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1.  MiRNA-99a directly regulates AGO2 through translational repression in hepatocellular carcinoma 
Zhang, J | Jin, H | Liu, H | lv, S | Wang, B | Wang, R | Liu, H | Ding, M | Yang, Y | Li, L | Zhang, J | Fu, S | Xie, D | Wu, M | Zhou, W | Qian, Q
Oncogenesis  2014;3(4):e97-.
The regulation network consisting of microRNAs (miRNAs) and their target genes remains largely elusive in hepatocellular carcinoma (HCC), especially the reciprocal loop between specific miRNAs and the miRNA processing machinery. In this study, we found that miR-99a was remarkably decreased in 111 of 152 (73.03%) primary HCC tissues and low-level expression of miR-99a was correlated with low tumor differentiation (P=0.001), liver cirrhosis (P=0.015), poor tumor-free survival (P=0.004) and overall survival (P=0.006) for HCC patients. By restoration of miR-99a, the HCC growth could be considerably inhibited both in vitro and in vivo. Subsequently, Argonaute-2 (Ago2), a central component of RNA-induced silencing complex, was found to be directly regulated by miR-99a via translational repression. Overexpression of Ago2 could partly impair the inhibitory effect of miR-99a on HCC cells in vitro. Then, we demonstrated that Ago2 was upregulated in HCC tissues at both RNA and protein levels and the expression of AGO2 protein and miR-99a was negatively correlated within detected HCC tissues (r=−0.727, P=0.004). Interestingly, the tumorigenicity of Ago2-knockdown HCC cells was severely impaired (4/10 vs 10/10, P<0.05), and this was in contrast to the miR-99a-overexpressing HCC cells. Functionally, the increased AGO2 protein could specifically facilitate oncogenic miR-21 to repress its targeted gene phosphatase and tensin homolog (Pten) in HCC, whereas leave the regulatory capacity of let-7a on its targeted oncogenes almost unaltered. In summary, our study has revealed a novel pathway for the tumor suppressor miR-99a to control tumor growth in HCC, via its downstream signaling of AGO2/miR-21/PTEN. In addition, this study provides potential strategies for HCC therapy by reintroduction of miRNA suppressors.
PMCID: PMC4007193  PMID: 24732044
Hepatocellular carcinoma; miR-99a; AGO2; PTEN
2.  Immunocapture of Prostate Cancer Cells with Anti-PSMA Antibodies in Microdevices 
Biomedical microdevices  2012;14(2):401-407.
Patients suffering from cancer can shed tumor cells into the bloodstream, leading to one of the most important mechanisms of metastasis. As such, the capture of these cells is of great interest. Circulating tumor cells are typically extracted from circulation through positive selection with the epithelial cell-adhesion molecule (EpCAM), leading to currently unknown biases when cells are undergoing epithelial-to-mesenchymal transition. For prostate cancer, prostate-specific membrane antigen (PSMA) presents a compelling target for immunocapture, as PSMA levels increase in higher-grade cancers and metastatic disease and are specific to the prostate epithelium. This study uses monoclonal antibodies J591 and J415—antibodies that are highly specific for intact extracellular domains of PSMA on live cells— in microfluidic devices for the capture of LNCaPs, a PSMA-expressing immortalized prostate cancer cell line, over a range of concentrations and shear stresses relevant to immunocapture. Our results show that J591 outperforms J415 and a mix of the two for prostate cancer capture, and that capture performance saturates following incubation with antibody concentrations of 10 micrograms per milliliter.
PMCID: PMC4074911  PMID: 22143878
CTC; microfluidic; PSMA; J591; circulating tumor cell; prostate cancer
3.  Phase 2 Trial of Paclitaxel Polyglumex with Capecitabine for Metastatic Breast Cancer 
American journal of clinical oncology  2012;10.1097/COC.0b013e31826e0550.
Capecitabine and paclitaxel are established effective treatments, alone and combined with other cytotoxic and targeted agents, for metastatic breast cancer (MBC). Paclitaxel polyglumex (a macromolecular conjugate of paclitaxel bound to poly-L-glutamic acid) has potential advantages over conventional paclitaxel, including little alopecia, short infusion time with no premedication, enhanced tumor permeability/retention effect, and improved tolerability. We therefore examined tolerability & efficacy of paclitaxel polyglumex with capecitabine in patients with MBC.
Patients and Methods
This was a single stage phase 2 study, with interim analysis conducted with endpoints of tumor response, adverse events (toxicities), time to progression & overall survival. The main eligibility criteria were: age >18, no prior MBC chemotherapy, ECOG performance score <2, disease measurable by RECIST criteria, no HER2 overexpression or amplification, no brain metastases or peripheral sensory neuropathy. Treatment consisted of paclitaxel polyglumex 135 mg/m2 by intravenous infusion on day 1 + capecitabine 825 mg/m2 orally twice daily days 1 - 14, repeated on a 3-week cycle. Forty one (41) evaluable patients were required to test null hypothesis that complete and partial tumor response rate (CR + PR) was at most 40% against the alternative of at least 60%. Paclitaxel polyglumex + capecitabine would be considered promising in this population if ≥21 responses were observed among first 41 evaluable patients.
