Search

Background Mutations in the human gene encoding the neuron-specific Eag1 (KV10.1; KCNH1) potassium channel are linked to congenital neurodevelopmental diseases. Disease-causing mutant Eag1 channels manifest aberrant gating function and defective protein homeostasis. Both the E3 ubiquitin ligase cullin 7 (Cul7) and the small acid protein 14-3-3 serve as binding partners of Eag1. Cul7 mediates proteasomal and lysosomal degradation of Eag1 protein, whereas over-expression of 14-3-3 notably reduces Eag1 channel activity. It remains unclear whether 14-3-3 may also contribute to Eag1 protein homeostasis. Results In human cell line and native rat neurons, disruptions of endogenous 14-3-3 function with the peptide inhibitor difopein or specific RNA interference up-regulated Eag1 protein level in a transcription-independent manner. Difopein hindered Eag1 protein ubiquitination at the endoplasmic reticulum and the plasma membrane, effectively promoting the stability of both immature and mature Eag1 proteins. Suppression of endogenous 14-3-3 function also reduced excitotoxicity-associated Eag1 degradation in neurons. Difopein diminished Cul7-mediated Eag1 degradation, and Cul7 knock-down abolished the effect of difopein on Eag1. Inhibition of endogenous 14-3-3 function substantially perturbed the interaction of Eag1 with Cul7. Further structural analyses suggested that the intracellular Per-Arnt-Sim (PAS) domain and cyclic nucleotide-binding homology domain (CNBHD) of Eag1 are essential for the regulatory effect of 14-3-3 proteins. Significantly, suppression of endogenous 14-3-3 function reduced Cul7-mediated degradation of disease-associated Eag1 mutant proteins. Conclusion Overall these results highlight a chaperone-like role of endogenous 14-3-3 proteins in regulating Eag1 protein homeostasis, as well as a therapeutic potential of 14-3-3 modulators in correcting defective protein expression of disease-causing Eag1 mutants.

Aims The prevalent human hepatocellular carcinoma (HCC) is a leading cause of global cancer-related mortality. The small molecular weight triepoxide derivative, 1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl) (teroxirone), has been proved effective against the proliferation of lung cancer cells. The purpose is to further examine if teroxirone regulate growth and metastatic potential of HCC cells with aims at disclosing more of the reaction mechanisms. Main methods Measurements of cell viability and flow cytometry were conducted to test sensitivities of teroxirone against HCC cells. The signaling pathway leading to apoptotic death was unraveled by Western blotting analysis. The metastatic progression was evaluated by cell-based phenotype assay that included migration, invasion, gelatin zymography and wound assay. The in vivo drug efficiency was done in immune-deficient mice with the established xenograft tumors. Key findings Teroxirone inhibited growth of HCC cells, but not hepatic cells. The drug induced apoptosis in HCC cells bearing mutant p53. Pretreatment of caspase-3 inhibitor restored cell viabilities by suppressing extrinsic pathway-mediated cell death. More experiments suggested that sub-apoptotic concentrations of teroxirone mitigated migration, invasion and wound healing of HCC cells. The drug reduced growth of the xenograft tumors as established in animal models by activating apoptotic death. Significance The findings asserted that teroxirone is an eligible addition to the existing options as an anticancer agent to eliminate HCC.

Therapy by targeting cancer stem cells (CSCs) is an eligible method to eradicate malignant human tumors. A synthetic triepoxide derivative, teroxirone, was reported effective against the growth of human lung cancer cells by injuring cellular mitochondria functions. And yet it remains unclear if the residual but malicious CSCs can be effectively dissipated following treatment. The current study further affirmed that teroxirone inhibited the propagation of CSCs as enriched from NSCLC cells by inducing p53 that lead to ultimate apoptosis. More evidence supported that the reduced stemness of the spheroids was associated with apoptotic death. The results consolidate the notion that teroxirone is a viable and effective therapeutic agent for eradicating human lung cancer.

Numerous approaches suggested that compounds with conjugated triazole moieties or benzoxazone pharmacores are effective to antagonize proliferation of human tumors. The current study reported that a synthetic triazole-conjugated benzoxazone, 4-((5-benzyl-1H-1,2,3-triazol-3-yl)-methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one (BTO), inhibited growth rates of human non-small cell lung cancer cells. The cytotoxicity can be enhanced with increasing drug concentrations. More evidence supported that the induced reactive oxygen species lead to ultimate apoptotic cell death by recruiting autophagy. The mechanistic pathway as elucidated involved tumor suppressor p53 activation and LC3-1 conversion followed by PARP and procaspase-3 cleavage. Autophagy inhibition reverted apoptotic death and restored cell viabilities. BTO suppressed the development of A549 cell xenograft tumors by activating autophagy and apoptosis simultaneously. As an efficient tumor growth inhibitor with relatively small molecular weight, BTO is a viable addition to the existing list of lung cancer treatment.

Hsieh CH, Lee LC, Leong WY, Yang TC, Yao CF, Fang K. Drug Des Devel Ther. 2016;10:2947–2957.On page 2950, Figure 1, part B has been updated.Read the original article&nbsp

As a practical and safe herbal medicine, the seeds of Brucea javanica (L.) Merr., were used to cure patients suffering from infectious diseases such as malaria. Recent advances revealed that the herb could also be a useful cancer therapy agent. The study demonstrated that aqueous B. javanica (BJ) extract attenuated the growth of human non-small-lung cancer cells bearing mutant L858R/T790M epidermal growth factor receptor (EGFR). The reduced cell viability in H1975 cells was attributed to apoptosis. Transfection of EGFR small hairpin RNA reverted the sensitivities. When nude mice were fed BJ extract, the growth of xenograft tumors, as established by H1975 cells, was suppressed. Additional histological examination and fluorescence analysis of the resected tissues proved that the induced apoptosis mitigated tumor growth. The work proved that the BJ extract exerted its effectiveness by targeting lung cancer cells carrying mutated EGFR while alleviating tumorigenesis. Aqueous BJ extract is a good candidate to overcome drug resistance in patients undergoing target therapy.

