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The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

In this article, we report a novel,nanoprecipitation-based methodfor preparing silica nanoparticles with thiol and amine cofunctionalization.(3-Mercaptopropyl)trimethoxysilane (MPTMS) and 3-aminopropyltrimethoxysilane(APTMS) were used as the organosilane precursors, which were subjectedto acid-catalyzed polycondensation in an organic phase containinga water-miscible solvent (e.g., dimethyl sulfoxide). A pale colloidalsolution could be immediately formed when the preincubated organicphase was directly injected into water. The initial composition ratiobetween MPTMS and APTMS is an important factor governing the formationof nanoparticles. Specifically, large, unstable micrometer-sized particleswere formed for preparation using MPTMS as the sole silane source.In contrast, when APTMS was used alone, no particles could be formed.By reducing the fraction of APTMS (or increasing that of MPTMS) inthe initial mixture of organosilanes, the formation of nanometer-sizedparticles occurred at a critical fraction of APTMS (i.e., 25%). Remarkably,a tiny fraction (e.g., 1%) of APTMS was sufficient to produce stablenanoparticles with a hydrodynamic diameter of about 200 nm. Otherfactors that would also affect particle formation were determined.Moreover, an interesting temperature effect on particle formationwas observed. The TEM micrographs show spherical nanospheres withmean sizes of 130–150 nm in diameter. The solid-state 29Si NMR spectra demonstrate that the hybrid silica materialscontain fully and partially condensed silicon structures. The bifunctionalizedsilica nanoparticles have positive zeta potentials whose magnitudesare positively correlated with the amount of APTMS. The total thiolcontent, however, is negatively correlated with the amount of APTMS.The cationic nanoparticles can bind an antisense oligonucleotide ina composition-dependent manner. [ABSTRACT FROM AUTHOR]

Amphiphilic drugs are therapeutic agents whose molecular structures contain both hydrophobic and hydrophilic portions. Here we report a systematic study on how amphiphilic drugs can assist in silica nanoprecipitation. 3-Mercaptopropyltrimethoxysilane (MPTMS) was used as the sole silica material and 12 amphiphilic drugs spanning a wide spectrum of therapeutic categories were included. MPTMS polycondensation was conducted in a DMSO-based organic phase. After a sufficient time, particle formation was induced by injecting a small amount of the organic phase into a water solution containing various amphiphiles. The results show that all amphiphilic drugs studied exerted concentration-dependent facilitating effect on nanoparticle formation. Under certain preparation conditions, the particle solution showed physical stability over a long period and the formed particles could be as small as 100 nm. By systematically varying drug concentrations and injection volumes, the ability of each amphiphile to promote nanoprecipitation can be quantified and compared, based on two novel indices: the area under the critical volume-concentration curve (AUC) and the critical stabilization concentration (CSC). We demonstrate that both ability indices significantly correlated with the drug's log P and critical micelle concentrations (CMC). Furthermore, we have optimized the aging and particle purification condition and extensively characterized our system through comprehensive TEM and zeta-potential measurements, as well as determinations for drug entrapment and release. In conclusion, we have established a quantitative structure-activity relationship for amphiphilic small-molecular drugs in their ability to interact with poly(mercaptopropyl)silsesquioxane species and form nanoparticles via solvent shifting. We speculate that both hydrophobic and electrostatic interactions play important roles in the formation and stabilization of nanoparticles.

Although the antitumor activity of the crude extract of wild bitter gourd (Momordica charantia L.) has been reported, its bioactive constituents and the underlying mechanism remain undefined. Here, we report that 3 β ,7 β -dihydroxy-25-methoxycucurbita-5,23-diene-19-al (DMC), a cucurbitane-type triterpene isolated from wild bitter gourd, induced apoptotic death in breast cancer cells through peroxisome proliferator-activated receptor (PPAR) γ activation. Luciferase reporter assays indicated the ability of DMC to activate PPAR γ , and pharmacological inhibition of PPAR γ protected cells from DMC's antiproliferative effect. Western blot analysis indicated that DMC suppressed the expression of many PPAR γ -targeted signaling effectors, including cyclin D1, CDK6, Bcl-2, XIAP, cyclooxygenase-2, NF- κ B, and estrogen receptor α , and induced endoplasmic reticulum stress, as manifested by the induction of GADD153 and GRP78 expression. Moreover, DMC inhibited mTOR-p70S6K signaling through Akt downregulation and AMPK activation. The ability of DMC to activate AMPK in liver kinase (LK) B1-deficient MDA-MB-231 cells suggests that this activation was independent of LKB1-regulated cellular metabolic status. However, DMC induced a cytoprotective autophagy presumably through mTOR inhibition, which could be overcome by the cotreatment with the autophagy inhibitor chloroquine. Together, the ability of DMC to modulate multiple PPAR γ -targeted signaling pathways provides a mechanistic basis to account for the antitumor activity of wild bitter gourd.

