145 and LNCaP Cells via ROS Production and PI3K/AKT Pathway Inhibition.

The reactive oxygen species (ROS) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway play critical roles in the pathogenesis of prostate cancer by modulating cell proliferation. Picropodophyllin (PPP), an inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), exerts significant antitumor effects via the PI3K/AKT signaling pathway. However, the effects of PPP on prostate cancer via ROS production and the PI3K/AKT signaling pathway remain elusive. Herein, we focused on examining the antitumor effects of PPP on DU145 and LNCaP human prostate cancer cells to determine the possible molecular mechanism. Our data indicated that the inhibitory effect of PPP on the proliferation of DU145 and LNCaP human prostate cancer cells was mediated by apoptosis induction and cell cycle blockade. Furthermore, PPP significantly influenced the expression of apoptosis-related, cell cycle, ROS production, and PI3K/AKT signaling proteins. These findings suggest that PPP can induce cell cycle arrest and apoptosis via the production of ROS and inhibition of PI3K/AKT signaling pathway, thereby suppressing the proliferation of prostate cancer cells.

A comprehensive insight into the antineoplastic activities and molecular mechanisms of deoxypodophyllotoxin: Recent trends, challenges, and future outlook.

Lignans constitute an important group of polyphenols, which have been demonstrated to potently suppress cancer cell proliferation. Numerous in vitro and in vivo studies indicate that deoxypodophyllotoxin as a natural lignan possesses potent anticancer activities against various types of human cancer. The purpose of current review is to provide the reader with the latest findings in understanding the anticancer effects and molecular mechanisms of deoxypodophyllotoxin. This review comprehensively describes the influence of deoxypodophyllotoxin on signaling cascades and molecular targets implicated in cancer cell proliferation and invasion. A number of various signaling molecules and pathways, including apoptosis, necroptosis, cell cycle, angiogenesis, vascular disruption, ROS, MMPs, glycolysis, and microtubules as well as NF-κB, PI3K/Akt/mTOR, and MAPK cascades have been reported to be responsible for the anticancer activities of deoxypodophyllotoxin. The results of present review suggest that the cyclolignan deoxypodophyllotoxin can be developed as a novel and potent anticancer agent, especially as an alternative option for treatment of resistant tumors to chemotherapy.

2, 4, 5-Trideoxyhexopyranosides Derivatives of 4'-Demethylepipodophyllotoxin: De novo Synthesis and Anticancer Activity.

BACKGROUND: Podophyllotoxin is a natural lignan which possesses anticancer and antiviral activities. Etoposide and teniposide are semisynthetic glycoside derivatives of podophyllotoxin and are increasingly used in cancer medicine. OBJECTIVE: The present work aimed to design and synthesize a series of 2, 4, 5-trideoxyhexopyranosides derivatives of 4'-demethylepipodophyllotoxin as novel anticancer agents. METHODS: A divergent de novo synthesis of 2, 4, 5-trideoxyhexopyranosides derivatives of 4'- demethylepipodophyllotoxin has been established via palladium-catalyzed glycosylation. The abilities of synthesized glycosides to inhibit the growth of A549, HepG2, SH-SY5Y, KB/VCR and HeLa cancer cells were investigated by MTT assay. Flow cytometric analysis of cell cycle with propidium iodide DNA staining was employed to observe the effect of compound 5b on cancer cell cycle. RESULTS: Twelve D and L monosaccharide derivatives 5a-5l have been efficiently synthesized in three steps from various pyranone building blocks employing de novo glycosylation strategy. Dmonosaccharide 5b showed the highest cytotoxicity on five cancer cell lines with the IC50 values ranging from 0.9 to 6.7 µM. It caused HepG2 cycle arrest at G2/M phase in a concentrationdependent manner. CONCLUSION: The present work leads to the development of novel 2, 4, 5-trideoxyhexopyranosides derivatives of 4'-demethylepipodophyllotoxin. The biological results suggest that the replacement of the glucosyl moiety of etoposide with 2, 4, 5-trideoxyhexopyranosyl is favorable to their cytotoxicity. D-monosaccharide 5b was observed to cause HepG2 cycle arrest at the G2/M phase in a concentration- dependent manner.

