Induction of MAPK- and ROS-dependent autophagy and apoptosis in gastric carcinoma by combination of romidepsin and bortezomib

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors can synergistically induce apoptotic cell death in certain cancer cell types but their combinatorial effect on the induction of autophagy remains unknown. Here, we investigated the combinatorial effects of a proteasome inhibitor, bortezomib, and an HDAC inhibitor, romidepsin, on the induction of apoptotic and autophagic cell death in gastric carcinoma (GC) cells. Isobologram analysis showed that low nanomolar concentrations of bortezomib/romidepsin could synergistically induce killing of GC cells. The synergistic killing was due to the summative effect of caspase-dependent intrinsic apoptosis and caspase-independent autophagy. The autophagic cell death was dependent on the activation of MAPK family members (ERK1/2 and JNK), and generation of reactive oxygen species (ROS), but was independent of Epstein-Barr virus infection. In vivo, bortezomib/romidepsin also significantly induced apoptosis and autophagy in GC xenografts in nude mice. This is the first report demonstrating the potent effect of combination of HDAC and proteasome inhibitors on the induction of MAPK- and ROS-dependent autophagy in addition to caspase-dependent apoptosis in a cancer type.published_or_final_versio

Reactivation of Epstein-Barr virus lytic cycle by histone deacetylase inhibitors

Epstein-Barr virus (EBV) is closely associated with certain lymphoid and epithelial malignancies such as Burkitt lymphoma, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). In the tumor cells, the virus persists in a tight latency, expressing a limited number of latent proteins. Reactivation of EBV lytic cycle from latency leads to expression of many more viral lytic proteins which may provide potential therapeutic targets for the EBV-associated cancers. Histone deacetylase (HDAC) inhibitors belong to an emerging class of anti-cancer agents which work through acetylation of different histone and non-histone proteins in cancer cells. Our previous work showed that various pan-HDAC inhibitors, which inhibit eleven HDAC isoforms, can preferentially reactivate EBV lytic cycle in EBV-positive epithelial rather than lymphoid cancers and mediate enhanced killing of EBV-positive NPC and GC cells through augmentation of apoptotic cell death. Recently, we found that a selective class I HDAC inhibitor, romidepsin, can potently induce EBV lytic cycle in NPC and GC cells and confer susceptibility of the induced cells to killing by an anti-viral agent, ganciclovir, in vitro and in vivo. The reactivation of EBV lytic cycle by romidepsin is related to the inhibition of HDAC-1, -2 and -3 isoforms and the activation of PKC-. Interestingly, our current findings suggest that acetylation of non-histone proteins might also play a role in the regulation of EBV lytic cycle upon HDAC inhibition. In this review, we discuss our recent findings on the mechanisms of EBV lytic cycle reactivation and propose possible strategies in using HDAC inhibitors for the treatment of EBV-associated cancers.published_or_final_versio

Bortezomib combines with suberoylanilide hydroxamic acid (SAHA) to synergistically induce caspase-dependent apoptosis and blocks SAHA's activation of EBV lytic cycle in nasopharyngeal carcinoma

Poster Session 1 - Vaccines and Anti-Viral Therapeutics: no. 3.13Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), could induce Epstein-Barr virus (EBV) lytic cycle and apoptosis in EBV-positive nasopharyngeal carcinoma (NPC) cells. In this study, we investigated the effects of combining a proteasome inhibitor, bortezomib, with SAHA in the treatment of NPC cells. Synergistic killing of a panel of EBV-positive NPC cells upon treatment with various combinations of bortezomib (0, 7.5, 15, 30, 60 and 120 nM) and SAHA (0, 0.625, 1.25, 2.5, 5 and 10 uM) was demonstrated by MTT assay and isobologram analysis. The synergistic killing was due to apoptosis as demonstrated by markedly increased sub-G1, annexin V-positive and TUNEL-positive cell populations. Strong proteolytic cleavage of PARP, caspase-3, -7 and -9 and increased reactive oxygen species …postprin

Combination of SAHA and bortezomib up-regulates CDKN2A and CDKN1A and induces apoptosis of Epstein-Barr virus-positive Wp-restricted Burkitt lymphoma and lymphoblastoid cell lines

Epstein-Barr virus (EBV) latent proteins exert anti-apoptotic effects on EBV-transformed lymphoid cells by down-regulating BCL2L11 (BIM), CDKN2A (p16(INK4A) ) and CDKN1A (p21(WAF1) ). However, the potential therapeutic effects of targeting these anti-apoptotic mechanisms remain unexplored. Here, we tested both in vitro and in vivo effects of the combination of histone deacetylase (HDAC) and proteasome inhibitors on the apoptosis of six endemic Burkitt lymphoma (BL) lines of different latency patterns (types I and III and Wp-restricted) and three lymphoblastoid cell lines (LCLs). We found that the combination of HDAC and proteasome inhibitors (e.g. SAHA/bortezomib) synergistically induced the killing of Wp-restricted and latency III BL and LCLs but not latency I BL cells. The synergistic killing was due to apoptosis, as evidenced by the high percentage of annexin V positivity and strong cleavage of PARP1 (PARP) and CASP3 (caspase-3). Concomitantly, SAHA/bortezomib up-regulated the expression of CDKN2A and CDKN1A but did not affect the level of BCL2L11 or BHRF1 (viral homologue of BCL2). The apoptotic effects were dependent on reactive oxygen species generation. Furthermore, SAHA/bortezomib suppressed the growth of Wp-restricted BL xenografts in nude mice. This study provides the rationale to test the novel application of SAHA/bortezomib on the treatment of EBV-associated Wp-restricted BL and post-transplant lymphoproliferative disorder.postprin

Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies

Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells’ responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells’ responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV lytic cycle reactivation from latency.published_or_final_versio

Inhibition of class I histone deacetylases by romidepsin potently induces Epstein-Barr virus lytic cycle and mediates enhanced cell death with ganciclovir

Pan-histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein-Barr virus (EBV) lytic cycle in EBV-associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC-1, -2 and -3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA-approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ∼0.5 to 5 nM (versus ∼800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21WAF1 , which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21WAF1 and induction of lytic cycle were abrogated by a specific inhibitor of PKC-δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC-1, -2 and -3 by romidepsin or shRNA knockdown could confer susceptibility of EBV-positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV-associated cancers.postprin

Differing coping mechanisms, stress level and anorectal physiology in patients with functional constipation

Aim: To investigate coping mechanisms, constipation symptoms and anorectal physiology in 80 constipated subjects and 18 controls. Methods: Constipation was diagnosed by Rome II criteria. Coping ability and anxiety/depression were assessed by validated questionnaires. Transit time and balloon distension test were performed. Results: 34.5% patients were classified as slow transit type of constipation. The total colonic transit time (56 h vs 10 h, P<0.0001) and rectal sensation including urge sensation (79 mL vs 63 mL, P = 0.019) and maximum tolerable volume (110 mL vs 95 mL, P = 0.03) differed in patients and controls. Constipated subjects had significantly higher anxiety and depression scores and lower SF-36 scores in all categories. They also demonstrated higher scores of 'monitoring' coping strategy (14±6 vs 9±3, P = 0.001), which correlated with the rectal distension sensation (P = 0.005), urge sensation (P=0.002), and maximum tolerable volume (P = 0.035). The less use of blunting strategy predicted slow transit constipation in both univariate (P = 0.01) and multivariate analysis (P = 0.03). Conclusion: Defective or ineffective use of coping strategies may be an important etiology in functional constipation and subsequently reflected in abnormal anorectal physiology. © 2005 The WJG Press and Elsevier Inc. All rights reserved.published_or_final_versio

Comparison between empirical prokinetics, Helicobacter test-and-treat and empirical endoscopy in primary-care patients presenting with dyspepsia: A one-year study

Aim: To investigate the optimal strategy to treat dyspeptic patients in primary care. Methods: Dyspeptic patients presenting to primary care outpatient clinics were randomly assigned to: (1) empirical endoscopy, (2) H pylori test-and-treat, and (3) empirical prokinetic treatment with cisapride. Early endoscopy was arranged if patients remained symptomatic after 2 wk. Symptom severity, quality-of-life (SF-36) as well as patient preference and satisfaction were assessed. All patients underwent endoscopy by wk 6. Patients were followed up for one year. Results: Two hundred and thirty four patients were recruited (163 female, mean age 49). 46% were H pylori positive. 26% of H pylori tested and 25% of empirical prokinetic patients showed no improvement at wk 2 follow-up and needed early endoscopy. 15% of patients receiving empirical cisapride responded well to treatment but peptic ulcer was the final diagnosis. Symptom resolution and quality-of-life were similar among the groups. Costs for the three strategies were HK$4343,$1771 and $1750 per patient. 66% of the patients preferred to have early endoscopy. Conclusion: The three strategies are equally effective. Empirical prokinetic treatment was the least expensive but peptic ulcers may be missed with this treatment. The H pylori test-and-treat was the most cost-effective option. © 2006 The WJG Press. All rights reserved.published_or_final_versio

Normal 24-hour ambulatory proximal and distal gastroesophageal reflux parameters in Chinese.

OBJECTIVE: To quantify normal proximal and distal oesophageal acid parameters in healthy Chinese. DESIGN: Observational study. SETTING: University teaching hospital, Hong Kong. SUBJECTS AND METHODS: Twenty healthy adults who were not on medication and were free from gastrointestinal symptoms were recruited by advertisement. Ambulatory oesophageal acid (pH5 minutes, 4/0; and the longest single acid exposure episode, 11.2/3.0 minutes. CONCLUSION: Physiological gastroesophageal reflux occurs in healthy Chinese. These initial data provide a preliminary reference range that could be utilised by laboratories studying Chinese subjects.published_or_final_versio

Aerobic Exercise and Yoga Improve NeuroCognitive Function in Women with Early Psychosis

Impairments of attention and memory are evident in early psychosis, and are associated with functional disability. In a group of stable, medicated women patients, we aimed to determine whether participating in aerobic exercise or yoga improved cognitive impairments and clinical symptoms. A total of 140 female patients were recruited, and 124 received the allocated intervention in a randomized controlled study of 12 weeks of yoga or aerobic exercise compared with a wait-list group. The primary outcomes were cognitive functions including memory and attention. Secondary outcome measures were the severity of psychotic and depressive symptoms, and hippocampal volume. Data from 124 patients were included in the final analysis based on the intention-to-treat principle. Both yoga and aerobic exercise groups demonstrated significant improvements in working memory (P<0.01) with moderate to large effect sizes compared to the wait-list control group. The yoga group showed additional benefits in verbal acquisition (P<0.01) and attention (P=0.01). Both types of exercise improved overall and depressive symptoms (all P≤0.01) after 12 weeks. Small increases in hippocampal volume were observed in the aerobic exercise group compared with wait-list (P=0.01). Both types of exercise improved working memory in early psychosis patients, with yoga having a larger effect on verbal acquisition and attention than aerobic exercise. The application of yoga and aerobic exercise as adjunctive treatments for early psychosis merits serious consideration. This study was supported by the Small Research Funding of the University of Hong Kong (201007176229), and RGC funding (C00240 / 762412) by the Authority of Research, Hong Kong.published_or_final_versio