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Paclitaxel rapidly became one of the most effective anticancer drugs. However, the production of paclitaxel is hindered by substantial challenges, particularly considering the significant quantities of drug required and the inherently low concentration of paclitaxel and its intermediates in plants. Paclitaxel is currently produced in a so-called semi-synthesis in which baccatin III is extracted from Taxus species and chemically converted to paclitaxel. Despite the fact that many of the intermediates of paclitaxel biosynthesis are yet to be experimentally determined, a set of recent papers-facilitated by the sequencing and assembly of three Taxus genomes-has uncovered the minimal gene sets for both baccatin III and paclitaxel biosynthesis. Here we summarize the key milestones towards our understanding of paclitaxel biosynthesis and highlight recent advancements made possible by genome-level analysis of potential key genes involved. We argue that these studies will ultimately pave the way towards the elucidation of the entire paclitaxel biosynthetic pathway and facilitate the industrial production of paclitaxel via synthetic biology approaches. However, several major challenges lie ahead before we can fully tap into the amazing curative potential that taxanes provide., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. Springer Nature Limited.)

Alternaria species (Deuteromycetes, Ascomycota) as ubiquitous fungi and prolific producers of a variety of toxic compounds are a part of microbiomes of plants, humans, and animals, mainly causing disease, allergic reactions, and toxicosis. However, some species have also been reported as endophytic microorganisms with highly bioactive metabolites. Our previous results indicate that potentially endophytic Alternaria species from Cupressaceae produce bioactive metabolites that possibly contribute to plant holobiont's health. Here, a possible mechanism behind this bioactivity is elucidated. As some endophytic fungi are reported to produce cytotoxic taxane diterpenoids, eight potentially endophytic Alternaria isolates from our collection were analyzed for the presence of the key genes of the paclitaxel (Taxol) biosynthetic pathway, i.e., taxadin synthase (ts), 10-deacetylbaccatin III-10-O-acetyltransferase (dbat), and C-13-phenylpropanoid side-chain CoA acyltransferase (bapt). The presence of all genes, i.e., ts, dbat, and bapt, was detected by PCR in six isolates and dbat and bapt in two isolates. Chemical analyses of the fermentation broths by TLC and HPLC chromatography and IR spectroscopy indicated the synthesis of the final product, i.e., paclitaxel. So, we introduce the synthesis of taxane diterpenoids as a possible mechanism by which Alternaria occupies the plant niches and protects the plant holobiont in the presence of competing microorganisms., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In the quest for innovative cancer therapeutics, paclitaxel remains a cornerstone in clinical oncology. However, its complex biosynthetic pathway, particularly the intricate oxygenation steps, has remained a puzzle in the decades following the characterization of the last taxane hydroxylase. The high divergence and promiscuity of enzymes involved have posed significant challenges. In this study, we adopted an innovative approach, combining in silico methods and functional gene analysis, to shed light on this elusive pathway. Our molecular docking investigations using a library of potential ligands uncovered TB574 as a potential missing enzyme in the paclitaxel biosynthetic pathway, demonstrating auspicious interactions. Complementary in vivo assays utilizing engineered S. cerevisiae strains as novel microbial cell factory consortia not only validated TB574's critical role in forging the elusive paclitaxel intermediate, T5αAc-1β,10β-diol, but also achieved the biosynthesis of paclitaxel precursors at an unprecedented yield including T5αAc-1β,10β-diol with approximately 40 mg/L. This achievement is highly promising, offering a new direction for further exploration of a novel metabolic engineering approaches using microbial consortia. In conclusion, our study not only furthers study the roles of previously uncharacterized enzymes in paclitaxel biosynthesis but also forges a path for pioneering advancements in the complete understanding of paclitaxel biosynthesis and its heterologous production. The characterization of T1βOH underscores a significant leap forward for future advancements in paclitaxel production using heterologous systems to improve cancer treatment and pharmaceutical production, thereby holding immense promise for enhancing the efficacy of cancer therapies and the efficiency of pharmaceutical manufacturing., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A recent leading-edge study by Jiang et al. identified two enzymes that are responsible for key reactions in the biosynthesis of baccatin III. The authors successfully reconstructed the baccatin III synthesis pathway with a minimal number of synthetic enzymes in tobacco leaves, laying the foundation for industrial-scale sustainable production of the anticancer drug paclitaxel., Competing Interests: Declaration of interests The authors declare no competing financial interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

UV-B is an important environmental factor that differentially affects plant growth and secondary metabolites. The effects of supplemental ultraviolet-B (sUV-B) exposure (T1, 1.40 kJ·m -2 ·day -1 ; T2, 2.81 kJ·m -2 ·day -1 ; and T3, 5.62 kJ·m -2 ·day -1 ) on the growth biomass, physiological characteristics, and secondary metabolites were studied. Our results indicated that leaf thickness was significantly ( p < 0.05) reduced under T3 relative to the control (natural light exposure, CK); The contents of 6-BA and IAA were significantly reduced ( p < 0.05); and the contents of ABA, 10-deacetylbaccatin III, and baccatin III were significantly ( p < 0.05) increased under T1 and T2. The paclitaxel content was the highest (0.036 ± 0.0018 mg·g -1 ) under T3. The cephalomannine content was significantly increased under T1. Hmgr gene expression was upregulated under T1 and T3. The gene expressions of Bapt and Dbtnbt were significantly ( p < 0.05) upregulated under sUV-B exposure, and the gene expressions of CoA , Ts , and Dbat were significantly ( p < 0.05) downregulated. A correlation analysis showed that the 6-BA content had a significantly ( p < 0.05) positive correlation with Dbat gene expression. The IAA content had a significantly ( p < 0.05) positive correlation with the gene expression of Hmgr , CoA , Ts , and Dbtnbt . The ABA content had a significantly ( p < 0.05) positive correlation with Bapt gene expression. Dbat gene expression had a significantly ( p < 0.05) positive correlation with the 10-deacetylbaccatin content. Hmgr gene expression was positively correlated with the contents of baccatin III and cephalomannine. Bapt gene expression had a significantly ( p < 0.01) positive correlation with the paclitaxel content. A factor analysis showed that the accumulation of paclitaxel content was promoted under T2, which was helpful in clarifying the accumulation of taxane compounds after sUV-B exposure.

