Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis.

Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester-forming products all were determined in SA-rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C-3' hydroxylation, and SmCYP98A14 preferentially catalyses C-3 hydroxylation in RA generation. In addition, relative to C-3' hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA-abundant species and versatility of CYP98A enzymes catalytic preference in meta-hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.

A phase III randomized, double-blind, placebo-controlled trial of the denosumab biosimilar QL1206 in postmenopausal Chinese women with osteoporosis and high fracture risk.

The current study evaluated the efficacy and safety of a denosumab biosimilar, QL1206 (60 mg), compared to placebo in postmenopausal Chinese women with osteoporosis and high fracture risk. At 31 study centers in China, a total of 455 postmenopausal women with osteoporosis and high fracture risk were randomly assigned to receive QL1206 (60 mg subcutaneously every 6 months) or placebo. From baseline to the 12-month follow-up, the participants who received QL1206 showed significantly increased bone mineral density (BMD) values (mean difference and 95% CI) in the lumbar spine: 4.780% (3.880%, 5.681%), total hip :3.930% (3.136%, 4.725%), femoral neck 2.733% (1.877%, 3.589%) and trochanter: 4.058% (2.791%, 5.325%) compared with the participants who received the placebo. In addition, QL1206 injection significantly decreased the serum levels of C-terminal crosslinked telopeptides of type 1 collagen (CTX): -77.352% (-87.080%, -66.844%), and N-terminal procollagen of type l collagen (P1NP): -50.867% (-57.184%, -45.217%) compared with the placebo over the period from baseline to 12 months. No new or unexpected adverse events were observed. We concluded that compared with placebo, QL1206 effectively increased the BMD of the lumbar spine, total hip, femoral neck and trochanter in postmenopausal Chinese women with osteoporosis and rapidly decreased bone turnover markers. This study demonstrated that QL1206 has beneficial effects on postmenopausal Chinese women with osteoporosis and high fracture risk.

Effects of ultra-strong static magnetic field on the gut microbiota of humans and mice.

To explore the effect of ultra-strong static magnetic field on gut microbiota, 16 T static magnetic field was used to study the changes in the structure and composition of human and mouse gut microbiota in this environment. In the mouse gut microbiota, at the genus level, the magnetic field significantly decreased the relative abundances of Escherichia-Shigella, Lactobacillus, Enterococcus, Burkholderia-Caballeronia-Paraburkholderia, Parasutterella, and Ralstonia and significantly increased those of Parabacteroides, Alloprevotella, Alistipes, Odoribacter, Bacteroides, Mucispirillum, Sutterella, and Prevotellaceae_UCG-001. Similarly, at the genus level, the relative abundances of Bacteroides, Parabacteroides, Romboutsia, and Streptococcus significantly decreased in the human gut microbiota. Contrary to the changing trend of the abundance in the mouse gut, the abundances of Bacteroides and Parabacteroides in the human gut were significantly reduced under magnetic field. The BugBase phenotypic prediction analysis showed that the relative abundances of five phenotypes, including anaerobism, mobile elements, potential pathogenicity, stress-tolerant, and biofilm formation, changed significantly in the mouse gut microbiota, while the relative abundances of two phenotypes, including Gram-positive and Gram-negative phenotypes, changed significantly in the human gut microbiota. The 16 T magnetic field could differently affect the composition, structure, and phenotypes of gut microbiota in human and mice, suggesting the importance of model selection in studying the biological effects of magnetic field.

Response of human gut microbiota under simulated microgravity.

The present study was conducted to investigate the influence of microgravity on human gut microbiota using 16S rRNA gene sequencing in vitro. The diamagnetic material magnetic levitation method was used to simulate weightless environment. The human gut microbiota was cultured under two different conditions: normal gravity (1 g), and simulated microgravity (0 g), which showed that both the richness (P = 0.04) and diversity (P = 0.0002) of human gut microbiota were significantly altered. As compared to the normal gravity, the simulated microgravity significantly reduced abundance of bacteria related to anti-inflammatory effects, such as Subdoligranulum, Faecalibacterium, Fusicatenibacter, Butyricicoccus, and Lachnospiraceae-NK4A136-0 group (P < 0.05), while significantly increased that of Alistipes and Eubacterium-Ventriosum-group (P < 0.05). Moreover, the Spearman's correlation analysis showed that there were more significantly correlated species (|r|≥ 0.5, P < 0.05) in normal gravity than that in the simulated microgravity. KEGG pathway analysis revealed that the microgravity significantly (P < 0.05) affected the metabolism of gut microbiota, such as the metabolism of pyrimidine, fatty acids, glyoxylate and dicarboxylate, peptidoglycan biosynthesis, and carbon fixation in photosynthetic organisms. These results suggested that the exposure to a microgravity environment might induce disturbances in human gut microbiota. KEY POINTS: • Using 16sRNA gene sequencing technology, it was found that magnetic levitation-simulated microgravity had varying degrees of influence on the abundance, diversity, species correlation, and KEGG pathways of human intestinal microbes. • Digital PCR can improve the detection rate of microorganisms with low abundance.

