Advances of antitumor drug discovery in traditional Chinese medicine and natural active products by using multi-active components combination.

The antitumor efficacy of Chinese herbal medicines has been widely recognized. Leading compounds such as sterols, glycosides, flavonoids, alkaloids, terpenoids, phenylpropanoids, and polyketides constitute their complex active components. The antitumor monomers derived from Chinese medicine possess an attractive anticancer activity. However, their use was limited by low bioavailability, significant toxicity, and side effects, hindering their clinical applications. Recently, new chemical entities have been designed and synthesized by combining natural drugs with other small drug molecules or active moieties to improve the antitumor activity and selectivity, and reduce side effects. Such a novel conjugated drug that can interact with several vital biological targets in cells may have a more significant or synergistic anticancer activity than a single-molecule drug. In addition, antitumor conjugates could be obtained by combining pharmacophores containing two or more known drugs or leading compounds. Based on these studies, the new drug research and development could be greatly shortened. This study reviews the research progress of conjugates with antitumor activity based on Chinese herbal medicine. It is expected to serve as a valuable reference to antitumor drug research and clinical application of traditional Chinese medicine.

Challenges and potential for improving the druggability of podophyllotoxin-derived drugs in cancer chemotherapy.

Covering: up to 2020As a main bioactive component of the Chinese, Indian, and American Podophyllum species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.

Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes.

BACKGROUND: Yeast strains that are tolerant to multiple environmental stresses are highly desired for various industrial applications. Despite great efforts in identifying key genes involved in stress tolerance of budding yeast Saccharomyces cerevisiae, the effects of de novo purine biosynthesis genes on yeast stress tolerance are still not well explored. Our previous studies showed that zinc sulfate addition improved yeast acetic acid tolerance, and key genes involved in yeast stress tolerance were further investigated in this study. RESULTS: Three genes involved in de novo purine biosynthesis, namely, ADE1, ADE13, and ADE17, showed significantly increased transcription levels by zinc sulfate supplementation under acetic acid stress, and overexpression of these genes in S. cerevisiae BY4741 enhanced cell growth under various stress conditions. Meanwhile, ethanol productivity was also improved by overexpression of the three ADE genes under stress conditions, among which the highest improvement attained 158.39% by ADE17 overexpression in the presence of inhibitor mixtures derived from lignocellulosic biomass. Elevated levels of adenine-nucleotide pool "AXP" ([ATP] + [ADP] + [AMP]) and ATP content were observed by overexpression of ADE17, both under control condition and under acetic acid stress, and is consistent with the better growth of the recombinant yeast strain. The global intracellular amino acid profiles were also changed by overexpression of the ADE genes. Among the changed amino acids, significant increase of the stress protectant γ-aminobutyric acid (GABA) was revealed by overexpression of the ADE genes under acetic acid stress, suggesting that overexpression of the ADE genes exerts control on both purine biosynthesis and amino acid biosynthesis to protect yeast cells against the stress. CONCLUSION: We proved that the de novo purine biosynthesis genes are useful targets for metabolic engineering of yeast stress tolerance. The engineered strains developed in this study with improved tolerance against multiple inhibitors can be employed for efficient lignocellulosic biorefinery to produce biofuels and biochemicals.

Structural Insights into the Inhibition of Tubulin by the Antitumor Agent 4ß-(1,2,4-triazol-3-ylthio)-4-deoxypodophyllotoxin.

The colchicine domain is widely recognized as the binding site of microtubule depolymerization agents for anticancer drug design. Almost all of the drugs targeting the colchicine domain have been confirmed to bind to the tubulin ß-subunit. Here we studied a crystal structure (2.3 Å) of the complex between tubulin and 4ß-(1,2,4-triazol-3-ylthio)-4-deoxypodophyllotoxin (compound 1S) with superior antitumor activity, which was designed on the basis of the colchicine domain and synthesized in our previous work. A distinct binding model of the colchicine domain was found in the complexes of tubulin with compound 1S. From a comparison of the crystal structures of tubulin-compound 1S and tubulin-colchicine complexes, the side chains of the T7 loop of ß-tubulin flip outward and the T5 loop of α-tubulin changes its conformation. It has been shown that the ß-subunit T7 loop reversibly participates in resistance to straightening that opposes microtubule assembly by flipping in and out. Together with the biochemical results from compound 1S, the structural data highlight the main contributors in the α-subunits and the colchicine domain ß-subunits: the dual-target binding sites in the α-T7 loop and ß-H7-T7 loop of tubulin. Compound 1S can synchronously bind to αß-tubulin. The structures also highlight common features for the design and development of novel potent microtubule destabilizing agents.