Baccatin III is a crucial precursor in the biosynthesis pathway of paclitaxel. Its main sources are extraction from Taxus or chemical synthesis using 10-deacetylbaccatin III (10-DAB) as substrate. However, these preparation approaches exhibit serious limitations, including the low content of baccatin III in Taxus and the complicated steps of chemical synthesis. Heterologous expression of 10-deacetylbaccatin III-10-O-acetyltransferase (TcDBAT) in microbial strains for biotransformation of 10-DAB is a promising alternative strategy for baccatin III production. Here, the promotion effects of glycerol supply and slightly acidic conditions with a low-temperature on the catalysis of recombinant TcDBAT strain were clarified using 10-DAB as substrate. Taxus needles is renewable and the content of 10-DAB is relatively high, it can be used as an effective source of the catalytic substrate 10-DAB. Baccatin III was synthesized by integrating the extraction of 10-DAB from renewable Taxus needles and in situ whole-cell catalysis in this study. 40 g/L needles were converted into 20.66 mg/L baccatin III by optimizing and establishing a whole-cell catalytic bioprocess. The method used in this study can shorten the production process of Taxus extraction for baccatin III synthesis and provide a reliable strategy for the efficient production of baccatin III by recombinant strains and the improvement of resource utilization rate of Taxus needles.

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9α-hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1β-dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5α-ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol., (© 2024. The Author(s).)

Key Message: Taxadiene synthase, taxadiene-5α-hydroxylase, and taxane 13α-hydroxylase genes were introduced into Nicotiana benthamiana, and the improved resistance to lepidoptera pest fall armyworm was reported. Fall armyworm (FAW) is a serious agricultural pest. Genetic engineering techniques have been used to create pest-resistant plant varieties for reducing pest damage. Paclitaxel is a diterpenoid natural metabolite with antineoplastic effects in medicine. However, the effects of taxanes on the growth and development of lepidoptera pests, such as the FAW, are unknown. Here, selected paclitaxel precursor biosynthesis pathway genes, taxadiene synthase, taxane 5α-hydroxylase, and taxane 13α-hydroxylase, were engineered in the heterologous host Nicotiana benthamiana plants. Bioassay experiments showed that the transgenic N. benthamiana plants displayed improved resistance to FAW infestation, with degeneration of gut tissues and induced expression of apoptosis-related genes. Cytotoxicity experiment showed that the paclitaxel precursor, 10-deacetylbaccatin III, is cytotoxic to Sf9 cells, causing cell cycle arrest at the G2/M phase and disorder of the cytoskeleton. Metabolome analysis showed that heterologous expression of taxane genes in N. benthamiana affected the digestive system, steroid hormone and purine metabolism pathways of FAW larvae. In summary, this study provides a candidate approach for FAW control., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated-β-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Paclitaxel (Taxol®) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10β-hydroxylase (T10βH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10β-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10βH, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23 mg/L. Ultimately, the titer of T10OH reached 3.89 mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. KEY POINTS: • The T10βH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. • The expression and activity of T10βH were improved through protein engineering. • De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89 mg/L., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Taxus leaves provide the raw industrial materials for taxol, a natural antineoplastic drug widely used in the treatment of various cancers. However, the precise distribution, biosynthesis, and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown. Matrix-assisted laser desorption/ionization-mass spectrometry imaging analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei, confirming the tissue-specific accumulation of different active metabolites. Single-cell sequencing was used to produce expression profiles of 8846 cells, with a median of 2352 genes per cell. Based on a series of cluster-specific markers, cells were grouped into 15 clusters, suggesting a high degree of cell heterogeneity in T. mairei leaves. Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways. According to the cell-type annotation, most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells; phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells (including the stomatal complex and guard cells); and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells. A number of novel and cell-specific transcription factors involved in secondary metabolite biosynthesis were identified, including MYB17, WRKY12, WRKY31, ERF13, GT&#95;2, and bHLH46. Our research establishes the transcriptional landscape of major cell types in T. mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Taxol, which is a widely used important chemotherapeutic agent, was originally isolated from Taxus stem barks. However, little is known about the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems. Here, we used MALDI-IMS analysis to visualize the taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. A single-cell T. mairei stem atlas was created, providing a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused an uneven taxoid distribution across T. mairei stems. We developed a single-cell strategy to screen novel transcription factors (TFs) involved in taxol biosynthesis regulation. Several TF genes, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68, were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. In summary, we generated a single-cell Taxus stem metabolic atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules' diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a βM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding., Competing Interests: AP, DL, YM, JE, SL, KB, FJ, KA, NG, SK, TM, FG, MO, MS, WF, JD No competing interests declared, (© 2023, Prota et al.)

Plant cell cultures of various yew species are a profitable source of taxoids (taxane diterpenoids) with antitumor activity. So far, despite intensive studies, the principles of the formation of different groups of taxoids in cultured in vitro plant cells have not been fully revealed. In this study, the qualitative composition of taxoids of different structural groups was assessed in callus and suspension cell cultures of three yew species ( Taxus baccata, T. canadensis , and T. wallichiana ) and two T. × media hybrids. For the first time, 14-hydroxylated taxoids were isolated from the biomass of the suspension culture of T. baccata cells, and their structures were identified by high-resolution mass spectrometry and NMR spectroscopy as 7β-hydroxy-taxuyunnanin C, sinenxane C, taxuyunnanine C, 2α,5α,9α,10β,14β-pentaacetoxy-4(20), 11-taxadiene, and yunnanxane. UPLC-ESI-MS screening of taxoids was performed in more than 20 callus and suspension cell lines originating from different explants and grown in over 20 formulations of nutrient media. Regardless of the species, cell line origin, and conditions, most of the investigated cell cultures retained the ability to form taxane diterpenoids. Nonpolar 14-hydroxylated taxoids (in the form of polyesters) were predominant under in vitro culture conditions in all cell lines. These results, together with the literature data, suggest that dedifferentiated cell cultures of various yew species retain the ability to synthesize taxoids, but predominantly of the 14-OH taxoid group compared to the 13-OH taxoids found in plants.