Chemical Composition, Anti-Breast Cancer Activity and Extraction Techniques of Ent-Abietane Diterpenoids from Euphorbia fischeriana Steud.

Ent-abietane diterpenoids are the main active constituents of Euphorbia fischeriana. In the continuing search for new anti-breast cancer drugs, 11 ent-abietane diterpenoids (1-11) were isolated from E. fischeriana. The structures of these compounds were clearly elucidated on the basis of 1D and 2D NMR spectra as well as HRESIMS data. Among them, compound 1 was a novel compound, compound 10 was isolated from Euphorbia genus for the first time, compound 11 was firstly discovered from E. fischeriana. These compounds exhibited varying degrees of growth inhibition against the MCF-10A, MCF-7, ZR-75-1 and MDA-MB-231 cell lines in vitro. The experimental data obtained permit us to identify the roles of the epoxy group, hydroxyl group and acetoxyl group on their cytotoxic activities. Extraction is an important means for the isolation, identification, and application of valuable compounds from natural plants. To maximize yields of ent-abietane diterpenoids of E. fischeriana, 17-hydroxyjolkinolide B, jolkinolide B, 17-hydroxyjolkinolide A and jolkinolide A were selected as quality controls to optimize the salting-out-assisted liquid-liquid extraction (SALLE) by response surface methodology (RSM). The optimized conditions for SALLE were 0.47 g sodium dihydrogen phosphate, 5.5 mL acetonitrile and 4.5 mL water at pH 7.5. The experimental values of 17-hydroxyjolkinolide B, jolkinolide B, 17-hydroxyjolkinolide A and jolkinolide A (2.134, 0.529, 0.396, and 0.148 mg/g, respectively) were in agreement with the predicted values, thus demonstrating the appropriateness of the model.

Anti-Cancer Activities of Diterpenoids Derived from Euphorbia fischeriana Steud.

Euphorbia fischeriana Steud is an essential oriental folk medicine used for healing cancer, edema and tuberculosis. Recently, its anticancer activitity has attracted more attention. A volume of research has indicated that diterpenoids are the major anticancer active constituents from this medicinal herb. In this review, we aimed to provide a summary of the promising anticancer diterpenoids from this plant; many diterpenoids mentioned in this article are newly discovered diterpenoids. According to the carbon skeleton and substituents, they can be classified into eight subtypes: ent-abietane, daphnane, tigliane, ingenane, ent-atisane, ent-rosane, ent-kaurane, and lathyrane. Futhermore, their key anticancer mechanisms and protein targets of these compounds will be discussed. These natural diterpenoids could provide a reservoir for drug discovery.

Novel PLCL nanofibrous/keratin hydrogel bilayer wound dressing for skin wound repair.

In this study, a novel poly(L-lactate-caprolactone) copolymer (PLCL) nanofibrous/keratin hydrogel bilayer wound dressing loaded with fibroblast growth factor (FGF-2) was prepared by the low-pressure filtration-assisted method. The ability of the keratin hydrogel in the bilayer dressing to mimic the dermis and that of the nanofibrous PLCL to mimic the epidermis were discussed. Keratin hydrogel exhibited good porosity and maximum water absorption of 874.09%. Compared with that of the dressing prepared by the coating method, the interface of the bilayer dressing manufactured by the low-pressure filtration-assisted method (filtration time: 20 min) was tightly bonded, and its bilayer dressing interface could not be easily peeled off. The elastic modulus of hydrogel was about 44 kPa, which was similar to the elastic modulus of the dermis (2-80 kPa). Additionally, PLCL nanofibers had certain toughness and flexibility suitable for simulating the epidermal structures. In vitro studies showed that the bilayer dressing was biocompatible and biodegradable. In vivo studies indicated that PLCL/keratin-FGF-2 bilayer dressing could promote re-epithelialization, collagen deposition, skin appendages (hair follicles) regeneration, microangiogenesis construction, and adipose-derived stem cells (ADSCs) recruitment. The introduction of FGF-2 resulted in a better repair effect. The bilayer dressing also solved the problems of poor interface adhesion of hydrogel/electrospinning nanofibers. This paper also explored the preliminary role and mechanism of bilayer dressing in promoting skin healing, showing that its potential applications as a biomedical wound dressing in the field of skin tissue engineering.