48 patients were enrolled between April 2006 - April 2007; all patients were evaluable. The median cycles administered was 6. Eighteen (18) patients (38%; 95% CI: 24-53%) had a confirmed tumor response (2 CR, 16 PR) by RECIST criteria. Fifteen (15; 38%, 95% CI: 23%-53%) responses occurred in first 41 patients, falling short of prespecified goal of 21 responses. Median duration of tumor response was 13.2 months. Three of the responders were progression free at last follow-up with a median follow-up of 43 months. Median progression-free survival was 5.1 months (95% CI: 4.0-7.6 months). Six-month progression free survival was 42% (95% CI: 30-58%). Median dose level administered = 135 mg/m2 paclitaxel polyglumex, 825 mg/m2 capecitabine for cycles 1-7. Most common severe (grade 3/4) toxicities (at least possibly related to study drug) were: leukopenia 9 (19%), neutropenia 8 (17%), neuro-sensory 4 (8%), skin reaction-hand/foot 4 (8%), dyspnea 2 (4%). Forrty-six% (22/47) of patients experienced a grade ≥3 toxicity and 8% (4/48) experienced a grade ≥4 toxicity. No alopecia was reported.
Although the trial failed to reach goal of 21 confirmed tumor responses among the first 41 evaluable patients, paclitaxel polyglumex and capecitabine is well tolerated and effective in MBC.
PMCID: PMC3593738  PMID: 23211220
4.  An apoptosis-independent role of SMAC in tumor suppression 
Oncogene  2012;32(19):2380-2389.
Reduced expression of the pro-apoptotic protein SMAC (second mitochondria-derived activator of caspase) has been reported to correlate with cancer progression, while its significance and underlying mechanisms are poorly understood. In this study, we investigated the role of SMAC in intestinal tumorigenesis using both human samples and animal models. Decreased SMAC expression was found to correlate with increased cIAP2 expression and higher grades of human colon cancer. In mice, SMAC deficiency significantly increased the incidence and size of colon tumors induced by azoxymethane (AOM)/dextran sulfate sodium salt (DSS), and highly enriched β-catenin hot spot mutations. SMAC deficiency also significantly increased the incidence of spontaneous intestinal polyps in APCMin/+ mice. Loss of SMAC in mice led to elevated levels of cIAP1 and cIAP2, increased proliferation and activation of the NF-κB p65 subunit in normal and tumor tissues. Unexpectedly, SMAC deficiency had little effect on the incidence of precursor lesions, or apoptosis induced by AOM or DSS, or in established tumors in mice. Furthermore, SMAC knockout enhanced TNFα-mediated NF-κB activation via cIAP2 in HCT 116 colon cancer cells. These results demonstrate an essential and apoptosis-independent function of SMAC in tumor suppression and provide new insights into the biology and targeting of colon cancer.
PMCID: PMC3751796  PMID: 22751125
SMAC; cIAP; NF-κB; proliferation; colon cancer
5.  Radiological and clinical features of adult non-puerperal mastitis 
Tan, H | Li, R | Peng, W | Liu, H | Gu, Y | Shen, X
The British Journal of Radiology  2013;86(1024):20120657.
To describe the radiological and clinical features of adult non-puerperal mastitis and to determine the most accurate method of preventing unnecessary surgical procedures.
Clinical and imaging findings were retrospectively reviewed in 51 females with non-puerperal mastitis, which was confirmed by biopsy/surgical pathology. All 51 patients had pre-operative MRI; 45 patients also had sonograms and 25 also had mammograms, pre-operatively.
Of the 51 cases with non-puerperal mastitis, 94.1% (48/51) were confirmed as having acute or chronic inflammation, and the other 3 had plasma cell mastitis; areola papillaris inflammation was found in 39.2% (20/51) of the cases. Overall, 6 of the 25 cases that were examined with mammography and 2 of the 45 cases that were examined with sonography appeared normal, but all 51 lesions were positively identified on MRI. Asymmetrical density (12/25) on mammograms and solitary or separated/contiguous, clustered, hypoechoic mass-like lesions (31/45) on ultrasound were the most common signs of non-puerperal mastitis. On enhanced MRI, 90.2% (46/51) of patients showed non-mass-like enhanced lesions. Multiple regional enhancements in the pattern of distribution (32/46) and separated or contiguous, clustered, rim-like enhancements in the pattern of internal enhancement (29/46) were the most common manifestations in non-mass-like enhanced lesions. Of the 51 patients, mastitis Type 1 and Type 2 in the time–signal intensity curve were detected in 47.1% and 51.0% of the patients, respectively. The breast imaging reporting and data system categories with the highest number of patients were Category 0 (9/25) on mammography, Category 4a on sonography (18/45) and Category 4a on MRI (29/51).
The findings from mammography and ultrasound are non-specific; therefore, using MR can be helpful in the diagnosis, especially in the presence of non-mass-like enhancements that are multiple, regional, separated, or contiguous, clustered and rim-like.
Advances in knowledge:
Mastitis is often neglected because of the lack of typical clinical signs and symptoms. This study has assessed and described the clinical features and imaging findings of adult non-puerperal mastitis on mammograms, sonograms and MRI and found that MRI is more specific in the diagnosis of disease.
PMCID: PMC3635790  PMID: 23392197
6.  A nanomaterial-based breath test for distinguishing gastric cancer from benign gastric conditions 
British Journal of Cancer  2013;108(4):941-950.
Upper digestive endoscopy with biopsy and histopathological evaluation of the biopsy material is the standard method for diagnosing gastric cancer (GC). However, this procedure may not be widely available for screening in the developing world, whereas in developed countries endoscopy is frequently used without major clinical gain. There is a high demand for a simple and non-invasive test for selecting the individuals at increased risk that should undergo the endoscopic examination. Here, we studied the feasibility of a nanomaterial-based breath test for identifying GC among patients with gastric complaints.
