HM30181A, a potent P-glycoprotein inhibitor, potentiates the absorption and in vivo antitumor efficacy of paclitaxel in an orthotopic brain tumor model.

Objective: Delivery of chemotherapeutic drugs to the brain has remained a major obstacle in the treatment of glioma, owing to the presence of the blood-brain barrier and the activity of P-gp, which pumps its substrate back into the systemic circulation. The aim of the present study was to develop an intravenous formulation of HM30181A (HM) to inhibit P-gp in the brain to effectively deliver paclitaxel (PTX) for the treatment of malignant glioma. Methods: Two formulations of solubilized HM were designed on the basis of different solid dispersion strategies: i) spray-drying [polyvinlypyrrolidone (PVP)-HM] and ii) solvent evaporation [HP-ß-cyclodextrin (cyclodextrin)-HM]. The P-gp inhibition of these 2 formulations was assessed on the basis of rhodamine 123 uptake in cancer cells. Blood and brain pharmacokinetic parameters were also determined, and the antitumor effect of cyclodextrin-HM with PTX was evaluated in an orthotopic glioma xenograft mouse model. Results: Although both PVP-HM and cyclodextrin-HM formulations showed promising P-gp inhibition activity in vitro, cyclodextrin-HM had a higher maximum tolerated dose in mice than did PVP-HM. Pharmacokinetic study of cyclodextrin-HM revealed a plasma concentration plateau at 20 mg/kg, and the mice began to lose weight at doses above this level. Cyclodextrin-HM (10 mg/kg) administered with PTX at 10 mg/kg showed optimal antitumor activity in a mouse model, according to both tumor volume measurement and survival time (P < 0.05). Conclusions: In a mouse orthotopic brain tumor model, the intravenous co-administration of cyclodextrin-HM with PTX showed potent antitumor effects and therefore may have potential for glioma therapy in humans.

Potent Antagonists of RORγt, Cardenolides from Calotropis gigantea, Exhibit Discrepant Effects on the Differentiation of T Lymphocyte Subsets.

RORγt is the master transcription factor of IL-17 cytokine expression and Th17 lymphocyte differentiation, which are responsible for the induction of many autoimmune diseases. Recently, RORγt has become an attractive target for drug development to treat these types of diseases, and the field of RORγt antagonist research is now extremely competitive. In our current study, molecular docking was applied to demonstrate that cardenolides, including uscharin, calactin, and calotropin derived from Calotropis gigantea, probably directly bind to RORγt. Therefore, the inhibitory effect was further validated using a luciferase reporter assay. Because RORγt is the key transcriptional factor for Th17 differentiation, the effects of these compounds on Th17 differentiation were studied by flow cytometry. The results showed that uscharin, calactin, and calotropin inhibited Th17 differentiation from 100 to 500 nM. Furthermore, uscharin had a better effect than digoxin, a well-known inverse agonist of RORγt, in reducing Th17 polarization. Additionally, the effects of the cardenolides on the differentiation of other Th lineages, including Th1, Th2, and Treg, were investigated. Uscharin suppressed Th1, Th2, and Treg cell differentiation, while calactin suppressed the differentiation of Th1 cells, and calotropin did not influence the other T cell subsets, indicating that calactin suppressed Th1 and Th17 differentiation, and calotropin selectively quenched Th17 polarization. Structural analysis of the three compounds showed that the selectivity of uscharin, calactin, and calotropin on the suppression of the different subsets of T cells is correlated to the minor differences in their chemical structures. Collectively, calactin and calotropin have greater potential to be developed as lead compounds than uscharin to treat autoimmune diseases mediated by Th17 and/or Th1 cells.

Enhanced anti-tumor efficacy and mechanisms associated with docetaxel-piperine combination- in vitro and in vivo investigation using a taxane-resistant prostate cancer model.

Docetaxel (DTX) is widely used for metastatic castrated resistant prostate cancer, but its efficacy is often compromised by drug resistance associated with low intracellular concentrations. Piperine (PIP) could enhance the bioavailability of other drugs via the inhibition of CYPs and P-gp activities. Thus, we hypothesize a positive effect with the DTX-PIP combination on the anti-tumor efficacy and intra-tumor DTX concentrations in taxane-resistant prostate cancer. ICR-NOD/SCID mice implanted with taxane-resistant human prostate cancer cells were administrated with saline as well as PIP and DTX separately or in combination. The tumor growth was monitored together with intra-tumor concentrations of DTX. The inhibitory effects on CYPs and P-gp were further assessed in mouse liver microsome and MDCK-MDR1 cells. Compared with DTX alone, DTX-PIP combination significantly inhibited the tumor growth (114% vs. 217%, p = 0.002) with corresponding significantly higher intra-tumor DTX concentrations (5.854 ± 5.510 ng/ml vs. 1.312 ± 0.754 ng/mg, p = 0.037). The percentage of DTX metabolism was significantly decreased from 28.94 ± 1.06% to 18.14 ± 2.22% in mouse liver microsome after administration of PIP for two weeks. DTX accumulation in MDCK-MDR1 cell was significantly enhanced in the presence of PIP. Further microarray analysis revealed that PIP inhibited P-gp as well as CYP1B1 gene expression and induced a significant gene expression change relating to inflammatory response, angiogenesis, cell proliferation, or cell migration. In conclusion, DTX-PIP combination significantly induces activity against taxane-resistant prostate tumor. Such effect appeared to be attributed to the inhibitory effect of PIP on CYPs and P-gp activity as well as gene expression changes relating to tumorigenesis and cellular responses.

