Journal: Toxins

Article Title: Combination of Rhamnetin and RXP03 Mitigates Venom-Induced Toxicity in Murine Models: Preclinical Insights into Dual-Target Antivenom Therapy.

doi: 10.3390/toxins17060280

Figure Lengend Snippet: Figure 1. Inhibition of venom enzymatic activities and associated biological effects by Rhamnetin and RXP03. (A) The action performance of different snake venoms (DA, PM, GBs, NA, TS) on the plate and the inhibitory efficiency. Well 1 was filled with venom. Wells 2 to 5 were mixtures of venom and different concentrations of Rhamnetin. Well 6 was the negative control. (B) Test the inhibitory efficiency of Rhamnetin on the activity of PLA2 using NOB as the substrate. (C) The inhibitory efficiency of RXP03 on the activity of SVMP. (D) Anti-venom hemorrhagic activity of the combination of Rhamnetin and RXP03. Error bars represent the standard error of the mean for n = 3 replicates.

Article Snippet: Both Rhamnetin (catalogue: HY-N7036, ≥95% by HPLC) and RXP03 (catalogue: HY-162038, ≥95% by HPLC) were purchased from MedChemExpress® (Wuhan, Hubei Province, China).

Techniques: Inhibition, Negative Control, Activity Assay

Journal: Toxins

Article Title: Combination of Rhamnetin and RXP03 Mitigates Venom-Induced Toxicity in Murine Models: Preclinical Insights into Dual-Target Antivenom Therapy.

doi: 10.3390/toxins17060280

Figure Lengend Snippet: Figure 2. Alleviation of venom-induced oxidative stress and muscle function impairment. (A) Snake venom-induced muscle hemorrhage and edema were significantly alleviated by drug treatment. The muscle’s ability to resist tension has returned to normal. (B–G) Inhibitory effect of Rhamnetin and RXP03 in venom-induced serum oxidative stress. Antioxidant capacity (T-SOD, GSH, CAT, T-AOC) recovered and oxidative products of muscle (ROS, MDA) were reduced under the intervention of drugs. Data shown as mean ± standard error of the mean (n = 3). * represents significantly different from the NS group; #, venom group vs. drug-treated group (** p ≤0.01, *** p ≤0.001, ## p ≤0.01, and ### p ≤0.001).

Article Snippet: Both Rhamnetin (catalogue: HY-N7036, ≥95% by HPLC) and RXP03 (catalogue: HY-162038, ≥95% by HPLC) were purchased from MedChemExpress® (Wuhan, Hubei Province, China).

Techniques: Muscles

Journal: Toxins

Article Title: Combination of Rhamnetin and RXP03 Mitigates Venom-Induced Toxicity in Murine Models: Preclinical Insights into Dual-Target Antivenom Therapy.

doi: 10.3390/toxins17060280

Figure Lengend Snippet: Figure 4. Inhibition of systemic toxicity of snake venom by Rhamnetin and RXP03. (A) Motor function trajectories of mice 12 h post-envenoming. Venom groups (DA, PM, GBs, NA, TS) show restricted movement vs. saline control (NS). (B) Kaplan–Meier survival curves over 24 h. Venom-only groups exhibited rapid lethality (20% survival), while drug-treated groups showed significant protection (85% survival, p < 0.001). (C–H,J) Serum biochemical markers: Drug intervention normalized venom- elevated levels of (C) ALT/AST (liver damage), (D) CK (myotoxicity), (E) LDH (systemic injury), (F,G) myoglobin/free hemoglobin (rhabdomyolysis), (H,J) UA/Scr (renal impairment). (I) Urinary protein. * represents significantly different from the NS group; #, venom group vs. drug-treated group (*** p ≤0.001, ## p ≤0.01, and ### p ≤0.001).

Article Snippet: Both Rhamnetin (catalogue: HY-N7036, ≥95% by HPLC) and RXP03 (catalogue: HY-162038, ≥95% by HPLC) were purchased from MedChemExpress® (Wuhan, Hubei Province, China).