Alveolar exhaled breath samples from 130 patients with gastric complaints (37 GC/32 ulcers / 61 less severe conditions) that underwent endoscopy/biopsy were analyzed using nanomaterial-based sensors. Predictive models were built employing discriminant factor analysis (DFA) pattern recognition, and their stability against possible confounding factors (alcohol/tobacco consumption; Helicobacter pylori) was tested. Classification success was determined (i) using leave-one-out cross-validation and (ii) by randomly blinding 25% of the samples as a validation set. Complementary chemical analysis of the breath samples was performed using gas chromatography coupled with mass spectrometry.
Three DFA models were developed that achieved excellent discrimination between the subpopulations: (i) GC vs benign gastric conditions, among all the patients (89% sensitivity; 90% specificity); (ii) early stage GC (I and II) vs late stage (III and IV), among GC patients (89% sensitivity; 94% specificity); and (iii) ulcer vs less severe, among benign conditions (84% sensitivity; 87% specificity). The models were insensitive against the tested confounding factors. Chemical analysis found that five volatile organic compounds (2-propenenitrile, 2-butoxy-ethanol, furfural, 6-methyl-5-hepten-2-one and isoprene) were significantly elevated in patients with GC and/or peptic ulcer, as compared with less severe gastric conditions. The concentrations both in the room air and in the breath samples were in the single p.p.b.v range, except in the case of isoprene.
The preliminary results of this pilot study could open a new and promising avenue to diagnose GC and distinguish it from other gastric diseases. It should be noted that the applied methods are complementary and the potential marker compounds identified by gas-chromatography/mass spectrometry are not necessarily responsible for the differences in the sensor responses. Although this pilot study does not allow drawing far-reaching conclusions, the encouraging preliminary results presented here have initiated a large multicentre clinical trial to confirm the observed patterns for GC and benign gastric conditions.
PMCID: PMC3590679  PMID: 23462808
gastric cancer; breath analysis; diagnosis; volatile organic compound; sensor
7.  Changes in tau phosphorylation levels in the hippocampus and frontal cortex following chronic stress 
Studies have indicated that early-life or early-onset depression is associated with a 2- to 4-fold increased risk of developing Alzheimers disease (AD). In AD, aggregation of an abnormally phosphorylated form of the tau protein may be a key pathological event. Tau is known to play a major role in promoting microtubule assembly and stabilization, and in maintaining the normal morphology of neurons. Several studies have reported that stress may induce tau phosphorylation. The main aim of the present study was to investigate possible alterations in the tau protein in the hippocampus and frontal cortex of 32 male Sprague-Dawley rats exposed to chronic unpredictable mild stress (CUMS) and then re-exposed to CUMS to mimic depression and the recurrence of depression, respectively, in humans. We evaluated the effects of CUMS, fluoxetine, and CUMS re-exposure on tau and phospho-tau. Our results showed that a single exposure to CUMS caused a significant reduction in sucrose preference, indicating a state of anhedonia. The change in behavior was accompanied by specific alterations in phospho-tau protein levels, but fluoxetine treatment reversed the CUMS-induced impairments. Moreover, changes in sucrose preference and phospho-tau were more pronounced in rats re-exposed to CUMS than in those subjected to a single exposure. Our results suggest that changes in tau phosphorylation may contribute to the link between depression and AD.
PMCID: PMC3982945  PMID: 24652321
Alzheimers disease; Depression; Tau; Chronic unpredictable mild stress (CUMS); Re-exposure to CUMS; Fluoxetine
8.  Taiwan Aneurysm Registry: Multivariate Analysis of Two-Month, One-Year, and Two-Year Outcomes after Endovascular and Microsurgical Treatment of Ruptured Aneurysms 
Interventional Neuroradiology  2013;19(1):35-42.
We compared the outcomes of endovascular coiling with microsurgical clipping of aneurysms in a Taiwanese population.
In an ambi-directional cohort design, patient baseline characteristics and clinical course after treatment for ruptured subarachnoid aneurysm were abstracted from medical records from three hospitals to examine and compare differences in post-operative outcomes between those treated with endovascular coiling and those treated with microsurgical clipping. Outcomes were measured, using the modified Rankin scale, two months, one year and two years postoperatively.
Of the 642 patients enrolled in the study, 281 underwent endovascular treatment and 361 underwent neurosurgery. The demographics and baseline characteristics of two groups were comparable except for a larger maximum target aneurysm lumen size (p=0.02) in the endovascular group. Patients who underwent the endovascular procedure tended to have a better quality of life than those who had neurosurgery (p<0.01). When the severity of symptom data was pooled into two groups (Rankin values 0-2 and 3-6) a statistically significant relationship was found between the severity of symptoms and age, Hunt and Hess grade, number of target aneurysms detected, and log of maximum target aneurysm lumen size (all p≤0.01). After controlling for potential confounding factors and using the lumped Rankin outcome data, no significant difference in outcome was found between the two procedures at either time point.
Our study indicated that endovascular coiling achieves results comparable to surgical clipping for patients with ruptured subarachnoid aneurysms in a Taiwanese population.
