Effects of total coumarins from Pileostegia tomentella on exosomal miRNA expression and angiogenesis in colorectal cancer cells.

CONTEXT: Pileostegia tomentella Hand. Mazz (Saxifragaceae) total coumarins (TCPT) show antitumour activity in colorectal cancer (CRC) with unknown mechanism of action. Tumour angiogenesis mediated by exosomes-derived miRNA exhibits the vital regulation of endothelial cell function in metastasis of CRC. OBJECTIVE: To investigate the effect of TCPT on exosomal miRNA expression and angiogenesis of CRC cells. MATERIALS AND METHODS: HT-29-derived exosomes were generated from human CRC cells (HT-29) or either treated with TCPT (100 µg/mL) for 24 h, followed by identification by transmission electron microscope, nanoparticle tracking analysis (NTA) and Western blot. Co-culture experiments for human umbilical vein endothelial cells (HUVECs) and exosomes were performed to detect the uptake of exosomes in HUVECs and its influence on HUVECs cells migration and lumen formation ability. Potential target miRNAs in exosomes were screened out by sequencing technology. Rescue assays of angiogenesis were performed by the transfecting mimics or inhibitors of targeted miRNA into HUVECs. RESULTS: HT-29-derived exosomes, after TCPT treatment (Exo-TCPT), inhibited the migration and lumen formation of HUVECs, reduced the expression levels of vascular marker (FLT-1, VCAM-1 and VEGFR-2) in HUVECs. Furthermore, the level of miR-375-3p was significantly upregulated in Exo-TCPT. Rescue assays showed that high expression of miR-375-3p in HUVECs inhibited migration and lumen formation abilities, which was consistent with the effects of Exo-TCPT, whereas applying miR-375-3p inhibitors displayed opposite effects. DISCUSSION AND CONCLUSION: TCPT exhibits anti-angiogenesis in CRC, possibly through upregulating exosomal miR-375-3p. Our findings will shed light on new target exosomes miRNA-mediated tumour microenvironment and the therapeutic application of Pileostegia tomentella in CRC.

Corrigendum: Honokiol ameliorates post-myocardial infarction heart failure through Ucp3-mediated reactive oxygen species inhibition.

[This corrects the article DOI: 10.3389/fphar.2022.811682.].

Honokiol Ameliorates Post-Myocardial Infarction Heart Failure Through Ucp3-Mediated Reactive Oxygen Species Inhibition.

Post-myocardial infarction heart failure (post-MI HF) is one of the leading global causes of death, and current prevention and treatment methods still cannot avoid the increasing incidence. Honokiol (HK) has previously been reported to improve myocardial ischemia/reperfusion injury and reverse myocardial hypertrophy by activating Sirt1 and Sirt3. We suspect that HK may also have a therapeutic effect on post-MI HF. In this study, we aimed to investigate the efficacy and mechanism of HK in the treatment of post-MI HF. We found that HK inhibited myocardial reactive oxygen species (ROS) production, reduced myocardial fibrosis, and improved cardiac function in mice after MI. HK also reduced the abnormality of mitochondrial membrane potential (MMP) and apoptosis of cardiomyocytes caused by peroxide in neonatal cardiomyocytes. RNAseq results revealed that HK restored the transcriptome changes to a certain extent and significantly enhanced the expression of mitochondrial inner membrane uncoupling protein isoform 3 (Ucp3), a protein that inhibits the production of mitochondrial ROS, protects cardiomyocytes, and relieves heart failure after myocardial infarction (MI). In cardiomyocytes with impaired Ucp3 expression, HK cannot protect against the damage caused by peroxide. More importantly, in Ucp3 knockout mice, HK did not change the increase in the ROS level and cardiac function damage after MI. Taken together, our results suggest that HK can increase the expression of the cardioprotective protein Ucp3 and maintain MMP, thereby inhibiting the production of ROS after MI and ameliorating heart failure.

Discovery of a Series of Hydroxamic Acid-Based Microtubule Destabilizing Agents with Potent Antitumor Activity.