Although bioproduction of Paclitaxel by endophytic fungi is highly considered as an alternative promising source, but its yield is usually very low in comparison with other taxoids. Different strategies i.e., chemical and physical elicitations have been developed in order to overcome the shortage of Paclitaxel production. Paclitaxel biosynthesis is started with terpenoid pathway followed by phenylpropanoid metabolism where a benzoylphenylisoserine moiety is attached to C13 of baccatin III skeleton. This point which is catalyzed by the function of PAM seems to be a bottleneck that limits the rate of Paclitaxel production. Whether phenylpropanoids pathway regulates the taxanes biosynthesis in Cryptosporiopsis tarraconensis endophytic fungus elicited with benzoic acid (BA) was hypothesized in the present paper. The involvement of certain signal molecules and key enzymes of terpenoid and phenylpropanoid metabolism were investigated. According to the results, application of BA promoted a signaling pathway which was started with increase of H2O2 and ABA and continued by increase of NO and MJ, and finally resulted in increase of both phenylpropanoids and taxanes. However, again the rate of Paclitaxel production was lower than other taxoids, and the latter was much lower than phenolics. Therefore, supplying benzoic acid provided the precursor for the common taxan ring production. It is unlikely that Paclitaxel production is merely controlled by side chain production stage. It is more likely that in C. tarraconensis endophytic fungus, similar to Taxus sp., the competition between phenylpropanoid and taxoid pathways for substrate ended in favor of the former. The interaction network which was constructed based on DSPC algorithm confirmed that most compounds with close proximity have shared metabolic pathway relationships. Therefore, it is unlikely that the feeding with a given precursor directly result in increase of a desired metabolite which is composed of different merits., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mohammadi Ballakuti, Ghanati. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The microtubule-targeting paclitaxel (PTX) and docetaxel (DTX) are widely used chemotherapeutic agents. However, the dysregulation of apoptotic processes, microtubule-binding proteins, and multi-drug resistance efflux and influx proteins can alter the efficacy of taxane drugs. In this review, we have created multi-CpG linear regression models to predict the activities of PTX and DTX drugs through the integration of publicly available pharmacological and genome-wide molecular profiling datasets generated using hundreds of cancer cell lines of diverse tissue of origin. Our findings indicate that linear regression models based on CpG methylation levels can predict PTX and DTX activities (log-fold change in viability relative to DMSO) with high precision. For example, a 287-CpG model predicts PTX activity at R 2 of 0.985 among 399 cell lines. Just as precise (R 2 =0.996) is a 342-CpG model for predicting DTX activity in 390 cell lines. However, our predictive models, which employ a combination of mRNA expression and mutation as input variables, are less accurate compared to the CpG-based models. While a 290 mRNA/mutation model was able to predict PTX activity with R 2 of 0.830 (for 546 cell lines), a 236 mRNA/mutation model could calculate DTX activity at R 2 of 0.751 (for 531 cell lines). The CpG-based models restricted to lung cancer cell lines were also highly predictive (R 2 ≥0.980) for PTX (74 CpGs, 88 cell lines) and DTX (58 CpGs, 83 cell lines). The underlying molecular biology behind taxane activity/resistance is evident in these models. Indeed, many of the genes represented in PTX or DTX CpG-based models have functionalities related to apoptosis (e.g., ACIN1, TP73, TNFRSF10B, DNASE1, DFFB, CREB1, BNIP3), and mitosis/microtubules (e.g., MAD1L1, ANAPC2, EML4, PARP3, CCT6A, JAKMIP1). Also represented are genes involved in epigenetic regulation (HDAC4, DNMT3B, and histone demethylases KDM4B, KDM4C, KDM2B, and KDM7A), and those that have never been previously linked to taxane activity (DIP2C, PTPRN2, TTC23, SHANK2). In summary, it is possible to accurately predict taxane activity in cell lines based entirely on methylation at multiple CpG sites., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Taxane agents are of particular interest in non-small cell lung carcinomas (NSCLC) treatment, while multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) limits their clinical efficacy. TM2, a chemically semi-synthesized taxane derivative, exerted significant anti-cancer efficacy in vitro and in vivo, especially against vincristine-resistant and adriamycin-resistant cancer cells. In this study, the anti-cancer effect of TM2 on drug-resistant NSCLC was evaluated both in vitro and in vivo, and the mechanism underlying its anti-MDR activity was further clarified. It was found that TM2 was significantly cytotoxic to cisplatin- and paclitaxel-resistant A549 (human non-small cell lung cancer) cells that overexpressing P-gp, resulting in IC 50 values of 0.19 µM and 0.12 µM. TM2 micelles (5 mg/kg, 10 mg/kg, 20 mg/kg, i.v., 21 days) inhibited the growth of MDR xenograft with the maximal inhibitory rate up to 80.4%. Moreover, TM2 caused cell cycle arrest in the G 2 -M phase and apoptosis in drug-resistant cells through promoting tubulin polymerization, which acted in a way similar to taxane agents. Notably, TM2 acted as a P-gp inhibitor with high binding affinity, which resulted in impaired efflux function through forming H-bonds and ATP hydrolysis to induce P-gp conformational alterations. These findings indicated that TM2 displays anti-MDR activity with the potential for the treatment of NSCLC, which can inhibit P-gp function and stabilize microtubule polymerization., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae., Results: Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses., Conclusions: This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains., (© 2022. The Author(s).)