Techniques: Inhibition, Saline, Control

Journal: Toxins

Article Title: Combination of Rhamnetin and RXP03 Mitigates Venom-Induced Toxicity in Murine Models: Preclinical Insights into Dual-Target Antivenom Therapy.

doi: 10.3390/toxins17060280

Figure Lengend Snippet: Figure 5. Rhamnetin and RXP03 prevention of venom-induced coagulopathy. Under the influence of venom, the coagulation function is disrupted and APTT (A), PT (B), and TT (C) significantly increase. The snake venom depletes FIB (D) in the blood of the poisoned mice. Drugs reverse this effect. The results were expressed as mean ± standard deviation (n = 3). * represents significantly different from the NS group; #, venom group vs. drug-treated group (** p ≤0.01, *** p ≤0.001, ## p ≤0.01, and ### p ≤0.001).

Article Snippet: Both Rhamnetin (catalogue: HY-N7036, ≥95% by HPLC) and RXP03 (catalogue: HY-162038, ≥95% by HPLC) were purchased from MedChemExpress® (Wuhan, Hubei Province, China).

Techniques: Coagulation, Standard Deviation

Journal: Acta Pharmacologica Sinica

Article Title: AZD9291 inactivates the PRC2 complex to mediate tumor growth inhibition

doi: 10.1038/s41401-019-0248-2

Figure Lengend Snippet: AZD9291 disrupts the EZH2–EED interaction by binding to EZH2 directly. a NanoBRET assay. HEK293T cells were co-transfected with pFN31K-EZH2 NanoLuc and pFC14K-EED HaloTag vectors for about 20 h then treated with AZD or astemizole (the highest concentration was 10 μM, diluted by 2 times) overnight, donor emission (460 nm) and acceptor emission (618 nm) were measured using microplate reader and the ratio values with milliBRET units (mBU) were calculated as RawBRET = 618 nm Em/460 nm Em × 1000. n = 3 replicates. b HEK293T cell was exposed to AZD (2.5 μM) or EED226 (2.5 μM) for 48 h. The cell lysates were incubated with anti-EZH2 or immunoglobulin G (lgG) antibodies. Immunoprecipitation was immunoblotted with the indicated antibodies. c Each reaction solution containing 2 μM EED, 5× SYPRO orange, and testing compounds (25 μM) in 20 μL thermal shift buffer. The melting temperature (Tm) was calculated by the Boltzmann fitting method. d MDA-MB-453 cells were pre-treated with 10 μM MG132 for 1 h before treatment with the 20 μM AZD for 4 h. Cell lysates were used for SDS PAGE and Western blotting. All error bars represent mean ± SD analyzed by unpaired two-tailed t test. ** P < 0.01. AZD, AZD9291. See also Figure S1

Article Snippet: AZD9291 (HY-15772) and EPZ6438 (HY-13803) were purchased from the Med Chem Express (Monmouth Junction, NJ, USA); Astemizole (ab144583, Abcam, Cambridge, UK); MG-132 (S2619, Selleckchem, Houston, TX, USA), gefitinib (S1025, Selleckchem), afatinib (S1011, Selleckchem), EED226 (S8496, Selleckchem), both of them claimed to have more than 98% purity of the compound based on a high-performance liquid chromatography analysis.

Techniques: Binding Assay, Transfection, Concentration Assay, Incubation, Immunoprecipitation, SDS Page, Western Blot, Two Tailed Test