Aqueous extract of Sanguisorba officinalis blocks the Wnt/ß-catenin signaling pathway in colorectal cancer cells.

Sanguisorba officinalis (the Chinese name is DiYu, DY) exerts significant anti-proliferative activities against colorectal cancer (CRC) cells. Since most of CRC result from the aberrant activation of the Wnt/ß-catenin signaling pathway, inhibitors of the Wnt pathway are considered as promising anti-CRC agents. This study aimed to investigate whether DY could be a potential herbal Wnt inhibitor, and the bioactive constituents and underlying molecular mechanisms for DY's inhibiting activities would be studied as well. Accordingly, the inhibitory activities of DY and its main components against the Wnt pathway were assessed using the single-luciferase reporter assay based on HEK293 cells. Additionally, the levels of key Wnt-related genes or proteins were measured to verify the inhibitory effects on the Wnt pathway of CRC cells. Finally, the underlying mechanisms accounting for the efficacy of candidate drugs were explored by the transcriptomic study. Results show that DY and its tannins (RZ), and saponins (ZG) significantly inhibited the Wnt pathway of HEK293 cells activated by wnt3a. However, their respective constituents were not effective as expected. Additionally, DY and RZ prominently down-regulated the levels of ß-catenin and Wnt-targeted genes including Axin2, c-Myc or CyclinD1 of three CRC cells. Transcriptomic profiling study suggests that the down-regulation of the mRNA levels of Wnt-related genes such as LPAR6 may be associated with the inhibitory effects of DY and RZ on the Wnt pathway of HT29 cells. Therefore, our studies first uncovered the blocking activity of DY on the Wnt pathway, providing evidence for the rationale of developing Wnt inhibitors from DY as anti-CRC agents.

Profiling of ribonucleotides and deoxyribonucleotides pools in response to DNA damage and repair induced by methyl methanesulfonate in cancer and normal cells.

The absolute and relative pool sizes of deoxyribonucleotides (dRNs) are essential in DNA replication fidelity, DNA damage and repair. We found in this study that although DNA damage induced by methyl methanesulfonate (MMS) seemed similar in cancer (HepG2) and normal (LO2) cells, more extensive alterations in ribonucleotides (RNs) and dRNs pools occurred in HepG2 cells indicating that HepG2 cells were more vigilant to DNA damage. After 10 h repair, RNs pools were still severely perturbed in LO2 cells. Compared to LO2 cells, deoxyribonucleotide triphosphates (dNTPs) pools in HepG2 cells elevated by more folds which could facilitate more efficient DNA repair and improve survival probability following DNA damage, although this should definitely lead to higher mutation rates. DNA repair was more efficient in HepG2 cells at S phase and it partly came to an end while DNA repair was still uncompleted in LO2 cells outside S phase. In conclusion, our results demonstrated that HepG2 and LO2 cells presented many differences in nucleotide metabolism, cell cycle checkpoints and DNA repair pathways in response to DNA damage, which could be potential targets for cancer treatment.

Oxidative stress and diabetes mellitus.

Increasing evidences have suggested that oxidative stress plays a major role in the pathogenesis of diabetes mellitus (DM). Oxidative stress also appears to be the pathogenic factor in underlying diabetic complications. Reactive oxygen species (ROS) are generated by environmental factors, such as ionizing radiation and chemical carcinogens, and also by endogenous processes, including energy metabolism in mitochondria. ROS produced either endogenously or exogenously can attack lipids, proteins and nucleic acids simultaneously in living cells. There are many potential mechanisms whereby excess glucose metabolites traveling along these pathways might promote the development of DM complication and cause pancreatic ß cell damage. However, all these pathways have in common the formation of ROS, that, in excess and over time, causes chronic oxidative stress, which in turn causes defective insulin gene expression and insulin secretion as well as increased apoptosis. Various methods for determining biomarkers of cellular oxidative stress have been developed, and some have been proposed for sensitive assessment of antioxidant defense and oxidative damage in diabetes and its complications. However, their clinical utility is limited by less than optimal standardization techniques and the lack of sufficient large-sized, multi-marker prospective trials.