Efficacy and Safety of Denosumab Biosimilar QL1206 Versus Denosumab in Patients with Bone Metastases from Solid Tumors: A Randomized Phase III Trial.

BACKGROUND: Denosumab has been approved for the treatment of bone metastases from solid tumors. QL1206 is the first denosumab biosimilar and needs to be compared with denosumab in a phase III trial. OBJECTIVE: This phase III trial aims to compare the efficacy, safety, and pharmacokinetics between QL1206 and denosumab in patients with bone metastases from solid tumors. METHODS: This randomized, double-blind, phase III trial was conducted in 51 centers in China. Patients aged 18-80 years, with solid tumors and bone metastases, and an Eastern Cooperative Oncology Group performance status of 0-2 were eligible. This study was divided into a 13-week double-blind period, a 40-week open-label period, and a 20-week safety follow-up period. In the double-blind period, patients were randomly assigned (1:1) to receive three doses of QL1206 or denosumab (120 mg subcutaneously every 4 weeks, each). Randomization was stratified by tumor types, previous skeletal-related events, and current systemic anti-tumor therapy. In the open-label period, up to ten doses of QL1206 could be given in both groups. The primary endpoint was percentage change in urinary N-telopeptide/creatinine ratio (uNTX/uCr) from baseline to Week 13. Equivalence margins were ± 0.135. Secondary endpoints included percentage change in uNTX/uCr at Week 25 and 53, percentage change in serum bone-specific alkaline phosphatase at Week 13, 25, and 53, and time to on-study skeletal-related events. The safety profile was evaluated based on adverse events and immunogenicity. RESULTS: From September 2019 to January 2021, in the full analysis set, 717 patients were randomly assigned to receive QL1206 (n = 357) or denosumab (n = 360). Median percentage changes in uNTX/uCr at Week 13 in two groups were - 75.2% and - 75.8%, respectively. Least-squares mean difference in the natural log-transformed ratio of uNTX/uCr at Week 13 to baseline between the two groups was 0.012 (90% confidence interval - 0.078 to 0.103), within the equivalence margins. There were no differences in the secondary endpoints between the two groups (all p > 0.05). Adverse events, immunogenicity, and pharmacokinetics were similar in the two groups. CONCLUSIONS: Denosumab biosimilar QL1206 had promising efficacy, tolerable safety, and pharmacokinetics equivalent to denosumab and could benefit patients with bone metastases from solid tumors. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04550949, retrospectively registered on 16 September, 2020.

Equivalent efficacy study of QL1101 and bevacizumab on untreated advanced non-squamous non-small cell lung cancer patients: a phase 3 randomized, double-blind clinical trial.

OBJECTIVE: This phase 3 study aimed to test equivalence in efficacy and safety for QL1101, a bevacizumab analogue in Chinese patients with untreated locally advanced non-squamous non-small cell lung cancer (NSCLC). METHODS: Eligible patients were randomly assigned 1:1 to receive carboplatin and paclitaxel in combination with either QL1101 or bevacizumab, 15 mg/kg every 3-week for 6 cycles. This was followed by maintenance treatment with single agent QL1101 every 3-week. The primary end-point was objective response rate (ORR), with secondary end-points being progression-free survival (PFS), overall survival (OS), disease control rate (DCR), and adverse events (AEs). RESULTS: Of 675 patients, 535 eligible patients were randomized to the QL1101 group (n = 269) and bevacizumab group (n = 266). ORRs were 52.8% and 56.8%, respectively, for the QL1101 and bevacizumab groups, with an ORR hazard ratio 0.93 (95% confidence interval: 0.8-0131.1). The PFS, OS, DCR, and AEs were comparable between the 2 groups, which remained the same after stratification according to epidermal growth factor receptor mutation or smoking history. CONCLUSIONS: QL1101 showed similar efficacy and safety profiles as compared to bevacizumab among Chinese patients with untreated locally advanced non-squamous NSCLC.