Aroma improvement by repeated freeze-thaw treatment during Tuber melanosporum fermentation.

The aroma attributes of sulfurous, mushroom and earthy are the most important characteristics of the aroma of Tuber melanosporum. However, these three aroma attributes are absent in the T. melanosporum fermentation system. To improve the quality of the aroma, repeated freeze-thaw treatment (RFTT) was adopted to affect the interplay of volatile organic compounds (VOCs). Using RFTT, not only was the score on the hedonic scale of the aroma increased from the "liked slightly" to the "liked moderately" grade, but the aroma attributes of sulfurous, mushroom and earthy could also be smelled in the T. melanosporum fermentation system for the first time. A total of 29 VOCs were identified, and 9 compounds were identified as the key discriminative volatiles affected by RFTT. Amino acid analysis revealed that methionine, valine, serine, phenylalanine, isoleucine and threonine were the key substrates associated with the biosynthesis of the 9 key discriminative VOCs. This study noted that amino acid metabolism played an important role in the regulation of the aroma of the T. melanosporum fermentation system.

Comparison of carbon-sulfur and carbon-amine bond in therapeutic drug: 4ß-S-aromatic heterocyclic podophyllum derivatives display antitumor activity.

Herein is a first effort to systematically study the significance of carbon-sulfur (C-S) and carbon-amine (C-NH) bonds on the antitumor proliferation activity of podophyllum derivatives and their precise mechanism of apoptosis. Compared with the derivative modified by a C-NH bond, the derivative modified by a C-S bond exhibited superior antitumor activity, the inhibition activity of target proteins tubulin or Topo II, cell cycle arrest, and apoptosis induction. Antitumor mechanistic studies showed that the death receptor and the mitochondrial apoptotic pathways were simultaneously activated by the C-S bond modified aromatic heterocyclic podophyllum derivatives with a higher cellular uptake percentage of 60-90% and induction of a higher level of reactive oxygen species (ROS). Only the mitochondrial apoptotic pathway was activated by the C-NH bond modified aromatic heterocyclic podophyllum derivatives, with a lower cellular uptake percentage of 40-50%. This study provided insight into effects of the C-S and C-NH bond modification on the improvement of the antitumor activity of Podophyllum derivatives.

Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing.

Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the ß-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for ß-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100 genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained15 targeted mutations and produced 2.0 g/L ß-carotene in fed-batch fermentation.

WIP1 stimulates migration and invasion of salivary adenoid cystic carcinoma by inducing MMP-9 and VEGF-C.

The wild-type p53 induced phosphatase 1 (WIP1) is an oncogene overexpressed in a variety of human cancers. Here, we demonstrated that WIP1 silencing reduced MMP-9 and VEGF-C expression as well as migration and invasion of salivary adenoid cystic carcinoma (ACC) cells. Overexpression of MMP-9 or VEGF-C restored migration and invasion in WIP1 knockdown cells, indicating that MMP-9 and VEGF-C are downstream targets of WIP1 signaling. Levels of cyclin D1 and c-Myc, targets of Wnt/ß-catenin pathway, were significantly decreased by WIP1 silencing. In addition, WIP1 expression was positively associated with metastasis and prognosis of ACC patients as well as with MMP-9 or VEGF-C in ACC tissues.

Determination of the nucleosides and nucleobases in Tuber samples by dispersive solid-phase extraction combined with liquid chromatography-mass spectrometry.