PMCID: PMC3601615  PMID: 23472721
aneurysm, hemorrhage, microsurgery, endovascular coiling, size, outcome, quality of life
9.  Language Lateralization Represented by Spatiotemporal Mapping of Magnetoencephalography 
Determination of hemispheric language dominance is critical for planning epilepsy surgery. We assess the usefulness of spatiotemporal source analysis of magnetoencephalography for determining language laterality.
Thirty-five patients with epilepsy were studied. The patients performed a semantic word-processing task during MEG recording. Epochs containing language-related neuromagnetic activity were averaged after preprocessing. The averaged data between 250 and 550 ms after stimulus were analyzed by using dynamic statistical parametric mapping. ROIs were obtained in the opercular and triangular parts of the inferior frontal gyrus, superior temporal gyrus, and supramarginal gyrus in both hemispheres. We calculated laterality indices according to 1) dSPM-amplitude method, based on the amplitude of activation in the ROIs, and 2) dSPM-counting method, based on the number of unit dipoles with activation over a threshold in the ROIs. The threshold was determined as half of the maximum value in all ROIs for each patient. A LI ≥0.10 or ≤−0.10 was considered left- or right-hemisphere dominance, respectively; a LI between −0.10 and 0.10 was considered bilateral. All patients underwent an intracarotid amobarbital procedure as part of presurgical evaluation.
The dSPM-counting method demonstrated laterality consistent with the IAP in 32 of 35 patients (91.4%), the remaining 3 (8.6%) demonstrated bilateral language representation, whereas the dSPM-amplitude method showed 18 (51.4%) concordant and 17 (48.6%) bilateral. No laterality opposite to the IAP was found.
Spatiotemporal mapping of language lateralization with the dSPM-counting method may reduce the necessity for an IAP in as many as 90% of patients.
PMCID: PMC3732392  PMID: 22878013
10.  PUMA mediates ER stress-induced apoptosis in portal hypertensive gastropathy 
Tan, S | Wei, X | Song, M | Tao, J | Yang, Y | Khatoon, S | Liu, H | Jiang, J | Wu, B
Cell Death & Disease  2014;5(3):e1128-.
Mucosal apoptosis has been demonstrated to be an essential pathological feature in portal hypertensive gastropathy (PHG). p53-upregulated modulator of apoptosis (PUMA) was identified as a BH3-only Bcl-2 family protein that has an essential role in apoptosis induced by a variety of stimuli, including endoplasmic reticulum (ER) stress. However, whether PUMA is involved in mucosal apoptosis in PHG remains unclear, and whether PUMA induces PHG by mediating ER stress remains unknown. The aim of the study is to investigate whether PUMA is involved in PHG by mediating ER stress apoptotic signaling. To identify whether PUMA is involved in PHG by mediating ER stress, gastric mucosal injury and apoptosis were studied in both PHG patients and PHG animal models using PUMA knockout (PUMA-KO) and PUMA wild-type (PUMA-WT) mice. The induction of PUMA expression and ER stress signaling were investigated, and the mechanisms of PUMA-mediated apoptosis were analyzed. GES-1 and SGC7901 cell lines were used to further identify whether PUMA-mediated apoptosis was induced by ER stress in vitro. Epithelial apoptosis and PUMA were markedly induced in the gastric mucosa of PHG patients and mouse PHG models. ER stress had a potent role in the induction of PUMA and apoptosis in PHG models, and the apoptosis was obviously attenuated in PUMA-KO mice. Although the targeted deletion of PUMA did not affect ER stress, mitochondrial apoptotic signaling was downregulated in mice. Meanwhile, PUMA knockdown significantly ameliorated ER stress-induced mitochondria-dependent apoptosis in vitro. These results indicate that PUMA mediates ER stress-induced mucosal epithelial apoptosis through the mitochondrial apoptotic pathway in PHG, and that PUMA is a potentially therapeutic target for PHG.
PMCID: PMC3973242  PMID: 24625987
PHG; PUMA; ER stress; mitochondria; apoptosis
11.  Induction of autophagy and senescence by knockdown of ROC1 E3 ubiquitin ligase to suppress the growth of liver cancer cells 
Yang, D | Li, L | Liu, H | Wu, L | Luo, Z | Li, H | Zheng, S | Gao, H | Chu, Y | Sun, Y | Liu, J | Jia, L
Cell Death and Differentiation  2012;20(2):235-247.
Regulator of Cullins-1 (ROC1) or RING box protein-1 (RBX1) is an essential RING component of Cullin-RING ligase (CRL). Our previous studies showed that ROC1 is required for the growth of several cancer cell lines while ROC1 siRNA silencing inactivates CRL, leading to cell cycle arrest, cell senescence and/or apoptosis. However, it is completely unknown whether ROC1 knockdown triggers autophagic response by inactivating CRL. Moreover, the role of ROC1 in liver cancer remains elusive. In this study, we reported that ROC1 knockdown significantly inhibited the growth of liver cancer cells by sequentially and independently inducing autophagy and p21-dependent cell senescence. Mechanism analysis revealed that ROC1 silencing triggered autophagy by inhibition of mammalian target of rapamycin (mTOR) activity due to accumulation of mTOR-inhibitory protein Deptor, a substrate of CRL. Consistently, Deptor knockdown significantly blocked autophagy response upon ROC1 silencing. Biologically, autophagy response upon ROC1 silencing was a survival signal, and blockage of autophagy pathway sensitized cancer cells to apoptosis. Finally, we demonstrated that ROC1 was overexpressed in hepatocellular carcinomas, which is associated with poor prognosis of liver cancer patients. These findings suggest that ROC1 is an appealing drug target for liver cancer and provide a proof-of-concept evidence for a novel drug combination of ROC1 inhibitor and an autophagy inhibitor for effective treatment of liver cancer by enhancing apoptosis.
