Assessment of polymeric mucin-drug interactions.

Mucosal-delivered drugs have to pass through the mucus layer before absorption through the epithelial cell membrane. Although there has been increasing interest in polymeric mucins, a major structural component of mucus, potentially acting as important physiological regulators of mucosal drug absorption, there are no reports that have systematically evaluated the interaction between mucins and drugs. In this study, we assessed the potential interaction between human polymeric mucins (MUC2, MUC5B, and MUC5AC) and various drugs with different chemical profiles by simple centrifugal method and fluorescence analysis. We found that paclitaxel, rifampicin, and theophylline likely induce the aggregation of MUC5B and/or MUC2. In addition, we showed that the binding affinity of drugs for polymeric mucins varied, not only between individual drugs but also among mucin subtypes. Furthermore, we demonstrated that deletion of MUC5AC and MUC5B in A549 cells increased the cytotoxic effects of cyclosporin A and paclitaxel, likely due to loss of mucin-drug interaction. In conclusion, our results indicate the necessity to determine the binding of drugs to mucins and their potential impact on the mucin network property.

Optimization of the fermentation media, mathematical modeling, and enhancement of paclitaxel production by Alternaria alternata after elicitation with pectin.

Alternaria alternata fungus is a potent paclitaxel producer isolated from Corylus avellana. The major challenge is the lack of optimized media for endophytic fungi productivity. In the effort to maximize the production of taxoids by A. alternata, several fermentation conditions, including pH (pH 4.0-7.0), different types and concentrations of carbon (fructose, glucose, sucrose, mannitol, sorbitol, and malt extract), and nitrogen (urea, ammonium nitrate, potassium nitrate, ammonium phosphate, and ammonium sulfate) were applied step by step. Based on the results, A. alternata in a medium containing sucrose 5% (w/v) and ammonium phosphate 2.5 mM at pH 6.0 showed a rapid and sustainable growth rate, the highest paclitaxel yield (94.8 µg gFW-1 vs 2.8 µg gFW-1 in controls), and the maximum content of amino acids. Additionally, the effect of pectin was evaluated on fungus, and mycelia harvested. Pectin significantly enhanced the growth and taxoid yield on day 21 (respectively 171% and 116% of their corresponding on day 7). The results were checked out by mathematical modeling as well. Accordingly, these findings suggest a low-cost, eco-friendly, and easy-to-produce approach with excellent biotechnological potential for the industrial manufacture of taxoids.

Paclitaxel - a Product of Fungal Secondary Metabolism or an Artefact?

Taxol (common name: paclitaxel) is an extremely important component of drugs for the treatment of various cancers. Thirty years after the discovery of its effectiveness, a metabolic precursor of Taxol (10-deacetylbaccatin III) is still primarily extracted from needles of European yew trees. In order to meet the considerable demand, hopes were pinned on the possibilities of biotechnological production from the very beginning. In 1993, as if by chance, Taxol was supposedly discovered in fungi that grow endobiotically in yew trees. This finding aroused hopes of biotechnological use to produce fungal Taxol in large quantities in fermenters. It never came to that. Instead, a confusing flood of publications emerged that claimed to have detected Taxol in more and more eukaryotic and even prokaryotic species. However, researchers never reproduced these rather puzzling results, and they could certainly not be applied on an industrial scale. This paper will show that some of the misguided approaches were apparently based on a seemingly careless handling of sparse evidence and on at least questionable publications. Apparently, the desired gold rush of commercial exploitation was seductive. Scientific skepticism as an indispensable core of good scientific practice was often neglected, and the peer review process has not exerted its corrective effect. Self-critical reflection and more healthy skepticism could help to reduce the risk of such aberrations in drug development. This article uses this case study as a striking example to show what can be learned from the Taxol case in terms of research ethics and the avoidance of questionable research practices.

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis.

Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7ß,9α,10ß,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1ß-dehydroxybaccatin IV (1 a), and a minor 4ß,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4ß,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4ß,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

Synthetic biology for Taxol biosynthesis and sustainable production.

Incomplete understanding of the biosynthetic pathway of the anticancer compound Taxol hinders its production by metabolic engineering. Recent reports by Jiang et al. and other groups now describe the missing steps in Taxol biosynthesis, notably including oxetane ring formation. These findings will promote the sustainable production of Taxol through synthetic biology.

Unique fluorescent probe for the recognition of late apoptosis via translocation from plasma membrane to nucleus.