A simple, fast and inexpensive method based on dispersive solid phase extraction (DSPE) combined with LC-MS was developed for simultaneous determination of 7 nucleosides and nucleobases (i.e., adenine, hypoxanthine, uridine, adenosine, guanine, guanosine, and inosine) in Tuber fruiting-bodies and fermentation mycelia. The DSPE procedure was firstly introduced to remove the protein interference from sample solutions, and D3520 macroporous resin was chosen as the DSPE sorbent because of its high removal capability on protein interferences, but low adsorption rate on analytes. Besides, key parameters on DSPE procedure (i.e., macroporous resin type, macroporous resin amount, methanol concentration, and vortex time) were optimized, and the protein removal efficacy could achieve about 95% after the process optimization. Though the method validation test, the DSPE-LC-MS method was confirmed to be precise, accurate and sensitive, and the column blinding problem was solved successfully. By using this established method, the total amount of nucleosides and nucleobases in the fermentation mycelia was determined to range from 4881.5 to 12,592.9µgg⁻¹, which was about 2-25 times higher than the fruiting-bodies (from 498.1 to 2274.1µgg⁻¹). The formulation of nucleosides and nucleobases in the fermentation mycelia maintained relatively constant, while the formulation in Tuber fruiting-bodies varied significantly with their species. Hierarchical cluster analysis (HCA) showed the formulation similarity of nucleosides and nucleobases between Tuber fermentation mycelia and the fruiting-bodies of Tuber indicum and Tuber himalayense. From the viewpoint of nucleosides and nucleobases, this work confirms the potentiality of Tuber fermentation mycelia as the alternative resource for its fruiting-bodies.

Quantitative analysis for the effect of plant oil and fatty acid on Tuber melanosporum culture by uniform design combined with partial least squares regression.

Uniform design and partial least squares regression were adopted to quantitatively analyze the effects of plant oil and fatty acid as well as their addition amount and addition time on the performance of Tuber melanosporum submerged fermentation. The regression models showed the optimal scheme was the addition of 1.2 mL soybean oil on day 9, which was validated by experiment. The maximal biomass of 25.89 +/- 1.01 g/L and extracellular polysaccharides (EPS) production of 6.51 +/- 0.68 g/L were obtained, which were enhanced by 18.5% and 86%, respectively. Palmitic acid was identified to be the key component to stimulate the cell growth and EPS biosynthesis, and stearic acid was beneficial for the production of intracellular polysaccharides. Not only the biomass but also EPS production obtained in this work are the highest reported in the batch fermentation of truffle.

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction.

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 muM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 +/- 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.

Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors.

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.

A novel three-stage light irradiation strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of ganoderic acid and Ganoderma polysaccharides.

A novel three-stage light irradiation strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of bioactive metabolites ganoderic acid (GA) and Ganoderma polysaccharides was developed. Significance of light quality, i.e., blue light (390-500 nm, lambda(max) = 470 nm), red light (560-700 nm, lambda(max) = 625 nm), and white light (400-740 nm, lambda(max) = 550 nm), was studied at first. Interestingly, there was a gradual decrease trend of GA content after the culture of day 2 when the maximal GA content was obtained, while GA content decreased slowly under white light irradiation after day 6. The dark environment was favorable to the specific GA biosynthesis (i.e., GA content) before day 6, and after that the optimum was white light irradiation. A relatively lower irradiation density of white light (i.e., 0.94 and 2.82 W/m(2)) was beneficial for the specific GA biosynthesis before day 6, while GA content was higher under higher irradiation density of white light (i.e., 4.70 and 9.40 W/m(2)) at the later-stage of cultivation. 4.70 W/m(2) white light irradiation culture was the best from the viewpoint of GA accumulation. Therefore, a two-stage light irradiation strategy by combing the first 2 days dark culture with the following 4.70 W/m(2) white light irradiation culture was developed. The highest GA production in the two-stage culture was 276.0 +/- 12.5 mg/L, which was increased by 19% compared to 4.70 W/m(2) white light irradiation culture (i.e., 232.4 +/- 15.8 mg/L) and by 178% compared to the dark culture (i.e., 99.4 +/- 1.0 mg/L). Although there still existed a gradual decrease trend of GA content after day 2 when the maximal GA content was obtained in the two-stage culture. Following three-stage light irradiation strategy was further demonstrated in order to turn around the sharp decrease of GA content after day 2. The first-stage was the 2-day dark culture; the second-stage was the following six-day 0.94 W/m(2) white light irradiation culture, and the third-stage was 4.70 W/m(2) white light irradiation culture until the end of fermentation. During the three-stage culture of G. lucidum, the gradual decrease trend of GA content after day 2 was turned around, which suggested that 0.94 W/m(2) white light irradiation was beneficial for the metabolic flux towards the GA biosynthesis. The maximal GA content of 3.1 +/- 0.1 mg/100 mg DW was obtained, which was higher by 41% compared to the two-stage culture. The maximal GA production (i.e., 466.3 +/- 24.1 mg/L) and productivity (i.e., 38.9 mg/L per day) in the three-stage culture were 69 and 101% higher than those obtained in the two-stage culture. This is the first report investigating the significance of light irradiation on the medicinal mushroom submerged fermentation. Such work is very helpful to other mushroom fermentations for useful metabolite production.