Journal: Acta Pharmacologica Sinica

Article Title: AZD9291 inactivates the PRC2 complex to mediate tumor growth inhibition

doi: 10.1038/s41401-019-0248-2

Figure Lengend Snippet: AZD9291 selectively inhibits H3K27 methylation and exhibits anti-tumor activity. a EZH2, EED, SUZ12 protein, H3K27me3 and other histone lysine methylation or acetylation marks levels in MDA-MB-453 and T47D breast cancer cells and DLBCL cell lines U2932 (EZH2 WT type) and DB (EZH2 mutant type) were shown by following treatment with the indicated concentrations of AZD or EPZ for 72 h. The β-actin and total histone H3 were shown as a loading control. b Cells were treated with the AZD (2.5 μM) or EPZ (2.5 μM) for 48 h. The mRNA level of PRC2 targeted genes (fold change) was obtained as normalized by that of the DMSO-treated group. n = 3 replicates. c Cells were treated with AZD or GSK126 at gradient concentrations for 7 days and IC 50 s measured using SRB assay or CCK-8 assay. d Colony-forming assay. Cells were treated with AZD or EPZ for about 2 weeks and cells growth changes were measured by counting colony numbers. n = 3 replicates. e The cell cycle was examined by flow cytometry after treated with AZD 2–2.5 μM for 48 h. f , g The influence of AZD on cancer cells metastasis was detected by wound-healing experiment ( f ), Transwell experiment and Matrigel invasion experiment ( g ) to evaluate the mobile ability of cancer cells. Scale bar, 2 mm. All error bars represent mean ± SD analyzed by unpaired two-tailed t test. * P < 0.05, ** P < 0.01, *** P < 0.001. AZD, AZD9291. EPZ, EPZ6438. See also Figure S2

Article Snippet: AZD9291 (HY-15772) and EPZ6438 (HY-13803) were purchased from the Med Chem Express (Monmouth Junction, NJ, USA); Astemizole (ab144583, Abcam, Cambridge, UK); MG-132 (S2619, Selleckchem, Houston, TX, USA), gefitinib (S1025, Selleckchem), afatinib (S1011, Selleckchem), EED226 (S8496, Selleckchem), both of them claimed to have more than 98% purity of the compound based on a high-performance liquid chromatography analysis.

Techniques: Methylation, Activity Assay, Mutagenesis, Control, Sulforhodamine B Assay, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: Acta Pharmacologica Sinica

Article Title: AZD9291 inactivates the PRC2 complex to mediate tumor growth inhibition

doi: 10.1038/s41401-019-0248-2

Figure Lengend Snippet: AZD9291 decreases EZH2 expression and inhibits genes expression which EZH2 activated. a , b Cells were harvested for detection of protein level by Western blotting. Cells were treated with cycloheximide (100 μM) combined with vehicle (DMSO) or AZD (5 μM) for the indicated times ( a ). After treatment with 5 μM AZD for 48 h, MDA-MB-453 cells were lysed and subjected to the EZH2 ubiquitination assay ( b ). c , d Cells were treated with AZD 2.5 μM for 48 h then samples were subjected to qRT-PCR assay. The mRNA expression of EZH2 ( c ) and EZH2 activated genes ( d ) were tested. Error bars represent mean ± SD analyzed by unpaired two-tailed t test. * P < 0.05, ** P < 0.01, *** P < 0.001. AZD, AZD9291. CHX, Cycloheximide

Article Snippet: AZD9291 (HY-15772) and EPZ6438 (HY-13803) were purchased from the Med Chem Express (Monmouth Junction, NJ, USA); Astemizole (ab144583, Abcam, Cambridge, UK); MG-132 (S2619, Selleckchem, Houston, TX, USA), gefitinib (S1025, Selleckchem), afatinib (S1011, Selleckchem), EED226 (S8496, Selleckchem), both of them claimed to have more than 98% purity of the compound based on a high-performance liquid chromatography analysis.