Cell apoptosis is a crucial physiological process playing central roles in key biological and pathological activities. However, the current fluorescent probes for the detection of late apoptosis were "off-on" probes, which were facilely interfered by false positive signals caused by inhomogeneous staining and other factors. Herein, a unique fluorescent probe (NPn) discriminating late apoptosis from early apoptosis and heathy status with two different sets of fluorescent signals have been prepared, to overcome the possible false positive signals. NPn was designed impermeable to biomembranes and simultaneously with high affinity to DNA/RNA, which localized on the plasma membranes of living and early apoptotic cells, while relocated to the nucleus in late apoptotic cells. The hydrophilic amine unit and small ion radius were responsive for its membrane impermeability, which was confirmed with two control molecules without amine group. Using the probe, we have successfully evaluated the cell apoptosis induced by ultraviolet irradiation, rotenone, colchicine, and paclitaxel, demonstrating its potential application in biological researches.

Expression of MHC II in DRG neurons attenuates paclitaxel-induced cold hypersensitivity in male and female mice.

Chemotherapy is often a life-saving treatment, but the development of intractable pain caused by chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting toxicity that restricts cancer survival rates. Recent reports demonstrate that paclitaxel (PTX) robustly increases anti-inflammatory CD4+ T cells in the dorsal root ganglion (DRG), and that T cells and anti-inflammatory cytokines are protective against CIPN. However, the mechanism by which CD4+ T cells are activated, and the extent cytokines released by CD4+ T cells target DRG neurons are unknown. Here, we are the first to detect major histocompatibility complex II (MHCII) protein in mouse DRG neurons and to find CD4+ T cells breaching the satellite glial cell barrier to be in close proximity to neurons, together suggesting CD4+ T cell activation and targeted cytokine release. MHCII protein is primarily expressed in small nociceptive neurons in male and female mouse DRG but increased after PTX in small nociceptive neurons in only female DRG. Reducing one copy of MHCII in small nociceptive neurons decreased anti-inflammatory IL-10 and IL-4 producing CD4+ T cells in naïve male DRG and increased their hypersensitivity to cold. Administration of PTX to male and female mice that lacked one copy of MHCII in nociceptive neurons decreased anti-inflammatory CD4+ T cells in the DRG and increased the severity of PTX-induced cold hypersensitivity. Collectively, our results demonstrate expression of MHCII protein in mouse DRG neurons, which modulates cytokine producing CD4+ T cells in the DRG and attenuates cold hypersensitivity during homeostasis and after PTX treatment.

Niche inflammatory signals control oscillating mammary regeneration and protect stem cells from cytotoxic stress.

Stem cells are known for their resilience and enhanced activity post-stress. The mammary gland undergoes frequent remodeling and is subjected to recurring stress during the estrus cycle, but it remains unclear how mammary stem cells (MaSCs) respond to the stress and contribute to regeneration. We discovered that cytotoxic stress-induced activation of CD11c+ ductal macrophages aids stem cell survival and prevents differentiation. These macrophages boost Procr+ MaSC activity through IL1ß-IL1R1-NF-κB signaling during the estrus cycle in an oscillating manner. Deleting IL1R1 in MaSCs results in stem cell loss and skewed luminal differentiation. Moreover, under cytotoxic stress from the chemotherapy agent paclitaxel, ductal macrophages secrete higher IL1ß levels, promoting MaSC survival and preventing differentiation. Inhibiting IL1R1 sensitizes MaSCs to paclitaxel. Our findings reveal a recurring inflammatory process that regulates regeneration, providing insights into stress-induced inflammation and its impact on stem cell survival, potentially affecting cancer therapy efficacy.

Oxetane Ring Formation in Taxol Biosynthesis Is Catalyzed by a Bifunctional Cytochrome P450 Enzyme.

Taxol is a potent drug used in various cancer treatments. Its complex structure has prompted extensive research into its biosynthesis. However, certain critical steps, such as the formation of the oxetane ring, which is essential for its activity, have remained unclear. Previous proposals suggested that oxetane formation follows the acetylation of taxadien-5α-ol. Here, we proposed that the oxetane ring is formed by cytochrome P450-mediated oxidation events that occur prior to C5 acetylation. To test this hypothesis, we analyzed the genomic and transcriptomic information for Taxus species to identify cytochrome P450 candidates and employed two independent systems, yeast (Saccharomyces cerevisiae) and plant (Nicotiana benthamiana), for their characterization. We revealed that a single enzyme, CYP725A4, catalyzes two successive epoxidation events, leading to the formation of the oxetane ring. We further showed that both taxa-4(5)-11(12)-diene (endotaxadiene) and taxa-4(20)-11(12)-diene (exotaxadiene) are precursors to the key intermediate, taxologenic oxetane, indicating the potential existence of multiple routes in the Taxol pathway. Thus, we unveiled a long-elusive step in Taxol biosynthesis.