Suspension culture process of MethA tumor cell for the production of heat-shock protein glycoprotein 96: process optimization in spinner flasks.

Heat-shock proteins (HSPs) act like "chaperones", making sure that the cell's proteins are in the right shape and in the right place at the right time. Heat-shock protein glycoprotein 96 (gp96) is a member of the HSP90 protein family, which chaperones a number of molecules in protein folding and transportation. Heat-shock protein gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Currently, heat-shock protein gp96 was only isolated from murine and human tissues and cell lines. An animal cell suspension culture process for the production of heat-shock protein gp96 by MethA tumor cell was developed for the first time in spinner flasks. Effects of culture medium and condition were studied to enhance the MethA tumor cell density and the production and productivity of heat-shock protein gp96. Initial glucose concentration had a significant effect on the heat-shock protein gp96 accumulation, and an initial glucose level of 7.0 g/L was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Cultures at an initial glutamine concentration of 3 and 6 mM were nutritionally limited by glutamine. At an initial glutamine concentration of 6 mM, the maximal viable cell density of 19.90 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 4.95 mg/L was obtained. The initial concentration of RPMI 1640 and serum greatly affected the MethA tumor cell culture process. Specifically cultures with lower initial concentration of RPMI 1640 resulted in lower viable cell density and lower heat-shock protein gp96 production. At an initial serum concentration of 8%, the maximal viable cell density of 19.18 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 5.67 mg/L was obtained. The spin rate significantly affected the cell culture process in spinner flasks, and a spin rate of 150 rpm was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Not only the cell density but also the production and productivity of heat-shock protein gp96 attained in this work are the highest reported in the culture of MethA tumor cell. This work offers an effective approach for producing heat-shock protein glycoprotein 96 from the cell culture process. The fundamental information obtained in this study may be useful for the efficient production of heat-shock protein by animal cell suspension culture on a large scale.

Submerged cultivation of medicinal mushrooms for production of valuable bioactive metabolites.

Mushrooms are abundant sources of a wide range of useful natural products. Nowadays, commercial mushroom products are from mushrooms collected from field cultivation, which is a time-consuming and labor-intensive process. Submerged cultivation of mushrooms has significant industrial potential, but its success on a commercial scale depends on cost compared with existing technology. Increasing product yields and development of novel production systems that address the problems associated with this new technology will certainly facilitate expansion. This article outlines the major valuable metabolites produced by mushroom cultivation and advances in submerged culture of mushrooms, taking Ganoderma lucidum, a popular folk and an oriental medicine used to treat many diseases, as a typical example. Our latest data on mushroom cultivation for efficient production of bioactive ganoderic acids and Ganoderma polysaccharides in bioreactors are presented.

Scale-up of a liquid static culture process for hyperproduction of ganoderic acid by the medicinal mushroom Ganoderma lucidum.

Scale-up of a liquid static culture process was studied for hyperproduction of ganoderic acid (GA) by a famous Chinese traditional medicinal mushroom, Ganoderma lucidum. Initial volumetric oxygen transfer coefficient (K(L)a) and area of liquid surface per liquid volume (A(s)) were identified as key factors affecting cell growth and GA accumulation in liquid static cultures of G. lucidum, on the basis of which a multilayer static bioreactor was designed. At a low initial K(L)a level of 2.1 h(-1), a thick layer of white mycelia was formed on the liquid surface, and an optimal production of total GA (i.e., GA production in the liquid and on the liquid surface) was obtained. Both the formation of white mycelia and production of GA on the liquid surface were enhanced with an increase of A(s) within the range as investigated (0.24-1.53 cm(2)/mL). At an A(s) value of 0.90 cm(2)/mL, the total GA production reached maximum. A successful scale-up from a 20-mL static T-flask to a 7.5-L three-layer static bioreactor was achieved based on initial K(L)a. The maximum biomass (20.8 +/- 0.1 g DW/L), GA content (4.96 +/- 0.13 mg/100 mg DW), and total GA production (976 +/- 35 mg/L) were attained in static bioreactors. Not only GA content but also its production obtained in this work were the highest ever reported.