Hydroxamic acid group is one of the characteristic pharmacophores of histone deacetylase (HDAC) inhibitors. But here, we discovered a series of hydroxamic acid-based microtubule destabilizing agents (MDAs), which were derived from shortening the length of the linker in HDAC6 inhibitor SKLB-23bb. Interestingly, the low nanomolar antiproliferative activity of these MDAs depended on the presence of hydroxamic acid groups, but their inhibitory effects on HDAC were lost. Among them, 12b showed favorable metabolism stability, high bioavailability, and potent antitumor activity in multidrug-resistant cell lines and A2780/T xenograft model. More importantly, in the patient-derived xenograft models of triple-negative breast cancer and osimertinib-resistant non-small-cell lung cancer, both 20 mg/kg oral and 10 mg/kg intravenous administration of 12b could induce more than 70% tumor inhibition without obvious toxicity. Overall, we discovered that 12b, as a novel MDA based on hydroxamic acid, could serve as a potential MDA for further investigation.

Design, synthesis and anti-inflammatory study of novel N-heterocyclic substituted Aloe-emodin derivatives.

A novel series of Aloe-emodin derivatives containing N-heterocyclic moieties was designed and synthesized. The structure-activity relationship studies (SARs) indicated that the replacement of hydroxyethyl and benzhydryl piperazine groups could improve efficacy. Compounds 12r and 14a-14c exhibited a higher inhibitory effect on LPS-induced nitric oxide (NO) production in RAW264.7 macrophages than Aloe-emodin did. Among them, 12r showed the most potent inhibition with an IC50 value of 5.66 ± 0.47 µM. Further toxicity and pharmacokinetic studies were carried out and 12r was found to be the most active structure with low toxicity risk and good metabolic properties. It could also decrease the levels of IL-1ß, TNF-α, PGE2 and inhibit the activation of nuclear factor-κB signalling pathway. Importantly, 12r showed oral bioavailability of up to 55.16% and attenuated the inflammatory symptoms in an ulcerative colitis mouse model in vivo. These results indicate that 12r is suitable for development as an anti-inflammatory agent.

[Constraction of triple negative breast cancer cell model containing EGFR promoter and luciferase reporter gene].

Objective To establish a triple negative breast cancer cell line stably expressing human epidermal growth factor receptor (EGFR) promoter and luciferase (Luc) reporter gene and to preliminarily verify its application. Methods Using genetic recombination technology, the lentiviral vector carrying Luc reporter and EGFR promoter sequence was designed and constructed to infect MDA-MB231 cells and obtain MDA-MB231-EGFR-Luc2 cell lines by the selection with puromycin. The Luc luminescence value after stimulating with EGFR activator EGF or inhibitor gefitinib regulating the EGFR promoter activities was detected. Results Gene sequencing and enzyme digestion verified that the lentiviral expression vector carrying Luc reporter vector recombined with EGFR promoter was successfully constructed. Lentivirus-infected MDA-MB231 cells were screened by puromycin, the MDA-MB231-EGFR-Luc2 cells stably expressing firefly Luc was obtained. EGF increased the Luc luminescence value of MDA-MB231-EGFR-Luc2 cells in a dose-dependent manner, while gefitinib did the opposite. Conclusion The cell line of MDA-MB231-EGFR-Luc2 containing EGFR promoter and Luc reporter gene has been successfully constructed, which provides a new cell model for high throughput screening of EGFR-targeting drugs.

Rac1: A potential radiosensitization target of human nasopharyngeal carcinoma CNE2 cells.

Radiotherapy has a high cure rate for early nasopharyngeal carcinoma(NPC). However, the radiation resistance of poorly differentiated NPC cells impacts the effectiveness of treatment of early-stage NPC patients. Here, we explored the relationship between Ras-related C3 botulinum toxin substrate 1(Rac1) expression and NPC radiosensitivity. In vitro and in vivo studies revealed that upregulation of Rac1, when combined with X-ray treatment, increased growth inhibition and induced remarkable morphological changes and apoptosis in CNE2 cells. Furthermore, rupturing of the cell and nuclear membranes, degeneration of the cristae and significant swelling of the mitochondria were observed, which were consistent with the high apoptotic rate. The Rac1(+) cells exhibited approximately 50% more migration compared with that of the NC and Rac1(-) cells. The overexpression of Rac1 can increase the radiation sensitivity of NPC CNE2 cells, and the mechanism may be closely related to the oxidative damage of mitochondria. Rac1 might be a potential target for radiosensitization in poorly differentiated NPC.