Early Over-Expressing of microRNA-145 Effectively Precludes the Development of Neuropathic Mechanical Hyperalgesia via Suppressing Nav1.8 in Diabetic Rats.

BACKGROUND: Painful diabetic neuropathy (PDN) is a common complication secondary to diabetes mellitus. Nav1.8 is an isoform of voltage-gated sodium channels and its expression regulation is closely related with PDN. MicroRNA-145 (miR-145) is involved in the occurrence and development of neuropathic pain. TargetScan software has revealed that Nav1.8 (SCN10A) is the major target of miR-145. However, its function between miR-145 and Nav1.8 in PDN is unknown. OBJECTIVES: We aim to explore the regulatory effect of miR-145 on the expression and function of Nav1.8, which plays a pivotal role in precluding the advancement of neuropathic mechanical hyperalgesia in diabetic pain. STUDY DESIGN: An experimental, animal study. SETTING: An animal research facility at Nanjing Maternal and Child Health Institute, China. METHODS: The paw mechanical withdrawal threshold (PMWT) of rats was assessed with the von Frey test. The adverse regulation of Nav1.8 by miR-145 was confirmed by a dual luciferase detection system in HEK293T cells. The mRNA level and expression of Nav1.8 in dorsal root ganglion (DRG) neurons were assessed with real-time polymerase chain reaction (real-time PCR), western blotting and immunofluorescence assays following intrathecal injection of agomiR-145 in vitro and in vivo. Whole-cell patch-clamping was applied to assess alterations in the tetrodotoxin-resistant (TTX-R) sodium current (Nav1.8) in DRGs. RESULTS: The PMWT was significantly decreased in rats following streptozotocin (STZ) injection on Day 7 and was maintained at a lower level on Day 28; this change was accompanied by changes in the expression of Nav1.8 in DRG neurons, which was increased 3 days after STZ injection and reached a maximal level on Day 14. The early knockdown of Nav1.8 with siRNA or agomiR-145 treatment on Day 8 effectively precluded the deterioration of pain behaviors in STZ-treated rats. The luciferase intensity was significantly decreased in HEK293T cells expressing wild-type SCN10A infected with miR-145 mimic. In addition, Nav1.8 overexpression was significantly repressed via overexpression of miR-145 in cultured DRG neurons, and neuronal hyperexcitability was concomitantly decreased. Furthermore, the intrathecal administration of agomiR-145 elicited a significant decrease in Nav1.8 expression in DRG neurons from STZ-treated rats on Day 14. LIMITATIONS: The causes of PDN are likely to be multifactorial and inflammatory markers, such as IL-6, IL-2, and TNF-?, are elevated in hyperglycemia and might be the precipitating factors that contribute to miR-145 dysregulation. The curative effect of miR-145 upregulation in reversal of pain behaviors at the stage of well-established PDN wasn't investigated in this study. CONCLUSION: Early infection with a lentiviral vector overexpressing miR-145 adversely regulated the expression and function of TTX-resistant Nav1.8 and abrogated the development of PDN. Therefore, miR-145 might be a potential therapeutic target for preventing PDN in the near future.

A randomized, double-blind, single-dose study to evaluate the biosimilarity of QL1101 with bevacizumab in healthy male subjects.