Techniques: Expressing, Western Blot, Ubiquitin Proteomics, Quantitative RT-PCR, Two Tailed Test

Journal: Acta Pharmacologica Sinica

Article Title: AZD9291 inactivates the PRC2 complex to mediate tumor growth inhibition

doi: 10.1038/s41401-019-0248-2

Figure Lengend Snippet: AZD9291 attenuates EZH2 mRNA via miR-34a. a miRNAs array assay. MDA-MB-453 cells were treated with AZD 2.5 μM for 48 h. Total miRNAs were analyzed and top 10 most changed miRNAs were listed. b – d Kaplan–Meier plots of overall survival between tumors with high and low miR-34a expression levels. Analysis of all the breast cancer cases with METABRIC database ( b ) and the triple-negative ( c ) and ER-positive ( d ) breast cancer cases with TCGA database. e Cells were treated with AZD for 48 h and the expression level of mature miR-34a was tested. f Cells were transfected with 100 nM miR-34a mimics or miR-NC for 48 h, the mRNA level of EZH2, EED and SUZ12 were tested. g Detection of proteins expression by Western blotting. Cells were harvested after transfected with 100 nM miR-34a mimics or miR-NC for 48 h. h The prediction of EZH2 targeted by miR-34a. The predicted binding sequences of EZH2 3′UTR and all the sequences of miR-34a (the seed sequence of miR-34 was underlined), the EZH2 3′UTR mutant sequences were shown. i HEK293 cells were subjected to dual-luciferase assay. Cells were transfected with 100 nM miR-34a or miR-NC for 48 h, the luciferase activity was tested. Luciferase activity was normalized to the ratio of firefly and Renilla to luciferase activities. Error bars represent mean ± SD analyzed by unpaired two-tailed t test. ** P < 0.01, *** P < 0.001. AZD, AZD9291

Article Snippet: AZD9291 (HY-15772) and EPZ6438 (HY-13803) were purchased from the Med Chem Express (Monmouth Junction, NJ, USA); Astemizole (ab144583, Abcam, Cambridge, UK); MG-132 (S2619, Selleckchem, Houston, TX, USA), gefitinib (S1025, Selleckchem), afatinib (S1011, Selleckchem), EED226 (S8496, Selleckchem), both of them claimed to have more than 98% purity of the compound based on a high-performance liquid chromatography analysis.

Techniques: Expressing, Transfection, Western Blot, Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

Journal: Acta Pharmacologica Sinica

Article Title: AZD9291 inactivates the PRC2 complex to mediate tumor growth inhibition

doi: 10.1038/s41401-019-0248-2

Figure Lengend Snippet: miR-34a has the same effects with AZD9291. a , b Cells were transfected with the miR-34a or miR-NC 100 nM for 48 h, then the influences of miR-34a on the cell cycle ( a ) and apoptosis ( b ) were examined by flow cytometry. c , d The influences of AZD and miR-34a on cancer cell metastasis. AZD 2.5 μM or miR-34a 100 nM treated MDA-MB-453 cells for 48 h were subjected to transwell experiment and matrigel invasion experiment ( c ) and wound-healing experiment ( d ). Scale bar, 2 mm. e , f qRT-PCR assay. MDA-MB-453 cells were treated with miR-34a or AZD 2.5 μM for 48 h then samples were subjected to qRT-PCR assay. The mRNA level of EZH2 activated genes ( e ) and inhibited genes ( f ) were tested. g Western blotting. Cells were harvested after transfected with 100 nM miR-34a mimics or anti-miR-34a alone or combined with AZD 2.5 μM for 48 h. h Dual-luciferase assay. HEK293 cells were treated with AZD 2.5 μM for 48 h, then the luciferase activity was tested. i A hypothetical model illustrating the silencing mechanism of AZD on PRC2 in cancer cells. All error bars represent mean ± SD analyzed by unpaired two-tailed t test. ** P < 0.01, *** P < 0.001. AZD, AZD9291

Article Snippet: AZD9291 (HY-15772) and EPZ6438 (HY-13803) were purchased from the Med Chem Express (Monmouth Junction, NJ, USA); Astemizole (ab144583, Abcam, Cambridge, UK); MG-132 (S2619, Selleckchem, Houston, TX, USA), gefitinib (S1025, Selleckchem), afatinib (S1011, Selleckchem), EED226 (S8496, Selleckchem), both of them claimed to have more than 98% purity of the compound based on a high-performance liquid chromatography analysis.

Techniques: Transfection, Flow Cytometry, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Two Tailed Test