Mechanistic analysis of carbon-carbon bond formation by deoxypodophyllotoxin synthase.

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.

Gamma-Aminobutyric Acid (GABA) Promotes Growth in Zebrafish Larvae by Inducing IGF-1 Expression via GABAA and GABAB Receptors.

Insulin-like growth factor-1 (IGF-1) primarily increases the release of gamma-aminobutyric acid (GABA) in neurons; moreover, it is responsible for the promotion of longitudinal growth in children and adolescents. Therefore, in this study, we investigated whether exogenous GABA supplementation activates IGF-mediated growth performance. Zebrafish larvae treated with GABA at three days post fertilization (dpf) showed a significant increase in the total body length from 6 to 12 dpf through upregulation of growth-stimulating genes, including IGF-1, growth hormone-1 (GH-1), growth hormone receptor-1 (GHR-1), and cholecystokinin A (CCKA). In particular, at 9 dpf, GABA increased total body length from 3.60 ± 0.02 to 3.79 ± 0.03, 3.89 ± 0.02, and 3.92 ± 0.04 mm at concentrations of 6.25, 12.5, and 25 mM, and the effect of GABA at 25 mM was comparable to 4 mM ß-glycerophosphate (GP)-treated larvae (3.98 ± 0.02 mm). Additionally, the highest concentration of GABA (50 mM) -induced death in 50% zebrafish larvae at 12 dpf. GABA also enhanced IGF-1 expression and secretion in preosteoblast MC3T3-E1 cells, concomitant with high levels of the IGF-1 receptor gene (IGF-1R). In zebrafish larvae, the GABA-induced growth rate was remarkably decreased in the presence of an IGF-1R inhibitor, picropodophyllin (PPP), which indicates that GABA-induced IGF-1 enhances growth rate via IGF-1R. Furthermore, we investigated the effect of GABA receptors on growth performance along with IGF-1 activation. Inhibitors of GABAA and GABAB receptors, namely bicuculline and CGP 46381, respectively, considerably inhibited GABA-induced growth rate in zebrafish larvae accompanied by a marked decrease in the expression of growth-stimulating genes, including IGF-1, GH-1, GHR-1, and CCKA, but not with an inhibitor of GABAC receptor, TPMPA. Additionally, IGF-1 and IGF-1R expression was impaired in bicuculline and CGP 46381-treated MC3T3-E1 cells, but not in the cells treated with TPMPA. Furthermore, treatment with bicuculline and CGP 46381 significantly downregulated GABA-induced IGF-1 release in MC3T3-E1 cells. These data indicate that GABA stimulates IGF-1 release via GABAA and GABAB receptors and leads to growth promotion performance via IGF-1R.

Picropodophyllotoxin Induces G1 Cell Cycle Arrest and Apoptosis in Human Colorectal Cancer Cells via ROS Generation and Activation of p38 MAPK Signaling Pathway.

Picropodophyllotoxin (PPT), an epimer of podophyllotoxin, is derived from the roots of Podophyllum hexandrum and exerts various biological effects, including anti-proliferation activity. However, the effect of PPT on colorectal cancer cells and the associated cellular mechanisms have not been studied. In the present study, we explored the anticancer activity of PPT and its underlying mechanisms in HCT116 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to monitor cell viability. Flow cytometry was used to evaluate cell cycle distribution, the induction of apoptosis, the level of reactive oxygen species (ROS), assess the mitochondrial membrane potential (Δψm), and multi-caspase activity. Western blot assays were performed to detect the expression of cell cycle regulatory proteins, apoptosis-related proteins, and p38 MAPK (mitogen-activated protein kinase). We found that PPT induced apoptosis, cell cycle arrest at the G1 phase, and ROS in the HCT116 cell line. In addition, PPT enhanced the phosphorylation of p38 MAPK, which regulates apoptosis and PPT-induced apoptosis. The phosphorylation of p38 MAPK was inhibited by an antioxidant agent (N-acetyl-L-cysteine, NAC) and a p38 inhibitor (SB203580). PPT induced depolarization of the mitochondrial inner membrane and caspase-dependent apoptosis, which was attenuated by exposure to Z-VAD-FMK. Overall, these data indicate that PPT induced G1 arrest and apoptosis via ROS generation and activation of the p38 MAPK signaling pathway.