UV-B radiation is a typical environmental stressor that can promote phytochemical accumulation in plants. Taxus species are highly appreciated due to the existence of bioactive taxoids (especially paclitaxel) and flavonoids. However, the effect of UV-B radiation on taxoid and flavonoid biosynthesis in Taxus cuspidata Sieb. et Zucc. is largely unknown. In the present work, the accumulation of taxoids and flavonoids in T. cuspidata plantlets was significantly induced by 12 and 24 h of UV-B radiation (3 W/m 2 ), and a large number of significantly differentially expressed genes were obtained via transcriptomic analysis. The significant up-regulation of antioxidant enzyme- and flavonoid biosynthesis-related genes (phenylalanine ammonia lyase 1, chalcone synthase 2, flavonol synthase 1, and flavonoid 3', 5'-hydroxylase 2), suggested that UV-B might cause the oxidative stress thus promoting flavonoid accumulation in T. cuspidata. Moreover, the expression of some genes related to jasmonate metabolism and taxoid biosynthesis (taxadiene synthase, baccatin III-3-amino 3-phenylpropanoyltransferase 1, taxadiene-5α-hydroxylase, and ethylene response factors 15) was significantly activated, which indicated that UV-B might initiate jasmonate signaling pathway that contributed to taxoid enhancement in T. cuspidata. Additionally, the identification of some up-regulated genes involved in lignin biosynthesis pathway indicated that the lignification process in T. cuspidata might be stimulated for defense against UV-B radiation. Overall, our findings provided a better understanding of some potential key genes associated with flavonoid and taxoid biosynthesis in T. cuspidata exposed to UV-B radiation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The activation of toll like receptors (TLR) potentially affect the inflammatory tumor microenvironment and thus is associated with tumor growth or inhibition. Cabazitaxel (CAB) has been effectively used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, the immune regulatory role of CAB in the tumor microenvironment is not clear. In this context, we for the first time assessed the immunotherapeutic role of CAB in the TLR3 signalling following activation of Poly I:C in mCRPC cells., Methods and Results: The cytotoxic and apoptotic effects of CAB with the induction of Poly I:C were determined by WST-1, Annexin V, acridine orange, RT-PCR analysis, ELISA assay and immunofluorescence staining in DU-145 mCRPC and HUVEC control cells. Our findings showed that CAB treatment with Poly I:C significantly suppressed the proliferation of DU-145 cells through the induction of apoptosis and caspase activation. Additionally, higher concentration of CAB mediated the activation of TLR3 via increased cytoplasmic and nuclear expression of TLR3, TICAM-1 and IRF-3 in mCRPC cells., Conclusions: Co-treatment of CAB and Poly I:C was more effective in mCRPC cells with less toxicity in control cells. However, further investigations are required to elucidate the molecular mechanisms of TLRs signalling upon CAB treatment at the molecular level to further validate the immunotherapeutic efficacy of CAB in mCRPC., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Taxoids such as paclitaxel (Taxol) are an important class of anticancer drugs that bind β-tubulin and stabilize cellular microtubules. To provide new chemical tools for studies of microtubules, we synthesized derivatives of paclitaxel modified at the 7-position with the small coumarin-derived fluorophore Pacific Blue (PB). Three of these Pacific Blue-Taxoids termed PB-Gly-Taxol, PB-β-Ala-Taxol, and PB-GABA-Taxol bind purified crosslinked microtubules with affinities of 34-265 nM, where the affinity can be tuned based on the length of an amino acid linker. When added to living cells in the presence of verapamil or probenecid as inhibitors of efflux, these compounds allow visualization of the microtubule network by confocal microscopy. We describe methods for the synthesis of these probes, determination of their affinities for crosslinked tubulin, and imaging of microtubules in living HeLa cells. We further describe their uptake by Caco-2 cells and two transporter-deficient Caco-2 knockout cell lines in the absence and presence of efflux inhibitors by flow cytometry. These studies revealed that p-glycoprotein (MDR1) and multidrug-resistance protein 2 (MRP2) are major mediators of efflux of these molecular probes. These compounds provide useful tools for studies of microtubules and cellular efflux transporters in living cells., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Microtubules play crucial role in process of mitosis and cell proliferation, which have been considered as attractive drug targets for anticancer therapy. The aim of this study was to discover novel and chemically diverse tubulin inhibitors for treatment of cancer. In this investigation, the multilayer virtual screening methods, including common feature pharmacophore model, structure-based pharmacophore model and molecular docking, were developed to screen BioDiversity database with 30,000 compounds. A total of 102 compounds were obtained by the virtual screening, and further filtered by diverse chemical clusters with desired properties and PAINS analysis. Finally, 50 compounds were selected and submitted to the biological evaluation. Among these hits, hits 8 and 30 with novel scaffolds displayed stronger antiproliferative activity on four human tumor cells including Hela, A549, MCF-7, and HepG2. Moreover, the two hits were subsequently submitted to molecular dynamic simulations of 90 ns with the aim of exploring the stability of ligand-protein interactions into the binding pocket, and further probing the mechanism of the interaction between tubulin and hits. The molecular dynamic simulation results revealed there had stronger interactions between tubulin and hits in equilibrium state. Therefore, the hits 8 and 30 have been well characterized as lead compounds for developing new tubulin inhibitors with potential anticancer activity., (© 2021 Wiley Periodicals LLC.)