PMCID: PMC3554346  PMID: 22935614
ROC1; Cullin-RING ligase; autophagy; senescence; Deptor
12.  Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma 
British Journal of Cancer  2012;108(1):72-81.
Sorafenib is the only drug approved for the treatment of hepatocellular carcinoma (HCC). The bioenergetic propensity of cancer cells has been correlated to anticancer drug resistance, but such correlation is unclear in sorafenib resistance of HCC.
Six sorafenib-naive HCC cell lines and one sorafenib-resistant HCC cell line (Huh-7R; derived from sorafenib-sensitive Huh-7) were used. The bioenergetic propensity was calculated by measurement of lactate in the presence or absence of oligomycin. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, and siRNA of hexokinase 2 (HK2) were used to target relevant pathways of cancer metabolism. Cell viability, mitochondrial membrane potential, and sub-G1 fraction were measured for in vitro efficacy. Reactive oxygen species (ROS), adenosine triphosphate (ATP) and glucose uptake were also measured. A subcutaneous xenograft mouse model was used for in vivo efficacy.
The bioenergetic propensity for using glycolysis correlated with decreased sorafenib sensitivity (R2=0.9067, among sorafenib-naive cell lines; P=0.003, compared between Huh-7 and Huh-7 R). DCA reduced lactate production and increased ROS and ATP, indicating activation of oxidative phosphorylation (OXPHOS). DCA markedly sensitised sorafenib-resistant HCC cells to sorafenib-induced apoptosis (sub-G1 (combination vs sorafenib): Hep3B, 65.4±8.4% vs 13±2.9% Huh-7 R, 25.3± 5.7% vs 4.3±1.5% each P<0.0001), whereas siRNA of HK2 did not. Sorafenib (10 mg kg−1 per day) plus DCA (100 mg kg−1 per day) also resulted in superior tumour regression than sorafenib alone in mice (tumour size: −87% vs −36%, P<0.001).
The bioenergetic propensity is a potentially useful predictive biomarker of sorafenib sensitivity, and activation of OXPHOS by PDK inhibitors may overcome sorafenib resistance of HCC.
PMCID: PMC3553537  PMID: 23257894
glycolysis; oxidative phosphorylation; sorafenib; dichloroacetate; hepatocellular carcinoma
13.  Association Between Use of Specific Drugs and Antiretroviral Adherence: Findings from MACH 14 
AIDS and behavior  2013;17(1):142-147.
To determine the association between individual substances of abuse and antiretroviral adherence, analyses require a large sample assessed using electronic data monitoring (EDM). In this analysis, EDM data from 1636 participants in 12 U.S. adherence-focused studies were analyzed to determine the associations between recent use of various substances and adherence during the preceding four weeks. In bivariate analyses comparing adherence among patients who had used a specific substance to those who had not, adherence was significantly lower among those who had recently used cocaine, other stimulants or heroin but not among those who had used cannabis or alcohol. In multivariate analyses controlling for sociodemographics, amount of alcohol use and recent use of any alcohol, cocaine, other stimulants and heroin each was significantly negatively associated with adherence. The significant associations of cocaine, other stimulants, heroin, and alcohol use with adherence suggest that these are important substances to target with adherence-focused interventions.
PMCID: PMC3549004  PMID: 22246513
HIV; AIDS; substance use; adherence; cannabis
14.  Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome 
Blood Cancer Journal  2014;4(1):e170-.
Therapeutic options are limited for elderly patients with acute myeloid leukemia (AML). A phase Ib/II study was undertaken to evaluate the maximum-tolerated dose (MTD) and preliminary efficacy of the pan-histone deacetylase inhibitor panobinostat (LBH589) in combination with azacitidine in patients with AML or high-risk myelodysplastic syndrome (MDS) naïve to intensive chemotherapy. Thirty-nine patients (AML=29, MDS=10) received azacitidine 75 mg/m2 subcutaneously (days 1–5) and oral panobinostat (starting on day 5, thrice weekly for seven doses) in 28-day cycles until toxicity or disease progression. Dose-limiting toxicities during the phase Ib stage were observed in 0/4 patients receiving 10 mg panobinostat, in 1/7 patients (fatigue) receiving 20 mg, in 1/6 patients (fatigue) receiving 30 mg and in 4/5 patients (fatigue, syncope, hyponatremia and somnolence) receiving 40 mg. In phase II, an additional 17 patients received panobinostat at a MTD of 30 mg. The overall response rate (ORR=CR+CRi+PR) in patients with AML was 31% (9/29) and that in patients with MDS was 50% (5/10). After a median follow-up of 13 months, the median overall survival was 8 and 16 months in patients with AML and MDS, respectively. Increased histone H3 and H4 acetylation was a useful early biomarker of clinical response. Combining panobinostat with azacitidine was tolerable and clinically active in high-risk MDS/AML patients, warranting further exploration.
PMCID: PMC3913937  PMID: 24413064
acute myeloid leukemia; myelodysplastic syndrome; epigenetic therapy; histone deacetylase inhibitor; azacitidine
15.  Distinct Clinical Outcomes of Non-Small Cell Lung Cancer Patients with Epidermal Growth Factor Receptor (EGFR) Mutations Treated with EGFR Tyrosine Kinase Inhibitors: Non-Responders versus Responders 
PLoS ONE  2013;8(12):e83266.
Treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been associated with favorable progression free survival (PFS) in patients with non-small cell lung cancers (NSCLC) harboring EGFR mutations. However, a subset of this population doesn't respond to EGFR-TKI treatment. Therefore, the present study aimed to elucidate survival outcome in NSCLC EGFR-mutant patients who were treated with EGFR TKIs.
Among the 580 consecutive NSCLC patients who were treated at our facility between 2008 and 2012, a total of 124 treatment-naïve, advanced NSCLC, EGFR-mutant patients treated with EGFR TKIs were identified and grouped into non-responders and responders for analyses.
Of 124 patients, 104 (84%) responded to treatment, and 20 (16%) did not; and the overall median PFS was 9.0 months. Notably, the PFS, overall survival (OS) and survival rates were significantly unfavorable in non-responders (1.8 vs. 10.3 months, hazard ratio (HR) = 29.2, 95% confidence interval (CI), 13.48–63.26, P<0.0001; 9.4 vs. 17.3 months, HR = 2.74, 95% CI, 1.52–4.94, P = 0.0008; and 58% vs. 82% in 6, 37% vs. 60% in 12, and 19 vs. 40% at 24 months, respectively). In multivariate analysis, treatment efficacy strongly affected PFS and OS, independent of covariates (HR = 47.22, 95% CI, 17.88–124.73, P<0.001 and HR = 2.74, 95% CI, 1.43–5.24, P = 0.002, respectively). However, none of the covariates except of the presence of EGFR exon 19 deletion in the tumors was significantly associated with better treatment efficacy.
A subset of NSCLC EGFR-mutant patients displayed unfavorable survival despite EGFR TKI administration. This observation reinforces the urgent need for biomarkers effectively predicting the non-responders and for drug development overcoming primary resistance to EGFR TKIs. In addition, optimal therapeutic strategies to prolong the survival of non-responders need to be investigated.
PMCID: PMC3871587  PMID: 24376677
16.  Histological subtype and smoking status, but not gender, are associated with epidermal growth factor receptor mutations in non-small-cell lung cancer 
Molecular and Clinical Oncology  2013;2(2):252-258.
Mutations in epidermal growth factor receptor (EGFR) commonly occur in non-small-cell lung cancer (NSCLC) patients characterized by female gender, never-smoker status and adenocarcinoma histology. The aim of this study was to determine whether gender is a confounding factor for EGFR mutations in NSCLC. To elucidate the confounding effect, Pearson’s χ2 test and logistic regression models were used to correlate these characteristics with EGFR mutations in 426 NSCLC patients treated at our institutes. Of those 426 NSCLC patients, 47% were females, 57% were non-smokers and 84% had adenocarcinomas. The multivariate logistic regression analysis demonstrated that never-smoker status [odds ratio (OR)=3.49, 95% confidence interval (CI): 1.99–6.13; P<0.001)] and adenocarcinoma (OR=9.43, 95% CI 3.62–24.56; P<0.001) were associated with EGFR mutations; however, gender was not (OR=1.25, 95% CI: 0.73–2.15; P=0.416). Furthermore, gender was not associated with EGFR mutation subtypes (OR=1.19, 95% CI: 0.56–2.50; P=0.650). The frequency of EGFR mutations among females and males was not different in non-smokers (64.8 vs. 55.8%, P=0.204) or ever-smokers (27.8 vs. 24.2%, P=0.775). Therefore, if the assessment for EGFR mutation status was limited to non-smoking females with adenocarcinoma, up to 40% of the patients harboring EGFR mutations would be precluded from the benefit of EGFR inhibitor therapy. Our results indicated that gender is a confounding factor for EGFR mutations in NSCLC and suggested that gender may not be associated with tumorigenesis in NSCLC-harboring EGFR mutations.
PMCID: PMC3917768  PMID: 24649342
gender; a confounding factor; epidermal growth factor receptor mutations; non-small-cell lung cancer
17.  Age-associated differences in gene expression in response to delayed anesthetic preconditioning 
Age  2011;34(6):1459-1472.
Evidence suggests that the protective benefits of anesthetic preconditioning (APC) are significantly attenuated in the aged myocardium. In this study, we investigated the effect of aging on gene expression in delayed APC. Hearts from Fischer 344 rats, age 4 or 24 months, were divided into five groups: control; ischemia/reperfusion (I/R); and delayed APC at 6, 12, and 24 h. Whole-genome array was studied using Affymetrix Rat Genome 230 2.0 array. Data were analyzed for significant ≥2.0-fold changes in gene expression. Microarray results were confirmed by quantitative real-time reverse transcription–polymerase chain reaction. Of the 28,000 genes represented on the Affymetrix Rat Genome 230 2.0 Microarray chip, 24 transcripts in 6 h APC, 28 in 12 h APC, and 28 in 24 h APC group displayed significant up-regulation in mRNA levels, and 70 transcripts in 6 h APC, 101 in 12 h APC, and 82 in 24 h APC displayed significant down-regulation in young rat hearts. These altered genes fall into functional categories of cell defense/death, cell structure, gene expression/protein synthesis, inflammatory response/growth/remodeling, and signaling/communication. Although alterations for some genes were in common, the numbers of changed genes in old rats were markedly and consistently lower than the young rats. Twenty-four hour delayed APC also significantly reduced infarct size and improved myocardial left ventricular function in young hearts, effects that were not observed in old rat hearts. We concluded that delayed APC profoundly and differentially affected gene expression profiles of the cardiomyocyte in an age-associated pattern. The impaired genomic response to delayed APC could underlie the loss of the protective benefits of preconditioning in aged hearts.
