Panobinostat sensitizes KRAS-mutant non-small-cell lung cancer to gefitinib by targeting TAZ.

Mutation of KRAS in non-small-cell lung cancer (NSCLC) shows a poor response to epidermal growth factor receptor (EGFR) inhibitors and chemotherapy. Currently, there are no direct anti-KRAS therapies available. Thus, new strategies have emerged for targeting KRAS downstream signaling. Panobinostat is a clinically available histone deacetylase inhibitor for treating myelomas and also shows potentiality in NSCLC. However, the therapeutic efficacy of panobinostat against gefitinib-resistant NSCLC is unclear. In this study, we demonstrated that panobinostat overcame resistance to gefitinib in KRAS-mutant/EGFR-wild-type NSCLC. Combined panobinostat and gefitinib synergistically reduced tumor growth in vitro and in vivo. Mechanistically, we identified that panobinostat-but not gefitinib-inhibited TAZ transcription, and the combination of panobinostat and gefitinib synergistically downregulated TAZ and TAZ downstream targets, including EGFR and EGFR ligand. Inhibition of TAZ by panobinostat or short hairpin RNA sensitized KRAS-mutant/EGFR-wild-type NSCLC to gefitinib through abrogating AKT/mammalian target of rapamycin (mTOR) signaling. Clinically, TAZ was positively correlated with EGFR signaling, and coexpression of TAZ/EGFR conferred a poorer prognosis in lung cancer patients. Our findings identify that targeting TAZ-mediated compensatory mechanism is a novel therapeutic approach to overcome gefitinib resistance in KRAS-mutant/EGFR-wild-type NSCLC.

Evodiamine Prevents Glioma Growth, Induces Glioblastoma Cell Apoptosis and Cell Cycle Arrest through JNK Activation.

Evodiamine (EVO) is an active medicinal compound derived from the traditional herbal medicine Evodia rutaecarpa. It has been reported that evodiamine has several beneficial biological properties, including anticancer and anti-inflammatory activities. However, the in vitro and in vivo anticancer activities of EVO against the growth of glioblastoma cells remain undefined. EVO induced significant decreases in the viability of U87 and C6 glioma cells, but not of primary astrocytes, according with the occurrence of apoptotic characteristics including DNA ladders, caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage, and hypodiploid cells. The disruption of the mitochondrial membrane potential (MMP) was detected, and it was found that the peptidyl caspase-9 inhibitor, Z-LEHD-FMK, significantly prevented glioma cells from EVO-induced apoptosis. Increased c-Jun N-terminal kinase (JNK) protein phosphorylation by EVO was observed, and the addition of JNK inhibitors, SP600125 and JNKI inhibited the EVO-induced apoptosis was inhibited. Additionally, EVO treatment induced G2/M arrest with increased polymerized tubulin protein expression in U87 and C6 cells. Elevated expressions of the cyclin B1, p53, and phosphorylated (p)-p53 proteins were detected in EVO-treated glioma cells, and these were inhibited by JNK inhibitors. An in vivo study showed that EVO significantly reduced the growth of gliomas elicited by the subcutaneous injection of U87 cells with increases in cyclin B1, p53, and p-p53 protein expressions in tumors. An analysis of eight EVO-related chemicals showed that alkyl groups at position 14 in EVO are important for its anti-glioma effects which involve both apoptosis and G2/M arrest. Evidence is provided that supports EVO induction of apoptosis and G2/M arrest via the activation of JNK-mediated gene expression and disruption of MMP in glioblastoma cells. EVO was shown to penetrate the blood-brain barrier; EVO is therefore predicted to be a promising compound for the chemotherapy of glioblastomas and deserves further investigations.

Protein Kinase RNA-Like Endoplasmic Reticulum Kinase-Mediated Bcl-2 Protein Phosphorylation Contributes to Evodiamine-Induced Apoptosis of Human Renal Cell Carcinoma Cells.