Background: Effective treatment of glioma requires a nanocarrier that can cross the blood-brain barrier (BBB) to target the tumor lesion. In the current study, elemene (ELE) and cabazitaxel (CTX) liposomes were prepared by conjugating liposomes with transferrin (Tf) and embedding the cell membrane proteins of RG2 glioma cells into liposomes (active-targeting biomimetic liposomes, Tf-ELE/CTX@BLIP), which exhibited effective BBB infiltration to target glioma., Results: The findings showed that Tf-ELE/CTX@BLIP was highly stable. The liposomes exhibited highly significant homologous targeting and immune evasion in vitro and a 5.83-fold intake rate compared with classical liposome (ELE/CTX@LIP). Bioluminescence imaging showed increased drug accumulation in the brain and increased tumor penetration of Tf-ELE/CTX@BLIP in orthotopic glioma model nude mice. Findings from in vivo studies indicated that the antitumor effect of the Tf-ELE/CTX@BLIP led to increased survival time and decreased tumor volume in mice. The average tumor fluorescence intensity after intravenous administration of Tf-ELE/CTX@BLIP was 65.2, 12.5, 22.1, 6.6, 2.6, 1.5 times less compared with that of the control, CTX solution, ELE solution, ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP groups, respectively. Histopathological analysis showed that Tf-ELE/CTX@BLIP were less toxic compared with administration of the CTX solution., Conclusion: These findings indicate that the active-targeting biomimetic liposome, Tf-ELE/CTX@BLIP, is a promising nanoplatform for delivery of drugs to gliomas., (© 2021. The Author(s).)

Lung cancer (LC) is one of the leading causes of cancer-related death. As one of the key features of tumor microenvironment, hypoxia conditions are associated with poor prognosis in LC patients. Upregulation of hypoxic-induced factor-1α (HIF-1α) leads to the activation of various factors that contribute to the increased drug resistance, proliferation, and migration of tumor cells. Apurinic/apyrimidinic endonuclease-1 (APEX1) is a multi-functional protein that regulates several transcription factors, including HIF-1α, that contribute to tumor growth, oxidative stress responses, and DNA damage. In this study, we explored the mechanisms underlying cell responses to hypoxia and modulation of APEX1, which regulate HIF-1α and downstream pathways. We found that hypoxia-induced APEX1/HIF-1α pathways regulate several key cellular functions, including reactive oxygen species (ROS) production, carbonic anhydrase 9 (CA9)-mediated intracellular pH, migration, and angiogenesis. Cephalomannine (CPM), a natural compound, exerted inhibitory effects in hypoxic LC cells via the inhibition of APEX1/HIF-1α interaction in vitro and in vivo. CPM can significantly inhibit cell viability, ROS production, intracellular pH, and migration in hypoxic LC cells as well as angiogenesis of HUVECs under hypoxia through the inhibition of APEX1/HIF-1α interaction. Taken together, CPM could be considered as a promising compound for LC treatment.

Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10β-hydroxylase (T10βOH) and a newly putative β-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

Breast cancer is the leading cause of death by malignancy among women worldwide. Clinical data and molecular characteristics of breast tumors are essential to guide clinician's therapeutic decisions. In the new era of precision medicine, that aims at personalizing the treatment for each patient, there is urgent need to identify robust companion biomarkers for new targeted therapies. This review focuses on ATIP3, a potent anti-cancer protein encoded by candidate tumor suppressor gene MTUS1 , whose expression levels are markedly down-regulated in breast cancer. ATIP3 is a microtubule-associated protein identified both as a prognostic biomarker of patient survival and a predictive biomarker of breast tumors response to taxane-based chemotherapy. We present here recent studies pointing out ATIP3 as an emerging anti-cancer protein and a potential companion biomarker to be combined with future personalized therapy against ATIP3-deficient breast cancer.

Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Taxol (paclitaxel), a plant-derived anticancer drug, has been among the most successful anticancer drugs of natural origin. Endophytic fungi have been proposed as a prominent alternative source for Taxol and its intermediate Baccatin III, however the very low yields remain a hinderance to their commercial utilization. Significant research efforts towards this end are underway globally. Here, we report the results on our earlier reported Taxol-producing endophytic fungus, Fusarium solani from the standpoint of spores as seed inoculum and media selection for enhanced Taxol and baccatin III yields. Spores produced on M1D medium with 94.76% viability were used for further media optimization for Taxol and Baccatin III production in five different liquid media under static and shaker condition at different cultivation days. Taxol and Baccatin III when quantified through competitive inhibition enzyme immunoassay (CIEIA), showed maximum production at 136.3 µg L -1 and 128.3 µg L -1 , respectively in the modified flask basal broth (MFBB) under shaking condition. Further, two important genes of this pathway, namely taxane 13α-hydroxylase (T13αH) and 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) have been identified in this fungus. These findings are hoped to assist in further manipulation and metabolic engineering of the parent F. solani strain towards the enhanced production of Taxol and baccatin III.

TAS-119 is a novel orally active, selective inhibitor of Aurora kinase A identified as a clinical candidate for efficacy testing in combination with taxanes. In vitro , TAS-119 enhanced cell growth inhibition of paclitaxel in multiple human cancer cell lines derived from various tissues, including paclitaxel-resistant cell lines. Interestingly, TAS-119 did not enhance paclitaxel antitumor activity in normal lung diploid fibroblast cell lines WI-38 and MRC5. In vivo , TAS-119 enhanced the antitumor efficacy of paclitaxel and docetaxel in multiple models at doses inhibitory to Aurora A in tumors. Moreover, the drug combination was well tolerated, and TAS-119 did not exaggerate clinically documented side effects of taxanes, neutropenia and neurotoxicity, in rats. The same TAS-119 concentration enhanced the cell growth inhibitory activity of three clinically approved taxanes, paclitaxel, docetaxel, and cabazitaxel. The degree of enhancement calculated as fold of change of the IC 50 value for each taxane was almost the same among the three taxanes. We conducted in vitro and in vivo experiments to develop an optimized combination therapy regimen for TAS-119 with paclitaxel/docetaxel. Using in vitro and in vivo models, we tested the drug administration order for TAS-119 combined with paclitaxel and the TAS-119 treatment duration. The best regimen in preclinical models was combining paclitaxel or docetaxel treatment with 4 days of TAS-119 dosing, which was initiated on the same day as the paclitaxel or docetaxel administration or one day later. This information provided guidance for the design of a clinical trial of TAS-119 and paclitaxel or docetaxel combination., (©2020 American Association for Cancer Research.)

Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatment of cancer. Taxus spp. biofactories have been used for taxane production, but the lack of knowledge about the taxane biosynthetic pathway and its molecular regulation hinders their optimal function. The difficulties in introducing foreign genes in Taxus spp. genomes hinder the study of the molecular mechanisms involved in taxane production, and a new approach is required to overcome them. In this study, a reliable, simple and fast method to obtain Taxus � media protoplasts was developed, allowing their manipulation in downstream assays for the study of physiological changes in Taxus spp. cells. Using this method, Taxus protoplasts were transiently transfected for the first time, corroborating their suitability for transfection assays and the study of specific physiological responses. The two assayed transcription factors (BIS2 and TSAR2) had a positive effect on the expression of several taxane-related genes, suggesting their potential use for the improvement of taxane yields. Furthermore, the results indicate that the developed method is suitable for obtaining T. � media protoplasts for transfection with the aim of unraveling regulatory mechanisms in taxane production., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

For patients with metastatic prostate cancer, the 5-year survival rate of 31% points to a need for novel therapies and improvement of existing modalities. We propose that p53 gene therapy and chemotherapy, when combined, will provide superior tumor cell killing for the treatment of prostate carcinoma. To this end, we have developed the AdRGD-PGp53 vector which offers autoregulated expression of p53, resulting in enhanced tumor cell killing in vitro and in vivo. Here, we combined AdRGD-PGp53 along with the chemotherapy drugs used in the clinical treatment of prostate carcinoma, mitoxantrone, docetaxel, or cabazitaxel. Our results indicate that all drugs increase phosphorylation of p53, leading to improved induction of p53 targets. In vitro experiments reveal that AdRGD-PGp53 sensitizes prostate cancer cells to each of the drugs tested, conferring increased levels of cell death. In a xenograft mouse model of in situ gene therapy, AdRGD-PGp53 treatment, when combined with cabazitaxel, drastically reduced tumor progression and increased survival rates to 100%. Strikingly, we used a sub-therapeutic dose of cabazitaxel thus avoiding leukopenia, yet still showed potent anti-tumor effects when combined with AdRGD-PGp53 in this mouse model. The AdRGD-PGp53 approach warrants further development for its application in gene therapy of prostate carcinoma.

Overexpression of ATP-binding cassette subfamily B member 1 (ABCB1)-encoded multidrug resistance protein 1 (MDR1) constitutes a major mechanism of cancer drug resistance including docetaxel (DTX) and cabazitaxel (CTX) resistance in castration-resistant prostate cancer (CRPC). However, no therapeutics that targets MDR1 is available at clinic for taxane sensitization. We report here that retinoic acid receptor-related orphan receptor γ (RORγ), a nuclear receptor family member, unexpectedly mediates MDR1/ABCB1 overexpression. RORγ plays an important role in controlling the functions of subsets of immune cells and has been an attractive target for autoimmune diseases. We found that its small-molecule antagonists are efficacious in resensitizing DTX and CTX cross-resistant CRPC cells and tumors to taxanes in both androgen receptor-positive and -negative models. Our mechanistic analyses revealed that combined treatment with RORγ antagonists and taxane elicited a robust synergy in killing the resistant cells, which involves a coordinated alteration of p53, Myc, and E2F-controlled programs critical for both intrinsic and extrinsic apoptosis, survival, and cell growth. Our results suggest that targeting RORγ with small-molecule inhibitors is a novel strategy for chemotherapy resensitization in tumors with MDR1 overexpression., (©2019 American Association for Cancer Research.)

Currently, chemotherapy is one of the mainstays of oncologic therapies. But the efficacy of chemotherapy is often limited by drug resistance and severe side effects. Consequently, it is becoming increasingly important to investigate the underlying mechanism and overcome the problem of anticancer chemotherapy resistance. The solute carrier organic anion transporter family member 1B3 (SLCO1B3), a functional transporter normally expressed in the liver, transports a variety of endogenous and exogenous compounds, including hormones and their conjugates as well as some anticancer drugs. The extrahepatic expression of SLCO1B3 has been detected in different cancer cell lines and cancer tissues. Recently, accumulating data indicates that the abnormal expression and function of SLCO1B3 are involved in resistance to anticancer drugs, such as taxanes, camptothecin and its analogs, SN-38, and Androgen Deprivation Therapy (ADT) in breast, prostate, lung, hepatic, and colorectal cancer, respectively. Thus, more investigations have been implemented to identify the potential SLCO1B3-related mechanisms of cancer drug resistance. In this review, we focus on the emerging roles of SLCO1B3 protein in the development of cancer chemotherapy resistance and briefly discuss the mechanisms of resistance. Elucidating the function of SLCO1B3 in chemoresistance may bring out novel therapeutic strategies for cancer treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Trees of the genus Taxus are highly valuable medicinal plants with multiple pharmacological effects on various cancer treatments. Paclitaxel from Taxus trees is an efficient and widely used anticancer drug, however, the accumulation of taxoids and other active ingredients can vary greatly among Taxus species. In our study, the metabolomes of three Taxus species have been investigated., Results: A total of 2246 metabolites assigned to various primary and secondary metabolic pathways were identified using an untargeted approach. Analysis of differentially accumulated metabolites identified 358 T. media-, 220 T. cuspidata-, and 169 T. mairei-specific accumulated metabolites, respectively. By searching the metabolite pool, 7 MEP pathway precursors, 11 intermediates, side chain products and derivatives of paclitaxel, and paclitaxel itself were detected. Most precursors, initiated intermediates were highly accumulated in T. mairei, and most intermediate products approaching the end point of taxol biosynthesis pathway were primarily accumulated in T. cuspidata and T. media. Our data suggested that there were higher-efficiency pathways to paclitaxel in T. cuspidata and T. media compared with in T. mairei. As an important class of active ingredients in Taxus trees, a majority of flavonoids were predominantly accumulated in T. mairei rather than T. media and T. cuspidata. The variations in several selected taxoids and flavonoids were confirmed using a targeted approach., Conclusions: Systematic correlativity analysis identifies a number of metabolites associated with paclitaxel biosynthesis, suggesting a potential negative correlation between flavonoid metabolism and taxoid accumulation. Investigation of the variations in taxoids and other active ingredients will provide us with a deeper understanding of the interspecific differential accumulation of taxoids and an opportunity to accelerate the highest-yielding species breeding and resource utilization.