Seung-Hun Kim,1,* Chun-Yen Liu,1,* Po-Wei Fan,1 Chang-Heng Hsieh,1 Hsuan-Yuan Lin,1 Ming-Chung Lee,2 Kang Fang1 1Department of Life Science, National Taiwan Normal University, Taipei, 2Brion Research Institute of Taiwan, New Taipei City, Taiwan *These authors contributed equally to this work Abstract: As a practical and safe herbal medicine, the seeds of Brucea javanica (L.) Merr., were used to cure patients suffering from infectious diseases such as malaria. Recent advances revealed that the herb could also be a useful cancer therapy agent. The study demonstrated that aqueous B. javanica (BJ) extract attenuated the growth of human non-small-lung cancer cells bearing mutant L858R/T790M epidermal growth factor receptor (EGFR). The reduced cell viability in H1975 cells was attributed to apoptosis. Transfection of EGFR small hairpin RNA reverted the sensitivities. When nude mice were fed BJ extract, the growth of xenograft tumors, as established by H1975 cells, was suppressed. Additional histological examination and fluorescence analysis of the resected tissues proved that the induced apoptosis mitigated tumor growth. The work proved that the BJ extract exerted its effectiveness by targeting lung cancer cells carrying mutated EGFR while alleviating tumorigenesis. Aqueous BJ extract is a good candidate to overcome drug resistance in patients undergoing target therapy. Keywords: Brucea javanica, target therapy, epidermal growth factor receptor, human lung, herbal medicine, apoptosis

Chang Heng Hsieh,1 Li-Ching Lee,1 Wai-Yin Leong,1 Tsai-Chen Yang,1 Ching-Fa Yao,2 Kang Fang1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan Abstract: Trinucleotide CAG repeat expansion in the coding region of genes has a propensity to form polyglutamine (polyQ) aggregates that contribute to neuronal disorders. Strategies in elevating autophagy to disintegrate the insoluble aggregates without injuring cells have become a major goal for therapy. In this work, a triazole derivative, OC-13, was found accelerating autophagic clearance of polyQ aggregation in human neuroblastoma cells following induction of the enhanced green fluorescence-conjugated chimeric protein that enclosed 79 polyQ repeats (Q79-EGFP). OC-13 accelerated autophagy development and removed nuclear Q79-EGFP aggregates. The increase of Beclin-1, turnover of LC3-I to LC3-II and degradation of p62 supported autophagy activation. Pretreatment of autophagy inhibitor, bafilomycin A1, not only suppressed autophagolysome fusion, but also impeded aggregate eradication. The study also showed that c-Jun N-terminal kinase/Beclin-1 pathway was activated during OC-13 treatment and c-Jun N-terminal kinase inhibitor impaired autophagy and final breakdown. Autophagic clearance of the insoluble aggregates demonstrated the feasibility of OC-13 in alleviating neuronal disorders because of expanded glutamine stretches. Keywords: autophagic flux, polyglutamine, aggregates clearance, triazole, JNK pathway, neuronal disorders, green fluorescence protein

Protein phosphatase 2A is one of four major classes of serine/threonine phosphatases. Overexpression of brain-specific regulatory subunit PPP2R2 in neuron cells is implicated in pathogenesis. The alternative splicing of PPP2R2B encodes two isoforms. They are subunit of cytoplasmic specific Bβ1 and mitochondria-targeted Bβ2. The two constructs were transfected into human neuroblastoma cells, SK-N-SH, respectively, and the stable clones overexpressing either Bβ1 or Bβ2 established. We have reported that Bβ2 clones are sensitive to reactive oxygen species (ROS) treatment by inducing autophagic cell death. To study more on the onset of neuropathogenesis under strain, both clones were exposed to different environmental stress, e.g. starvation and endoplasmic reticulum (ER) stress. To learn how PPP2R2B overexpression responds to starvation, cells were incubated in Hank’s buffered salt solution of deprived nutrient. Cell death was induced in Bβ1 clones after 6 h starvation, but not in Bβ2 clones. The pharmacological inhibitor, Bafilomycin A1, rescued the cell death while suppressing autophagy. On the other hand, to assess how cells respond to ER stress, the cells were treated with 0.1 μM of N-glycosylation inhibitor, tunicamycin (TM). In contrast with Bβ1, the apoptotic cell death appeared in Bβ2 after 48 h treatment. The formation of autophagolysosome was detected in Bβ2 following 12 h treatment with TM as evidenced by lysotracker and GFP-LC3 staining for fluorescence microscopy analysis. The autophagy inhibitor, 3-methyladenine, salvaged the final apoptosis. The stable cell lines with ectopically transfected PPP2R2B genes encoding isoforms of brain-specific regulatory subunit exhibit distinct apoptosis under different stressors. The induced autophagic apoptotic cell death is related to mitochondrial membrane potential drop and ROS generation. Disturbance of autophagy alleviates the induced cell death. The results promised a good model for understanding the onset in pathogenesis under stress in neuron cells with aberrant PPP2R2B expression.