PURPOSE: This is the first study to compare the pharmacokinetics of QL1101, a proposed bevacizumab biosimilar, with Avastin® sourced from Roche Diagnostics GmbH. METHODS: In this double-blind, single-dose, parallel-group study, healthy male subjects were randomized 1:1 to receive QL1101 or Avastin® 3 mg/kg intravenously. Pharmacokinetic assessments were conducted for 85 days, with additional safety and immunogenicity assessments until day 90. Primary study endpoints were area under the concentration-time curve (AUC) from time zero to infinity (AUC0-∞), AUC from time zero to the last quantifiable concentration (AUC0-last), and maximum serum concentration (Cmax). Pharmacokinetic equivalence was shown if the 90% confidence intervals (CIs) of the geometric mean ratios (GMRs) of the C0-max, AUC0-last, and AUC0-∞ were within the predefined bioequivalence margin of 80-125.00%. RESULTS: A total of 82 subjects were randomized to the following groups: 42 to QL1101 and 40 to Avastin®. The 90% CIs of the GMRs of AUC0-∞, AUC0-last, and Cmax of QL1101 and Avastin® were (97.8%, 107.0%), (94.5%, 106.9%), and (94.1%, 107.3%), respectively, which were all within the bioequivalence margin. The incidence of adverse events was 90.5% and 95.0% in the QL1101 and Avastin® groups, respectively. Mean serum concentration-time profiles, secondary pharmacokinetic parameters, and safety and immunogenicity profiles were comparable across the two treatment groups. CONCLUSIONS: The study demonstrated the pharmacokinetic equivalence of QL1101 to Avastin®. QL1101 (3 mg/kg, iv) is safe and tolerable in healthy Chinese subjects. These data support the further clinical evaluation of QL1101 as a bevacizumab biosimilar.

Chloroquine inhibits cell growth in human A549 lung cancer cells by blocking autophagy and inducing mitochondrial­mediated apoptosis.

Chloroquine (CQ) has been revealed to exhibit antitumor activity in several human tumors including lung cancer as mono­ or add­on therapy. The antitumor effect of CQ appears to depend on the tumor type, stage and genetic context. Few studies have focused on the mechanism concerning the antitumor effect of CQ monotherapy and the cause and effect relationship among autophagy, apoptosis and CQ in human lung A549 cells. Therefore, the present study aimed to identify the antitumor effect of CQ monotherapy and analyze the possible mechanism. In the present study, we demonstrated that CQ suppressed human A549 cell growth in a dose­ and time­dependent manner. CQ­mediated growth inhibition in A549 cells was characterized by the targeting of the PI3K/AKT pathway, thus, inducing mitochondria­mediated apoptosis at relatively higher concentrations by downregulating Bcl­2 expression, increasing the expression level of Bax, decreasing mitochondrial membrane potential, releasing cytochrome c from the mitochondria into the cytosol, activating caspase­3 and cleaving PARP. Collectively, these findings may offer a new rationale for using CQ as a lung cancer therapy drug in clinical practice.

TOX3 protein expression is correlated with pathological characteristics in breast cancer.

TOX3 is a newly identified gene that has been observed to correlate with breast cancer by genome-wide association studies (GWAS) in recent years. In addition, it has been noted that single-nucleotide polymorphisms (SNPs) in the TOX3 gene have a strong correlation with estrogen receptor (ER)-positive tumors. However, the role of TOX3 in breast carcinoma development is still unclear. There are limited studies on the subject of TOX3 mRNA expression in breast tumors and little information on the variation of TOX3 protein expression in relation to the clinical pathological features in breast cancer and healthy tissues. In this study, we characterize the protein expression of TOX3 in breast tumors with respect to various clinical and pathological characteristics and explore the correlation between TOX3 protein expression and ER-positive tumors. A breast cancer tissue microarray containing 267 human breast tumors and 25 healthy controls, breast cancer cell lines (ZR-75-1, MDA-MB-231, MCF-7 and Bcap-37) with positive or negative ER expression, tumor tissues and matched controls were used to analyze the protein expression levels of TOX3 by immunohistochemistry, western blot analysis and quantitative polymerase chain reaction. Among the 267 breast tumor specimens, ER expression was detected in 66 tumor tissues. The expression levels of TOX3 increased in breast carcinoma tissue compared with controls, and were higher in advanced carcinoma (T3 and T4), lymph node metastases tissues (N2) and stage III tissues. Furthermore, TOX3 protein expression was more intense in ER-positive tumors, but did not demonstrate a statistical significance. However, it was significantly increased in ER-positive breast cancer cell lines (ZR-75-1, MCF-7 and Bcap-37) compared with the MDA-MB-231 cell line, which had ER-negative expression. Our findings provide support to the hypothesis that TOX3 has a strong correlation with the development of breast cancer. The current study is likely to assist in investigating the mechanisms involved in breast cancer development.

Fucosyltransferase 8 expression in breast cancer patients: A high throughput tissue microarray analysis.