Enhanced Aryltetralin Lignans Production in Linum Adventi-Tious Root Cultures.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside.

Selective elimination of CML stem/progenitor cells by picropodophyllin in vitro and in vivo is associated with p53 activation.

Chronic myeloid leukemia (CML) is a hematologic malignancy originating from BCR-ABL oncogene-transformed hematopoietic stem cells (HSCs) known as leukemia stem cells (LSCs). Therefore, targeting LSCs is of primary importance to eradicate CML. The present study demonstrates that picropodophyllin (PPP) effectively induces apoptosis and inhibits colony formation in CML stem/progenitor cells as well as quiescent CML progenitors resistant to imatinib therapy, while sparing normal hematopoietic cells in vitro. Administration of PPP in vivo markedly diminishes CML stem/progenitor cells in a transgenic mouse model of CML by inhibition of cell proliferation and enhancement of apoptosis in LSK cells, and significantly improves survival of CML mice. Furthermore, PPP treatment preferentially leads to transcriptional activation of p53 in CML but not normal CD34+ cells, upregulation of p53 protein in LSCs-enriched Sca-1+ cells from CML mice, and increased phosphorylation of p53 and upregulation of Bax protein in Ku812 cells. These results suggest that the inhibitory effects of PPP on CML stem/progenitor cells are associated with selective activation of p53 pathway and propose that PPP is a potent agent that selectively targets CML LSCs, and may be of value in the CML therapy.

Synthesis of (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin via a multi-enzyme cascade in E. coli.

BACKGROUND: The aryltetralin lignan (-)-podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. Over the years, the commercial demand for this compound rose notably because of the high clinical importance of its semi-synthetic chemotherapeutic derivatives etoposide and teniposide. To satisfy this demand, (-)-podophyllotoxin is conventionally isolated from the roots and rhizomes of Sinopodophyllum hexandrum, which can only grow in few regions and is now endangered by overexploitation and environmental damage. For these reasons, targeting the biosynthesis of (-)-podophyllotoxin precursors or analogues is fundamental for the development of novel, more sustainable supply routes. RESULTS: We recently established a four-step multi-enzyme cascade to convert (+)-pinoresinol into (-)-matairesinol in E. coli. Herein, a five-step multi-enzyme biotransformation of (-)-matairesinol to (-)-deoxypodophyllotoxin was proven effective with 98 % yield at a concentration of 78 mg/L. Furthermore, the extension of this cascade to a sixth step leading to (-)-epipodophyllotoxin was evaluated. To this end, seven enzymes were combined in the reconstituted pathway involving inter alia three plant cytochrome P450 monooxygenases, with two of them being functionally expressed in E. coli for the first time. CONCLUSIONS: Both, (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin, are direct precursors to etoposide and teniposide. Thus, the reconstitution of biosynthetic reactions of Sinopodophyllum hexandrum as an effective multi-enzyme cascade in E. coli represents a solid step forward towards a more sustainable production of these essential pharmaceuticals.

PICT1 is critical for regulating the Rps27a-Mdm2-p53 pathway by microtubule polymerization inhibitor against cervical cancer.

In our previous study, it showed that P-3F, a podophyllotoxin derivative, causes the increased level of p53 expression by enhancing p53 stability, resulting from blockage of the Mdm2-p53 feedback loop via nucleolus-to-nucleoplasm translocation of Rps27a in human cervical cancer HeLa cell line. However, the mechanism of regulating Rps27a localization remains to be studied. In the current study, it has been demonstrated that the level of protein interacting with carboxyl terminus 1 (PICT1), originally identified as a tumor suppressor, was decreased in a concentration-dependent manner in response to P-3F, leading to inhibition of human cervical cancer cell lines proliferation. Also remarkably, reduction of serine phosphorylation of STMN1 at position 16 induced by P-3F was required in the downregulation of PICT1, in which p53 activity was likely to be directly involved. Note as well that, PICT1 also played an important role in p53 stability enhancement by inhibiting Mdm2-mediated p53 ubiquitination due to Rps27a translocation from the nucleolus to the nucleoplasm to interact with Mdm2 following treatment with P-3F. Collectively, these findings indicated that P-3F, a microtubule polymerization inhibitor, promotes the decreased level of PICT1 expression, which is critical for regulating the Rps27a-Mdm2-p53 pathway against cervical cancer.