Parkin enhances sensitivity of paclitaxel to nasopharyngeal carcinoma by activating BNIP3/NIX-mediated mitochondrial autophagy.

As a malignant head and neck cancer, nasopharyngeal carcinoma (NPC) has high morbidity. Parkin expression has been reported to be reduced in NPC tissues and its upregulation could enhance paclitaxel-resistant cell cycle arrest. This study was performed to explore the possible mechanism of Parkin related to B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3)/BNIP3-like (NIX)-mediated mitochondrial autophagy in NPC cells. Initially, after Parkin overexpression or silencing, cell viability and proliferation were evaluated by lactate dehydrogenase and colony formation assays. JC-1 staining was used to assess the mitochondrial membrane potential. In addition, the levels of cellular reactive oxygen species (ROS) and mitochondrial ROS were detected using DCFH-DA staining and mitochondrial ROS (MitoSOX) red staining. The expression of proteins was measured using Western blot. Results showed that Parkin overexpression inhibited, whereas Parkin knockdown promoted the proliferation of paclitaxel-treated NPC cells. Besides, Parkin overexpression induced, whereas Parkin knockdown inhibited mitochondrial apoptosis in paclitaxel-treated NPC cells, as evidenced by the changes of Cytochrome C (mitochondria), Cytochrome C (cytoplasm), BAK, and Bcl-2 expression. Moreover, the levels of ROS, mitochondrial membrane potential, and LC3II/LC3I in paclitaxel-treated C666-1 cells were hugely elevated by Parkin overexpression and were all declined by Parkin knockdown in CNE-3 cells. Furthermore, Parkin upregulation activated, whereas Parkin downregulation inactivated BNIP3/NIX signaling. Further, BNIP3 silencing or overexpression reversed the impacts of Parkin upregulation or downregulation on the proliferation and mitochondrial apoptosis of paclitaxel-treated NPC cells. Particularly, Mdivi-1 (mitophagy inhibitor) or rapamycin (an activator of autophagy) exerted the same effects on NPC cells as BNIP3 silencing or overexpression, respectively. Collectively, Parkin overexpression activated BNIP3/NIX-mediated mitochondrial autophagy to enhance sensitivity to paclitaxel in NPC.

Upregulation of miR-129-5p increases the sensitivity to Taxol through inhibiting HMGB1-mediated cell autophagy in breast cancer MCF-7 cells

Although Taxol has improved the survival of cancer patients as a first-line chemotherapeutic agent, an increasing number of patients develop resistance to Taxol after prolonged treatment. The potential mechanisms of cancer cell resistance to Taxol are not completely clear. It has been reported that microRNAs (miRNAs) are involved in regulating the sensitivity of cancer cells to various chemotherapeutic agents. In this study, we aimed to explore the role of miR-129-5p in regulating the sensitivity of breast cancer cells to Taxol. Cell apoptosis and autophagy, and the sensitivity of MCF-7 cells to Taxol were assessed with a series of in vitro assays. Our results showed that the inhibition of autophagy increased the Taxol-induced apoptosis and the sensitivity of MCF-7 cells to Taxol. Up-regulation of miR-129-5p also inhibited autophagy and induced apoptosis. Furthermore, miR-129-5p overexpression increased the sensitivity of MCF-7 cells to Taxol. High mobility group box 1 (HMGB1), a target gene of miR-129-5p and a regulator of autophagy, was negatively regulated by miR-129-5p. We found that interference of HMGB1 enhanced the chemosensitivity of Taxol by inhibiting autophagy and inducing apoptosis in MCF-7 cells. Taken together, our findings suggested that miR-129-5p increased the chemosensitivity of MCF-7 cells to Taxol through suppressing autophagy and enhancing apoptosis by inhibiting HMGB1. Using miR-129-5p/HMGB1/autophagy-based therapeutic strategies may be a potential treatment for overcoming Taxol resistance in breast cancer.