We investigated the anticancer mechanism of evodiamine (EVO) against the viability of human A498 renal cell carcinoma (RCC) cells in vitro and in vivo. The in vitro study showed that EVO decreased the viability of A498 cells with the occurrence of apoptotic characteristics such as hypodiploid cells, DNA ladders, chromatin-condensed cells, and cleaved caspase (Casp)-3/poly(ADP ribose) polymerase (PARP) proteins. Pharmacological studies using chemical inhibitors of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) indicated that phosphorylation of the c-Jun N-terminal kinase (JNK) protein participated in EVO-induced cell death of A498 cells, and application of the JNK inhibitor, SP600125 (SP), inhibited EVO-induced cleavage of the Casp-3/PARP proteins and chromatin condensation according to Giemsa staining. EVO disruption of the mitochondrial membrane potential (MMP) with increased protein levels of the phosphorylated Bcl-2 protein (p-Bcl-2) was prevented by JNK inhibitors in A498 cells. A structure-activity relationship study showed that a methyl group at position 14 in EVO was important for its apoptotic effects and increased p-Bcl-2 protein in A498 cells. Furthermore, significant increases in the phosphorylated endoplasmic reticular stress protein, protein kinase RNA-like endoplasmic reticulum kinase (p-PERK at Thr980), by EVO were detected in A498 cells, and the PERK inhibitor, GSK2606414, significantly suppressed EVO-induced apoptosis, p-JNK, p-PERK, and cleaved PARP proteins. The in vivo study showed that EVO significantly reduced RCC growth elicited by a subcutaneous injection of A498 cells, and an increased protein level of p-PERK was observed according to an immunohistochemical analysis. Apoptosis by EVO was also demonstrated in other RCC cells such as 786-O, ACHN, and Caki-1 cells. This is the first study to demonstrate the anti-RCC effect of EVO via apoptosis in vitro and in vivo, and activation of JNK and PERK to induce Bcl-2 protein phosphorylation, which led to disruption of the MMP.

Hydrogel microspheres for stabilization of an antioxidant enzyme: effect of emulsion cross-linking of a dual polysaccharide system on the protection of enzyme activity.

Catalase is an antioxidant enzyme abundant in natural resources. However, the enzyme is usually inactivated by gastric acid and digestive enzymes after oral ingestion. In this study, carboxymethyl chitosan (CM-chitosan) and hyaluronic acid (HA) conjugate hydrogel microspheres have been prepared by an emulsion cross-linking technique to retain the activity of catalase in simulated gastrointestinal (GI) fluids. Cross-linking reduced the swelling capability and increased the resistance toward hyaluronidase digestion of prepared HA-CM-chitosan hydrogel microspheres. Catalase entrapped in the hydrogel microspheres exhibited superior stability over a wide pH range (pH 2.0 and 6.0-8.0) as compared to the native enzyme. The entrapped catalase was also protected against degradation by digestive enzymes. Following the treatments, the catalase-loaded microspheres, in contrast to native catalase, could effectively decrease the intracellular H2O2 level and protect HT-29 colonic epithelial cells against H2O2-induced oxidative damage to preserve cell viability. These results suggested that the HA-CM-chitosan hydrogel microspheres can be used for entrapment, protection and intestinal delivery of catalase for H2O2 scavenging.

Nanoparticle-induced tight-junction opening for the transport of an anti-angiogenic sulfated polysaccharide across Caco-2 cell monolayers.

Fucoidan has the ability to inhibit angiogenesis by human umbilical vein endothelial cells (HUVECs). However, a major clinical limitation is its poor oral availability because fucoidan is a hydrophilic macromolecule. In this study, an oversulfation reaction of fucoidan has been performed to enhance its anti-angiogenic activities. The synthesized, oversulfated fucoidan (OFD) was characterized by Fourier transform infrared spectroscopy. The oversulfate content of OFD was estimated to be 41.7% by using a BaCl2 gelatin method. Nanoparticles (NPs) composed of chitosan (CS) and OFD were prepared by a polycation-polyanion complex method. The mean particle sizes of prepared CS/OFD NPs were in the range of 172-265nm with a negative or positive surface charge, depending on the relative concentrations of CS to OFD used. The self-assembled NPs with pH-sensitive characteristics could be used as a pH-switched nanocarrier for oral delivery of the antiangiogenic macromolecule, OFD, in response to simulated gastrointestinal (GI) tract media. Evaluation of test NPs in enhancing the intestinal paracellular transport of OFD suggested that the NPs with a positive surface charge could transiently open the tight junctions between Caco-2 cells and thus increase the paracellular permeability. Tight-junction opening and restoration were examined by monitoring the redistribution of ZO-1 tight-junction proteins using confocal laser scanning microscopy (CLSM). The transported OFD significantly inhibits the tube formation of HUVECs via competitive binding of OFD and basic fibroblast growth factor (bFGF) to bFGF receptors (bFGFRs).

Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.