PMCID: PMC3528372  PMID: 22009153
Anesthetic; Preconditioning; Delayed; Gene; Expression; Myocardium
18.  Single-cell clones of liver cancer stem cells have the potential of differentiating into different types of tumor cells 
Liu, H | Zhang, W | Jia, Y | Yu, Q | Grau, G E | Peng, L | Ran, Y | Yang, Z | Deng, H | Lou, J
Cell Death & Disease  2013;4(10):e857-.
Cancer stem cells (CSCs) are believed to be a promising target for cancer therapy because these cells are responsible for tumor development, maintenance and chemotherapy resistance. Finding out the critical factors regulating CSC fate is the key for target therapy of CSCs. Just as normal stem cells are regulated by their microenvironment (niche), CSCs are also regulated by cells in the tumor microenvironment. However, whether various tumor microenvironments can induce CSCs to differentiate into different cancer cells is not clear. Here, we show that single-cell-cloned CSCs, accidentally obtained from a human liver cancer microvascular endothelial cells, express classic stem cell markers, genes associated with self-renewal and pluripotent factors and possess colony-forming ability in vitro and the ability of serial transplantation in vivo. The single-cell-cloned CSCs treated with the different tumor cell/tissue-derived conditioned culture medium, which is a mimic of carcinoma microenvironment, could differentiate into corresponding tumor cells and express specific markers of the respective type of tumor cells at the gene, protein and cell levels, respectively. Interestingly, this multilineage differentiation potential of single-cell-cloned liver CSCs sharply declined after the specific knockdown of octamer-binding transcription factor 4 (Oct4) alone, even though they were under the same induction conditions (carcinoma microenvironments). These data support the hypothesis that single-cell-cloned liver CSCs have the potential of differentiating into different types of tumor cells, and the tumor microenvironment does play a crucial role in deciding differentiation directions. Simultaneously, Oct4 in CSCs is indispensable in this process. These factors are promising targets for liver CSC-specific therapy.
PMCID: PMC3824650  PMID: 24136221
CSCs; carcinoma/cancer microenvironments; multilineage differentiation potential; Oct4; microvascular endothelial cells
19.  The immunogenicity of virus-derived 2A sequences in immunocompetent individuals 
Gene therapy  2013;20(9):958-962.
Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.
PMCID: PMC3766470  PMID: 23698740
2A sequences; polycistronic vectors; T cell gene transfer; immunogenicity
20.  Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor 
Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.
PMCID: PMC3854432  PMID: 24068195
BMP2; bFGF; Adenovirus; T2A sequence; BMPCs; Osteogenic differentiation
21.  Nutrigenomic targeting of carbohydrate craving behavior: Can we manage obesity and aberrant craving behaviors with neurochemical pathway manipulation by Immunological Compatible Substances (nutrients) using a Genetic Positioning System (GPS) Map? 
Medical hypotheses  2009;73(3):427-434.
Genetic mediated physiological processes that rely on both pharmacological and nutritional principles hold great promise for the successful therapeutic targeting of reduced carbohydrate craving, body-friendly fat loss, healthy body recomposition, and overall wellness. By integrating an assembly of scientific knowledge on inheritable characteristics and environmental mediators of gene expression, we review the relationship of genes, hormones, neurotransmitters, and nutrients as they correct unwanted weight gain coupled with unhappiness. In contrast to a simple one-locus, one-mechanism focus on pharmaceuticals alone, we hypothesize that the use of nutrigenomic treatment targeting multi-physiological neurological, immunological, and metabolic pathways will enable clinicians to intercede in the process of lipogenesis by promoting lipolysis while attenuating aberrant glucose cravings. In turn, this approach will enhance wellness in a safe and predictable manner through the use of a Genetic Positioning System (GPS) Map. The GPS Map, while presently incomplete, ultimately will serve not only as a blueprint for personalized medicine in the treatment of obesity, but also for the development of strategies for reducing many harmful addictive behaviors and promoting optimal health by using substances compatible with the body’s immune system.
PMCID: PMC3758908  PMID: 19450935
22.  Elastase induced lung epithelial cell apoptosis and emphysema through placenta growth factor 
Cell Death & Disease  2013;4(9):e793-.
Chronic pulmonary obstructive disease (COPD) is the fourth leading cause of death worldwide, however, the pathogenic factors and mechanisms are not fully understood. Pulmonary emphysema is one of the major components of COPD and is thought to result from oxidative stress, chronic inflammation, protease–antiprotease imbalance and lung epithelial (LE) cell apoptosis. In our previous studies, COPD patients were noted to have higher levels of placenta growth factor (PlGF) in serum and bronchoalveolar lavage fluid than controls. In addition, transgenic mice overexpressing PlGF developed pulmonary emphysema and exposure to PlGF in LE cells induced apoptosis. Furthermore, intratracheal instillation of porcine pancreatic elastase (PPE) on to PlGF wild type mice induced emphysema, but not in PlGF knockout mice. Therefore, we hypothesized that PPE generates pulmonary emphysema through the upregulation of PlGF expression in LE cells. The elevation of PlGF then leads to LE cell apoptosis. In the present study, we investigated whether PPE induces PlGF expression, whether PlGF induces apoptosis and whether the downstream mechanisms of PlGF are related to LE cell apoptosis. We found that PPE increased PlGF secretion and expression both in vivo and in vitro. Moreover, PlGF-induced LE cell apoptosis and PPE-induced emphysema in the mice were mediated by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways. Given these findings, we suggest that the increase in PlGF and PlGF-induced JNK and p38 MAPK pathways contribute to PPE-induced LE cell apoptosis and emphysema. Regulatory control of PlGF and agents against its downstream signals may be potential therapeutic targets for COPD.