Production of the anticancer drug Taxol and its precursors in heterologous hosts is more sustainable than extraction from tissues of yew trees or chemical synthesis. Although attempts to engineer the Taxol pathway in microbes have made significant progress, challenges such as functional expression of plant P450 enzymes remain to be addressed. Here, we introduce taxadiene synthase, taxadiene-5α-hydroxylase, and cytochrome P450 reductase in a high biomass plant Nicotiana benthamiana. Using a chloroplastic compartmentalized metabolic engineering strategy, combined with enhancement of isoprenoid precursors, we show that the engineered plants can produce taxadiene and taxadiene-5α-ol, the committed taxol intermediates, at 56.6 μg g -1  FW and 1.3 μg g -1  FW, respectively. In addition to the tools and strategies reported here, this study highlights the potential of Nicotiana spp. as an alternative platform for Taxol production.

10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) is a key rate-limiting enzyme of the Taxol biosynthetic pathway, which is uncharacterized in Taxol-producing endophytic fungi. Here, an open reading frame of DBAT was cloned from the Taxol-producing endophytic fungus Lasiodiplodia theobromae (LtDBAT). The LtDBAT enzyme was heterologously expressed and purified by the affinity and gel filtration chromatography methods. The molecular weight of the purified protein was 49 kDa and its identity was confirmed by western blot. The purified LtDBAT enzyme was capable of catalyzing 10-deacetylbaccatin III into baccatin III, as shown by liquid chromatography-mass spectroscopy. The mass spectra of baccatin III were identical to the authentic baccatin III. The LtDBAT enzyme was characterized and the kinetic parameters of catalysis were determined. In addition, localization of LtDBAT was performed by using confocal microscopy and the result showed that the enzyme was localized in lipid droplets. Together, this study provides biochemical insights into the fungal recombinant DBAT enzyme that is involved in the Taxol biosynthetic pathway. In the near future, engineering of the LtDBAT enzyme and the Taxol biosynthetic pathway in endophytic fungi could be an eco-friendly and economically feasible alternative source for production of Taxol and its precursors., (© FEMS 2019.)

This is the first study to isolate the taxoid taxuyunnanin C (group of 14-hydroxylated taxoids) from the biomass of suspension cell culture of the Canadian yew (Taxus canadensis). According to available data, this is the first report of the presence of nonpolar (polyacylated) forms of 14-hydroxylated taxoids, including taxuyunnanin C, in T. canadensis.

Background: Taxol is an efficient anticancer drug; however, the accumulation of taxoids can vary hugely among Taxus species. The mechanism underlying differential accumulation of taxoids is largely unknown. Thus, comparative analysis of the transcriptomes in three Taxus species, including T. media, T. mairei and T. cuspidata, was performed., Results: KEGG enrichment analysis revealed that the diterpenoid biosynthesis and cytochrome P450 pathways were significantly enriched in different comparisons. Differential expressions of these taxol biosynthesis related genes might be a potential explanation for the interspecific differential accumulation of taxol and its derivatives. Besides, the sequences of several MEP pathway-associated genes, such as DXS, DXR, MCT, CMK, MDS, HDS, HDR, IPPI, and GGPPS, were re-assembled based on independent transcriptomes from the three Taxus species. Phylogenetic analysis of these MEP pathway-associated enzymes also showed a high sequence similarity between T. media and T. cuspidata. Moreover, 48 JA-related transcription factor (TF) genes, including 10 MYBs, 5 ERFs, 4 RAPs, 3 VTCs, and 26 other TFs, were analyzed. Differential expression of these JA-related TF genes suggested distinct responses to exogenous JA applications in the three Taxus species., Conclusions: Our results provide insights into the expression pattern and sequence similarity of several taxol biosynthesis-related genes in three Taxus species. The data give us an opportunity to reveal the mechanism underlying the variations in the taxoid contents and to select the highest-yielding Taxus species.

Covering: up to May 2018The verticillene family of 6,12-membered ring-fused diterpenes are found in plants, liverworts, corals and insects. Carbocations derived from verticillene hydrocarbons are central intermediates in the biosynthesis of the taxane and the phomactin families of polycyclic natural products. This perspective delineates these unique biosynthetic interrelationships, which are reinforced by recent biomimetic synthesis investigations, alongside quantum chemical calculations and targeted engineering studies of the taxadiene synthase (TXS) cascade.