Synthesis and screening of novel anthraquinone-quinazoline multitarget hybrids as promising anticancer candidates.

Aim: The EGF receptor (EGFR) is overexpressed in multiple epithelial-derived cancers and is considered to be a vital target closely associated with cancer therapy. In this study, a series of novel anthraquinone-quinazoline hybrids targeting several vital sites for cancer therapy were designed and synthesized. Methodology & results: Most of the synthesized hybrids demonstrated excellent antiproliferative activity and downregulation of the expression of EGFR. The most promising compound 7d showed the strongest antiproliferation activity; this compound significantly downregulated the expression of p-EGFR protein, induced a remarkable apoptosis effect, promoted the rearrangement of F-actin filaments and destruction of cytoskeleton, induced DNA damage and enhanced radiosensitivity of A549 cells. Conclusion: The novel anthraquinone-quinazoline hybrid 7d emerges as an anticancer drug candidate with promising multitargeted biological activities.

Anti-Tumor Study of H6, a 4-Substituted Coumarins Derivative, Loaded Biodegradable Self-Assembly Nano-Micelles In Vitro and In Vivo.

In our previous study, we identified a class of 4-substituted coumarins as a powerful microtubule inhibitors binding to the colchicine site of ß-tubulin. H6 showed potent anti-proliferative ability with IC50 values from 7 to 47 nM, and remarkable ability to reduce tumor growth in several xenograft models including taxol resistant tumor models. However, the extremely hydrophobicity limited its clinical application. In this study, to improve the anticancer activity and reduce the toxicity of H6, we successfully prepared MPEG-PCL with different proportions and H6-loaded polymeric micelles (H6/MPEG2kPCL2k micelles) by a simple thin-film hydration method. The prepared H6/MPEG-PCL micelles had a drug loading of 3.79 ± 0.001%, an encapsulation efficiency of 98.00 ± 0.41%, a mean particle size of 30.45 ± 0.18nm and a polydispersity index (PDI) of 0.096 ± 0.009. Computer simulation results revealed a good compatibility of H6 and MPEG2k-PCL2k copolymer. In in vitro release study and pharmacokinetic study showed H6 micelles can release H6 over an extended period. Furthermore, H6 micelles possessed comparative effect as free H6 in inhibiting cell growth, preventing cell migration, and inducing apoptosis. Mechanism study identified that H6 is a novel reversible microtubule inhibitor. In in vivo studies, H6 micelles exhibited tumor growth inhibition on two pulmonary metastatic tumor models (B16/F10 and 4T1). Importantly, H6 micelles significantly improved the solubility, reduced the toxicity, extended the half-life of drugs, and augmented the therapeutic window. All these results imply that H6 micelles have great potential for suppression of tumor metastasis.

Novel Anthraquinone Compounds Induce Cancer Cell Death through Paraptosis.

Novel anthraquinone compounds that induce ER stress and paraptosis-like cell death were designed and synthesized. Compound 4a is the first organic micromolecule to kill tumor cells by only paraptosis, and its mechanism of action has been further explored. Paraptosis does not appear to involve either phosphatidylserine translocation associated with apoptosis or cell cycle arrest. The bisbenzyloxy and N-(2-hydroxyethyl)formamide structures may be two critical pharmacophores for paraptosis. Bisbenzyloxy can induce ER stress, and the N-(2-hydroxyethyl)formamide structure can increase the ratio of LC3II/I and cytoplasmic vacuolization and facilitates paraptosis. Some antitumor drugs fail to eradicate malignant cell lines with impaired apoptotic pathways; paraptosis may be a route to kill such cells and provides a new potential strategy for cancer chemotherapy.