PMCID: PMC3789187  PMID: 24008737
placenta growth factor; chronic pulmonary obstructive disease; emphysema; apoptosis
Phosphodiesterases (PDEs) limit vasodilation in response to a variety of signaling cascades by metabolizing the cyclic nucleotides cAMP and cGMP. The objective of this study was to test the hypothesis that NO regulates expression of PDE3A, a cGMP-inhibited PDE. Incubation of rat pulmonary artery smooth muscle cells (rPaSMCs) with the NO-donor compound S-nitroso-glutathione (GSNO) increased PDE3A gene expression in a dose- and time-dependent manner. NO-donors increased PDE3A protein levels. Total and milrinone inhibitable cAMP PDE activity were increased 2.8±0.1- and 2.0±0.1-fold respectively in extracts of rPaSMCs exposed to GSNO. The effects of GSNO on PDE3A gene expression were mimicked by the soluble guanylate cyclase (sGC) activators YC-1 and BAY 41-2272 and blocked by the sGC inhibitor ODQ. Incubation of rPaSMC with interleukin-1β and tumor necrosis factor-α induced PDE3A gene expression, an effect which was inhibited by L-NIL, an antagonist of NO synthase 2, or ODQ. Actinomycin D, an inhibitor of RNA polymerase, blocked the GSNO-induced increase of PDE3A mRNA levels, whereas cycloheximide, an inhibitor of protein translation, did not. These observations suggest that NO modulates PDE3A gene expression via mechanisms dependent upon cGMP synthesis and gene transcription. Prolonged exposure to NO may alter the sensitivity of vascular smooth muscle to cGMP- or cAMP-dependent vasodilators, as well as PDE isoform-selective inhibitors.
PMCID: PMC3757338  PMID: 21224496
nitric oxide; cyclic guanosine monophosphate; GSNO; phosphodiesterase 3A; soluble guanylate cyclase; vasodilation
24.  Pediatric Hodgkin Lymphoma: Are We Over-Scanning Our Patients? 
Pediatric hematology and oncology  2012;29(5):415-423.
Despite the favorable outcome of most pediatric patients with Hodgkin lymphoma (HL), there is rising concern about risks of carcinogenesis from both diagnostic and therapeutic radiation exposure for patients treated on study protocols. Although previous studies have investigated radiation exposure during treatment, radiation from post-treatment surveillance imaging may also increase the likelihood of secondary malignancies. All diagnostic imaging examinations involving ionizing radiation exposure performed for surveillance following completion of therapy were recorded for 99 consecutive pediatric patients diagnosed with HL from 2000 to 2010. Cumulative radiation dosage from these examinations and the frequency of relapse detection by these examinations were recorded. In the first 2 years following completion of therapy, patients in remission received a median of 11 examinations (range 0–26). Only 13 of 99 patients relapsed, 11 within 5 months of treatment completion. No relapse was detected by 1- or 2-view chest radiographs (n = 38 and 296, respectively), abdomen/pelvis computed tomography (CT) scans (n = 211), or positron emission tomography (PET) scans alone (n = 11). However, 10/391 (2.6%) of chest CT scans, 4/364 (1.1%) of neck CT scans, and 3/47 (6.4%) of PET/CT scans detected relapsed disease. Thus, only 17 scans (1.3%) detected relapse in a total of 1358 scans. Mean radiation dosages were 31.97 mSv for Stage 1, 37.76 mSv for Stage 2, 48.08 mSv for Stage 3, and 51.35 mSv for Stage 4 HL. Approximately 1% of surveillance imaging examinations identified relapsed disease. Given the very low rate of relapse detection by surveillance imaging stipulated by current protocols for pediatric HL patients, the financial burden of the tests themselves, the high cure rate, and risks of second malignancy from ionizing radiation exposure, modification of the surveillance strategy is recommended.
PMCID: PMC3685486  PMID: 22632168
Hodgkin disease; late effects; radiology
25.  A robust general phase retrieval method for medical applications 
From medical imaging perspective the robustness of a phase retrieval method is of critical importance. In this presentation we compare the robustness of two general phase retrieval methods, namely the transport of intensity equation inversion (TIE-inversion) method and the attenuation partition based (AP-based) method. We showed that the TIE-inversion method, regardless if being assisted with the Tikhonov regularization, failed to retrieve the phase maps in two experimental studies. The failure exposes this method’s weakness as being unstable against the noise. In contrast, the sample phase maps are retrieved successfully by using the AP-based method. The stark performance differences of the two methods are rooted in their different techniques dealing with the singularity problem. This comparison shows that the robust AP-based phase retrieval method will be superior to the TIE-inversion method for medical imaging applications where radiation doses are stringently limited.
PMCID: PMC3721370  PMID: 23894250
Medical-image reconstruction methods and algorithms; computer-aided so; X-ray radiography and digital radiography (DR)

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