The present study investigated the feasibility of improving the tumor‑targeting efficacy and decreasing the toxicity of liposomal cabazitaxel (Cab) with aptamer modification. The process involved preparing aptamer (TLS1c)‑modified liposomes and studying the behavior of the liposomes in vitro and in vivo. TLS1c as an aptamer, which has high specificity for BNL 1ME A.7R.1 (MEAR) cells, was conjugated with Cab liposomes (Cab/lipo) to enhance MEAR tumor tissue targeting. Confocal laser scanning microscopy and flow cytometry analyses demonstrated that the fluorescence of the liposomes modified with the aptamer was notably stronger compared with that of the unmodified liposomes. Furthermore, the biodistribution data of the modified liposomes tested in tumor‑bearing mice revealed high specificity of biotinylated TLS1c‑modified Cab/lipo (BioTL‑Cab/lipo) for tumor tissues. Furthermore, the modified liposomes demonstrated decreased cytotoxicity and simultaneously retained potent inhibition against tumor growth. It is likely that the specific binding of the aptamer (TLS1c) to the targeted cells (MEAR) facilitates the binding of the liposomes to the targeted cells. Therefore, BioTL‑Cab/lipo may be considered as a promising delivery system to improve cell targeting and reduce drug toxicity in the treatment of cancer.

The β-glycoside hydrolases (LXYL-P1-1 and LXYL-P1-2) from Lentinula edodes (strain M95.33) can specifically hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) to form 10-deacetyltaxol for the semi-synthesis of Taxol. Our previous study showed that both the I368T mutation in LXYL-P1-1 and the T368E mutation in LXYL-P1-2 could increase the enzyme activity, which prompted us to investigate the effect of the I368E mutation on LXYL-P1-1 activity. In this study, the β-xylosidase and β-glucosidase activities of LXYL-P1-1 I368E were 1.5 and 2.2 times higher than those of LXYL-P1-1. Most importantly, combination of I368E and V91S exerted the cumulative effects on the improvement of the enzyme activities and catalytic efficiency. The β-xylosidase and β-glucosidase activities of the double mutant LXYL-P1-1 V91S/I368E were 3.2 and 1.7-fold higher than those of LXYL-P1-1 I368E . Similarly, the catalytic efficiency of LXYL-P1-1 V91S/I368E on 7-β-xylosyl-10-deacetyltaxol was 1.8-fold higher than that of LXYL-P1-1 I368E due to the dramatic increase in the substrate affinity. Molecular docking results suggest that the V91S and I368E mutation might positively promote the interaction between enzyme and substrate through altering the loop conformation near XDT and increasing the hydrogen bonds among Ser 91 , Trp 301 , and XDT. This study lays the foundation for exploring the relationship between the structure and function of the β-glycoside hydrolases.

Drug receptor site occupancy is a central pharmacology parameter that quantitatively relates the biochemistry of drug binding to the biology of drug action. Taxanes and epothilones bind to overlapping sites in microtubules (MTs) and stabilize them. They are used to treat cancer and are under investigation for neurodegeneration. In cells, they cause concentration-dependent inhibition of MT dynamics and perturbation of mitosis, but the degree of site occupancy required to trigger different effects has not been measured. We report a live cell assay for taxane-site occupancy, and relationships between site occupancy and biological effects across four drugs and two cell lines. By normalizing to site occupancy, we were able to quantitatively compare drug activities and cell sensitivities independent of differences in drug affinity and uptake/efflux kinetics. Across all drugs and cells tested, we found that inhibition of MT dynamics, postmitotic micronucleation, and mitotic arrest required successively higher site occupancy. We also found interesting differences between cells and drugs, for example, insensitivity of the spindle assembly checkpoint to site occupancy. By extending our assay to a mouse xenograft tumor model, we estimated the initial site occupancy required for paclitaxel to completely prevent tumor growth as 80%. The most important cellular action of taxanes for cancer treatment may be formation of micronuclei, which occurs over a broad range of site occupancies., Competing Interests: The authors declare no conflict of interest.

The combined use of elicitors can be an effective way to increase the production of secondary metabolites (SMs) in plant cell, tissue and organ cultures. This study investigated the effects of a salicylic acid (SA) pretreatment and different glucose levels on the growth, biochemical traits and taxane production in a Taxus baccata callus culture. For this purpose, after a pretreatment with SA (5 μM), three-month-old calli were cultured on B5 medium fortified with different concentrations of glucose (0, 0.5, 1, 2 and 3%), and they were compared with calli cultured on a B5 medium supplemented only with glucose. When the calli were harvested at 21 days, their fresh weight (g), dry weight (g) and cell viability (%) had decreased significantly (p < 0.05) with the higher glucose concentrations. The glucose treatment increased the hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) content, and caused oxidative stress in treated tissues. The lower H 2 O 2 content and oxidative stress was associated with an increased antioxidant enzyme activity in the SA-pretreated samples, which resulted in less membrane damage and improved growth and cell viability under the glucose treatment compared to the control. By reducing the activity of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO), the SA pretreatment reduced the production and oxidation of phenolic compounds under the glucose treatment; this decrease was associated with less browning of tissues and higher viability. Increases in taxol (5.1-fold) and total taxanes (3.5-fold) in the SA-pretreated calli cultured on the medium containing 2% glucose, compared to the control, indicated that the two treatments had a significant effect on taxane production in the T. baccata callus culture., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Plant cell biofactories represent a promising solution to the increasing demand for plant-derived compounds, but there are still limiting factors that prevent optimal production, including the loss of yield during in vitro maintenance. Our results reveal a clear correlation between genomic methylation levels and a progressive decline in taxane production in Taxus spp. cell cultures. A comparative study of two cell lines, one 10 years old and low productive and the other new and high productive, revealed important differences in appearance, growth, taxane accumulation and expression levels of several taxane biosynthetic genes. Differences in taxane content and gene expression profile indicate an altered pathway regulation and that the BAPT gene, located in the center of the expression network of taxane biosynthetic genes, is active in a potentially flux-limiting step. The methylation patterns of the BAPT gene were studied in both cell lines by bisulfite sequencing, which revealed high rates of CHH methylated cytosines on the core promoter. Using a bioinformatics approach, this hotspot was identified as a Y-patch promoter element. The Y-patch may play a key role in the epigenetic regulation of the taxane biosynthetic pathway, which would open up novel genetic engineering strategies toward stable and high productivity.