Purpose: In this study, we investigated the biochemical pharmacology of pirenoxine (PRX) and catalin under in vitro selenite/calcium- and ultraviolet (UV)-induced lens protein turbidity challenges. The systemic effects of catalin were determined using a selenite-induced cataractogenesis rat model., Methods: In vitro cataractogenesis assay systems (including UVB/C photo-oxidation of lens crystallins, calpain-induced proteolysis, and selenite/calcium-induced turbidity of lens crystallin solutions) were used to screen the activity of PRX and catalin eye drop solutions. Turbidity was identified as the optical density measured using spectroscopy at 405 nm. We also determined the in vivo effects of catalin on cataract severity in a selenite-induced cataract rat model. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to analyze the integrity of crystallin samples., Results: PRX at 1,000 μM significantly delayed UVC-induced turbidity formation compared to controls after 4 h of UVC exposure (p<0.05), but not in groups incubated with PRX concentrations of <1,000 μM. Results were further confirmed by SDS-PAGE. The absolute γ-crystallin turbidity induced by 4 h of UVC exposure was ameliorated in the presence of catalin equivalent to 1~100 μM PRX in a concentration-dependent manner. Samples with catalin-formulated vehicle only (CataV) and those containing PRX equivalent to 100 μM had a similar protective effect after 4 h of UVC exposure compared to the controls (p<0.05). PRX at 0.03, 0.1, and 0.3 μM significantly delayed 10 mM selenite- and calcium-induced turbidity formation compared to controls on days 0~4 (p<0.05). Catalin (equivalent to 32, 80, and 100 μM PRX) had an initial protective effect against selenite-induced lens protein turbidity on day 1 (p<0.05). Subcutaneous pretreatment with catalin (5 mg/kg) also statistically decreased the mean cataract scores in selenite-induced cataract rats on post-induction day 3 compared to the controls (1.3±0.2 versus 2.4±0.4; p<0.05). However, catalin (equivalent to up to 100 μM PRX) did not inhibit calpain-induced proteolysis activated by calcium, and neither did 100 μM PRX., Conclusions: PRX at micromolar levels ameliorated selenite- and calcium-induced lens protein turbidity but required millimolar levels to protect against UVC irradiation. The observed inhibition of UVC-induced turbidity of lens crystallins by catalin at micromolar concentrations may have been a result of the catalin-formulated vehicle. Transient protection by catalin against selenite-induced turbidity of crystallin solutions in vitro was supported by the ameliorated cataract scores in the early stage of cataractogenesis in vivo by subcutaneously administered catalin. PRX could not inhibit calpain-induced proteolysis activated by calcium or catalin itself, and may be detrimental to crystallins under UVB exposure. Further studies on formulation modifications of catalin and recommended doses of PRX to optimize clinical efficacy by cataract type are warranted.

Background: Folate receptors (FRs) are cellular surface markers for numerous solid tumors and myeloid leukemias. The aim of this study was to develop an antisense oligodeoxyribonucleotide (ODN) carrier targeting FR-overexpressing cancer cells using folate (FA) as the targeting moiety. G3139, a phosphorothioate antisense ODN against human bcl2 mRNA, was evaluated in this study., Materials and Methods: G3139-containing liposomes were prepared using an ethanol dilution method. For the targeted formulation, 0.5 mol% of folate-PEG-DSPE was incorporated as a targeting ligand into cationic liposomes composed of DC-Chol/egg PC/PEG-DSPE at 25:65:10 mol/mol. Particle size and surface charge were measured and cellular uptake was assessed by fluorescence microscopy and flow cytometry. The ODN-containing formulations were evaluated in FR+ KB cells for Bcl2 down-regulation measured by Western blot. The cytotoxicity of the formulations was determined by MTT assay., Results: The G3139-containing liposomes had an average diameter of 80-90 nm with high ODN entrapment efficiency (70-80%). Incorporation of the folate ligand did not significantly alter the particle size and entrapment efficiency. The formulation exhibited colloidal stability in a serum-containing environment. In uptake studies, the folate-targeted formulation showed ligand concentration-dependent uptake that was up to 6-fold more efficient than that of the non-targeted formulation (p < 0.05). The uptake could be blocked by an excess amount of free folate, thus indicating an FR-dependent mechanism., Conclusion: FR-targeted G3139-containing liposomes showed promising transfection activity in KB cells. FR-targeted formulations were capable of specific targeting to FR-overexpressing cell lines and optimizing the amount of folate ligand in the liposomal formulation can result in more efficient antisense delivery.