Autophagy induction by IGF1R inhibition with picropodophyllin and linsitinib.

Induction of macroautophagy (hereafter termed autophagy) is a strategy to improve the outcome of antineoplastic therapies by facilitating the induction of immunogenic cancer cell death and the consequent immune recognition of malignant cells. We analyzed 65,000 distinct compounds by means of a phenotypic discovery platform for autophagy induction and identified the IGF1R (insulin like growth factor 1 receptor) inhibitor picropodophyllin (PPP) as a potent inducer of autophagic flux. We found that PPP acts on-target, as an inhibitor of the tyrosine kinase activity of IGF1R and enhances the release of adenosine triphosphate, ATP, from stressed and dying cancer cells in vitro, thereby improving the therapeutic efficacy of chemoimmunotherapy in cancer-bearing mice. This PPP effect was phenocopied by another IGF1R inhibitor, linsitinib. Moreover, in human triple-negative breast cancer, phosphorylation of IGF1R correlates with reduced autophagy, an unfavorable local immune profile and poor prognosis. In summary, IGF1R inhibition may constitute a novel strategy for the treatment of cancer in the context of chemoimmunotherapy.

Activation of IGF-1 pathway and suppression of atrophy related genes are involved in Epimedium extract (icariin) promoted C2C12 myotube hypertrophy.

The regenerative effect of Epimedium and its major bioactive flavonoid icariin (ICA) have been documented in traditional medicine, but their effect on sarcopenia has not been evaluated. The aim of this study was to investigate the effects of Epimedium extract (EE) on skeletal muscle as represented by differentiated C2C12 cells. Here we demonstrated that EE and ICA stimulated C2C12 myotube hypertrophy by activating several, including IGF-1 signal pathways. C2C12 myotube hypertrophy was demonstrated by enlarged myotube and increased myosin heavy chains (MyHCs). In similar to IGF-1, EE/ICA activated key components of the IGF-1 signal pathway, including IGF-1 receptor. Pre-treatment with IGF-1 signal pathway specific inhibitors such as picropodophyllin, LY294002, and rapamycin attenuated EE induced myotube hypertrophy and MyHC isoform overexpression. In a different way, EE induced MHyC-S overexpression can be blocked by AMPK, but not by mTOR inhibitor. On the level of transcription, EE suppressed myostatin and MRF4 expression, but did not suppress atrogenes MAFbx and MuRF1 like IGF-1 did. Differential regulation of MyHC isoform and atrogenes is probably due to inequivalent AKT and AMPK phosphorylation induced by EE and IGF-1. These findings suggest that EE/ICA stimulates pathways partially overlapping with IGF-1 signaling pathway to promote myotube hypertrophy.

Triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives induced apoptosis in HeLa cells by up-regulating TMEM133.

Heterocycle modification has been widely and successfully employed in the antitumor drugs. However, the different antitumor efficacy was corelated with the heterocycle substituted, and the genetic mechanism underlying these effects has not been elucidated. In this study, the intrinsic regularity between different types of heterocycle-substituted DMEP derivative compounds and the mechanisms of their antitumor activity was preliminarily disclosed. Triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives induced more severe DNA damage and higher levels of 26S proteasomal Topo IIß degradation, though inhibited the recruition of γH2AX to resist the DNA damage. The reduced DNA repair led to higher up-regulation of cell cycle arrest proteins, and ultimately DNA damage mediated-ATM/ATR apoptotic pathways and specifically activated DNA damage response gene TMEM133, which induced apoptosis through up-regulation of G2/M cell cycle arrest-related genes. Over-expression and knock-out of TMEM133 demonstrated that TMEM133 is essential for inhibition of the tumor cell growth during treatment with triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives.