A novel Rhein derivative: Activation of Rac1/NADPH pathway enhances sensitivity of nasopharyngeal carcinoma cells to radiotherapy.

Radiation resistance and recurrent have become the major factors resulting in poor prognosis in the clinical treatment of patients with nasopharyngeal carcinoma (NPC). New strategies to enhance the efficacy of radiotherapy have been focused on the development of radiosensitizers and searching for directly targets that modulated tumor radiosensitivity. A novel potential radiosensitizer 1,8-Dihydroxy -3-(2'-(4″-methylpiperazin-1″-yl) ethyl-9,10-anthraquinone -3-carboxylate (RP-4) was designed and synthesized based on molecular docking technology, which was expected to regulate the radiosensitivity of tumor cells through targeting Rac1. In order to assess the radiosensitization activity of RP-4 on NPC cells, the highly differentiated CNE1 and poorly differentiated CNE2 cells NPC lines were employed. According to the results, RP-4 showed higher binding affinity toward the interaction with Rac1 than lead compounds. We found that RP-4 could inhibit cell viability and proliferation in CNE1 and CNE2 cells and significantly induced apoptosis after non-toxic concentration of RP-4 combined with 2Gy irradiation. RP-4 could effectively modulated the radiosensitivity both CNE1 cells and CNE2 cells through activating Rac1/NADPH signaling pathway and its downstream JNK/AP-1 pathway. What's more, Rac1/NADPH signaling pathway were significantly activated in Rac1-overexpressed CNE1 and CNE2 cells after treated with RP-4. Taken together, Rac1 and its downstream pathway may probably be the direct targets of RP-4 in regulating radiosensitivity of NPC cells, our finding provided a novel strategy for the development of therapeutic agents in response to tumorous radiation resistance.

Effects of Rubiadin isolated from Prismatomeris connata on anti-hepatitis B virus activity in vitro.

Prismatomeris connata was a kind of Rubiaceae plant for treatment of hepatitis, hepatic fibrosis and silicosis. Whereas, the effective components of Prismatomeris connata remains unexplored. The aim of this study was to investigate the inhibitory effects and mechanisms of Rubiadin isolated from Prismatomeris connata against HBV using HepG2.2.15 cells. The levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay. HBV DNA was qualified q-PCR. Rubiadin was isolated by silica gel column. The structure of the compound was elucidated by HPLC, FT-IR, 1 H-NMR, 13 C-NMR and identified as 1,3-Dihydroxy-2-methyl-9, 10-anthraquinone. Rubiadin significantly decreased HBeAg,HBcAg secretion level and inhibit HBV DNA replication. Rubiadin inhibits the proliferation of the cells and HBx protein expression in a dose-dependent manner. The intracellular calcium concentration was significantly reduced. These results demonstrated that Rubiadin could inhibit HepG2.2.15 cells proliferation, reduce the level of HBx expression, and intracellular free calcium, which might become a novel anti-HBV drug candidate.

Inhibition of Anaphase-Promoting Complex by Silence APC/CCdh1 to Enhance Radiosensitivity of Nasopharyngeal Carcinoma Cells.

The aim of this study was to investigate the possibility of APC/CCdh1 as a potential therapeutic target in the radiosensitivity of nasopharyngeal carcinoma (NPC) cell CNE-1, and explain the role of APC subunits after silence of Cdh1 combined with radiotherapy. Transfection with Cdh1 shRNA significantly increased the radiosensitivity of CNE-1 cells and the radiation enhancement ratio (RER) of sh-Cdh1 cells was 1.76. Knockdown of Cdh1 in CNE-1 cells increased irradiation induced apoptosis and G2/M phase cell cycle arrest. The levels of CDC20 and CylinB1 increased and the levels of Ku70 and APC3 decreased after irradiation. APC/CCdh1 is involved in regulation of radiosensitivity in human NPC CNE-1 cells. Our study may provide a promising therapeutic strategy for NPC by targeting Cdh1. J. Cell. Biochem. 118: 3150-3157, 2017. © 2016 Wiley Periodicals, Inc.