The aim of this study was to compare the expression of fucosyltransferase 8 (FUT8) in breast cancer tissue and to investigate the relationship between this marker with tumor progression and its applicability to differential diagnosis. An immunohistochemical study was performed for FUT8 using the tissue microarray technique. In addition, the mRNA and protein levels of FUT8 in the tissue were also tested by real-time PCR and Western blot. There was a significant difference in cytoplasmic expression of FUT8 between breast cancer tissue and matched normal tissue (p<0.001). The percent of FUT8 staining in breast cancer tissues ranging from negative, weak positive, positive and strong positive were 2.7%, 40.2%, 54% and 3.2%, respectively. High FUT8 protein expression correlated with lymphatic metastasis (p=0.008) and with stage status (p=0.039). We detected that reduced FUT8 expression correlated with disease-free survival (p=0.02) and overall survival (p=0.04) of breast cancer patients. Expression of FUT8 can stratify breast cancer tissue and may be considered a prognostic marker for breast cancer patients.

12-Deoxyphorbol 13-palmitate inhibits the expression of VEGF and HIF-1α in MCF-7 cells by blocking the PI3K/Akt/mTOR signaling pathway.

Vascular endothelial growth factor (VEGF) is an essential component for angiogenesis, and hypoxia-inducible factor-1α (HIF-1α), which controls the switch of glycolytic and oxidative metabolism, activates the transcription of VEGF. 12-Deoxyphorbol 13-palmitate (DP) is a compound isolated from the roots of Euphorbia fischeriana, and has been revealed to possess anticancer activity. In the present study, we found that DP is an effective inhibitor of VEGF and HIF-1α in MCF-7 cells. DP markedly reduced cell viability as determined by MTT assay. ELISA, western blotting and RT-qPCR assays indicated that DP significantly decreased the protein and mRNA expression of VEGF and the protein expression of HIF-1α, while HIF-1α mRNA remained unchanged. In addition, the entrance of HIF-1α into the nucleus was blocked after DP treatment as detected by immunofluorescence analysis. In a further study, we proved that the effects mentioned above were associated with constitutive interference of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. DP effectively inhibited the phosphorylation of PI3K and its downstream factors p-Akt and p-mTOR, oppositely enhanced the expression of TSC1 (hamartin) and TSC2 (tuberin), which could be reversed by the co-treatment with the PI3K inhibitor wortmannin. Moreover, the addition of wortmanin further downregulated the protein levels of VEGF and HIF-1α. The results revealed that DP inhibited the expression of VEGF and HIF-1α through the PI3K/Akt/mTOR signaling pathway, confirming that DP may be a potential therapeutic candidate for breast cancer.

[Establishment of breast cancer MDA-MB-231 cell line stably over-expressing human TOX high mobility group box family member 3].

OBJECTIVE: To construct the lentiviral expression vector of human TOX high mobility group box family member 3 (TOX3) gene and the MDA-MB-231 cell line which stably over-expresses TOX3 gene. METHODS: TOX3 gene was synthesized by the gene synthesis method and amplified by PCR, and then cloned into pLVEF-1a/GFP-Puro vector to construct pLVEF-1a/GFP-Puro-TOX3 lentiviral vector. After restriction enzyme analysis and sequence identification, the lentiviral vector was packaged and the titer was detected. The human breast cancer MDA-MB-231 cells were transfected with the recombinant lentiviral vector and cultured selectively by puromycin to acquire stably transfected cells. MDA-MB-231 cells which expressed GFP were observed by fluorescence microcopy. And the expression levels of TOX3 mRNA and protein in transfected MDA-MB-231 cells were detected by real-time quantitative PCR(qRT-PCR) and Western blotting, respectively. RESULTS: Restriction enzyme digestion and sequence analysis demonstrated that the lentiviral expression vectors of pLVEF-1a/GFP-Puro and pLVEF-1a/GFP-Puro-TOX3 were successfully constructed, and the viral titers were respectively 2×10(8) TU/mL and 1×10(8) TU/mL after lentiviral packaging. And after being transfected, more than 95% cells expressed GFP under a fluorescence microscope. The results of qRT-PCR and Western blotting showed that, when compared with the MDA-MB-231-NC negative control group, the expression of TOX3 mRNA and protein significantly increased in the MDA-MB-231-TOX3 group. CONCLUSION: The study successfully constructed lentiviral expression vector of TOX3 gene and obtained MDA-MB-231 cell line stably over-expressing TOX3 gene by transfection with the recombinant vector.