Podophyllotoxin: History, Recent Advances and Future Prospects.

Podophyllotoxin, along with its various derivatives and congeners are widely recognized as broad-spectrum pharmacologically active compounds. Etoposide, for instance, is the frontline chemotherapeutic drug used against various cancers due to its superior anticancer activity. It has recently been redeveloped for the purpose of treating cytokine storm in COVID-19 patients. Podophyllotoxin and its naturally occurring congeners have low bioavailability and almost all these initially discovered compounds cause systemic toxicity and development of drug resistance. Moreover, the production of synthetic derivatives that could suffice for the clinical limitations of these naturally occurring compounds is not economically feasible. These challenges demanded continuous devotions towards improving the druggability of these drugs and continue to seek structure-optimization strategies. The discovery of renewable sources including microbial origin for podophyllotoxin is another possible approach. This review focuses on the exigency of innovation and research required in the global R&D and pharmaceutical industry for podophyllotoxin and related compounds based on recent scientific findings and market predictions.

Novel podophyllotoxin and benzothiazole derivative induces transitional morphological and functional changes in HaCaT cells.

Podophyllotoxin (PPT) is an antimitotic drug used topically in the treatment of anogenital warts. Due to its toxicity it cannot be administered systemically as an anticancer agent. However, modified PPT derivatives such as etoposide and teniposide are used clinically as systemic agents. Thus, we invented novel PPT derivative KL3 that was synthesized by photocyclization. Earlier we have shown that KL3 has an anticancer effect in various cell lines. Here we compared the toxicity of KL3 vs PPT on non-cancerous normal human keratinocytes (HaCaT) and peripheral blood mononuclear cells (PBMC) showing that KL3 is less toxic than PPT to non-cancerous cells. At concentrations that neither induced cell death, nor affected cell cycle, KL3 in HaCaT cells evoked transient ultrastructural features of ER stress, swelling of mitochondria and elongation of cytoplasmic processes. Those changes partially reversed with prolonged incubation while features of autophagy were induced. PPT in equivalent concentrations induced HaCaT cell death by cell cycle arrest, intrinsic apoptosis and finally disintegration of cell membranes followed by secondary necrosis. In conclusion, we show that the KL3 derivative of PPT in contrast to PPT allows repair of normal keratinocytes and triggers mechanisms that restore non-tumor cell homeostasis.

Deoxypodophyllotoxin Inhibits Cell Growth and Induces Apoptosis by Blocking EGFR and MET in Gefitinib-Resistant Non-Small Cell Lung Cancer.

As one of the major types of lung cancer, non-small cell lung cancer (NSCLC) accounts for the majority of cancer-related deaths worldwide. Treatments for NSCLC includes surgery, chemotherapy, and targeted therapy. Among the targeted therapies, resistance to inhibitors of the epidermal growth factor receptor (EGFR) is common and remains a problem to be solved. MET (hepatocyte growth factor receptor) amplification is one of the major causes of EGFR-tyrosine kinase inhibitor (TKI) resistance. Therefore, there exists a need to find new and more efficacious therapies. Deoxypodophyllotoxin (DPT) extracted from Anthriscus sylvestris roots exhibits various pharmacological activities including anti-inflammation and anti-cancer effects. In this study we sought to determine the anti-cancer effects of DPT on HCC827GR cells, which are resistant to gefitinib (EGFR-TKI) due to regulation of EGFR and MET and their related signaling pathways. To identify the direct binding of DPT to EGFR and MET, we performed pull-down, ATP-binding, and kinase assays. DPT exhibited competitive binding with ATP against the network kinases EGFR and MET and reduced their activities. Also, DPT suppressed the expression of p-EGFR and p-MET as well as their downstreat proteins p-ErbB3, p-AKT, and p-ERK. The treatment of HCC827GR cells with DPT induced high ROS generation that led to endoplasmic-reticulum stress. Accordingly, loss of mitochondrial membrane potential and apoptosis by multi-caspase activation were observed. In conclusion, these results demonstrate the apoptotic effects of DPT on HCC827GR cells and signify the potential of DPT to serve as an adjuvant anti-cancer drug by simultaneously inhibiting EGFR and MET.