Yung-Che Chen

Temporal and spatial expression of bone morphogenetic proteins in extracorporeal shock wave-promoted healing of segmental defect

Extracorporeal shock wave (ESW) is a noninvasive acoustic wave, which has recently been demonstrated to promote bone repair. The actual healing mechanism triggered by ESW has not yet been identified. Bone morphogenetic proteins (BMP) have been implicated as playing an important role in bone development and fracture healing. In this study, we aimed to examine the involvement of BMP-2, BMP-3, BMP-4, and BMP-7 expression in ESW promotion of fracture healing. Rats with a 5-mm segmental femoral defect were given ESW treatment using 500 impulses at 0.16 mJ/mm(2). Femurs and calluses were subjected to immunohistochemistry and RT-PCR assay 1, 2, 4, and 8 weeks after treatment. Histological observation demonstrated that fractured femurs received ESW treatment underwent intensive mesenchymal cell aggregation, hypertrophic chondrogenesis, and endochondral/intramembrane ossification, resulting in the healing of segmental defect. Aggregated mesenchymal cells at the defect, chondrocytes at the hypertrophic cartilage, and osteoblasts adjunct to newly formed woven bone showed intensive proliferating cell nuclear antigen expression. ESW treatment significantly promoted BMP-2, BMP-3, BMP-4, and BMP-7 mRNA expression of callus as determined by RT-PCR, and BMP immunoreactivity appeared throughout the bone regeneration period. Mesenchymal cells and immature chondrocytes showed intensive BMP-2, BMP-3, and BMP-4 immunoreactivity. BMP-7 expression was evident on osteoblasts located at endochondral ossification junction. Our findings suggest that BMP play an important role in signaling ESW-activated cell proliferation and bone regeneration of segmental defect.

Toll-like receptor 2 gene polymorphisms, pulmonary tuberculosis, and natural killer cell counts

BackgroundTo investigate whether the toll-like receptor 2 polymorphisms could influence susceptibility to pulmonary TB, its phenotypes, and blood lymphocyte subsets.MethodsA total of 368 subjects, including 184 patients with pulmonary TB and 184 healthy controls, were examined for TLR2 polymorphisms over locus -100 (microsatellite guanine-thymine repeats), -16934 (T>A), -15607 (A>G), -196 to -174 (insertion>deletion), and 1350 (T>C). Eighty-six TB patients were examined to determine the peripheral blood lymphocyte subpopulations.ResultsWe newly identified an association between the haplotype [A-G-(insertion)-T] and susceptibility to pulmonary TB (p = 0.006, false discovery rate q = 0.072). TB patients with systemic symptoms had a lower -196 to -174 deletion/deletion genotype frequency than those without systemic symptoms (5.7% vs. 17.7%; p = 0.01). TB patients with the deletion/deletion genotype had higher blood NK cell counts than those carrying the insertion allele (526 vs. 243.5 cells/μl, p = 0.009). TB patients with pleuritis had a higher 1350 CC genotype frequency than those without pleuritis (12.5% vs. 2.1%; p = 0.004). TB patients with the 1350 CC genotype had higher blood NK cell counts than those carrying the T allele (641 vs. 250 cells/μl, p = 0.004). TB patients carrying homozygous short alleles for GT repeats had higher blood NK cell counts than those carrying one or no short allele (641 vs. 250 cells/μl, p = 0.004).ConclusionsTLR2 genetic polymorphisms influence susceptibility to pulmonary TB. TLR2 variants play a role in the development of TB phenotypes, probably by controlling the expansion of NK cells.

hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells

Telomerase activity is suppressed in normal somatic tissues but is activated in most cancer cells. We have previously found that all six telomerase subunit proteins, including hTERT and hsp90 are needed for full enzyme activity. Telomerase activity has been reported to be upregulated by protein kinase C (PKC), but the mechanism is not clear. In this study, we examined how PKC regulates telomerase activity in head and neck cancer cells. PKC inhibitor, bisindolylmaleimide I (BIS), inhibited telomerase activity but had no effect on the expressions of telomerase core subunits. RNA interference (RNAi) and in vitro phosphorylation studies revealed that PKC isoforms α, β, δ, ɛ, ζ specifically involved in telomerase regulation, and the phosphorylation target was on hTERT. Treatment with the hsp-90 inhibitor novobiocin dissociated hsp90 and hTERT as revealed by immunoprecipitation and immunoblot analysis and reduced telomerase activity. Treatment with the PKC activator SC-10 restored the association of hsp90 and hTERT and reactivate telomerase, suggesting that hTERT phosphorylation by PKC is essential for telomerase holoenzyme integrity and function. Analysis on clinical normal and tumour tissues reveal that the expressions of PKC α, β, δ, ɛ, ζ were higher in the tumour tissues, correlated with telomerase activity. Disruption of PKC phosphorylation by BIS significantly increased chemosensitivity to cisplatin. In conclusion, PKC isoenzymes α, β, δ, ɛ, ζ regulate telomerase activity in head and neck cancer cells by phosphorylating hTERT. This phosphorylation is essential for telomerase holoenzyme assembly, leading to telomerase activation and oncogenesis. Manipulation of telomerase activity by PKC inhibitors is worth exploring as an adjuvant therapeutic approach.

Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers

Infection with high-risk types (type 16 or type 18) of human papillomaviruses (HPVs) increases a patients risk of cervical cancer. Given the importance of the cervix and the severe side effects resulting from traditional cancer therapies, this study aimed to achieve targeted inhibition of viral oncogenes in tumor cells using small interfering RNAs (siRNA). To accomplish this, we developed nine siRNAs against either the E6 or E7 genes of HPV-16 or HPV-18 in several combinations, yielding siRNAs targeting 16E6, 16E7, 18E6 and 18E7. We measured the effectiveness of the siRNAs by examining E6 or E7 mRNA expression after transfection of the siRNAs into HPV-positive CaSki (HPV-16) or HeLa (HPV-18) cell lines. We found that the HPV-siRNAs significantly reduced cell growth and colony formation in both cell lines. Flow cytometry analysis revealed a significant increase in apoptosis. The siRNAs had no effect on cell growth, colony formation or apoptosis in HPV-negative C33A cells, demonstrating a lack of off-target effects. In addition, an in vivo xenograft study showed that intra-tumoral injection of the siRNAs reduced tumor growth in BALB/c nude mice. In conclusion, we have developed highly specific and potent HPV-siRNAs that successfully suppress tumor growth and induce apoptosis in HPV-positive cervical cancer cells. siRNA treatment has potential for further development as an adjuvant therapy for cervical cancer.

Inspiratory muscle training in bronchiectasis patients: a prospective randomized controlled study:

Objective: To investigate the efficacy and feasibility of home-based inspiratory muscle training in patients with bronchiectasis. Design: A prospective, single-blind, randomized, controlled study. Setting: Outpatient clinic of a tertiary care medical centre. Methods: Twenty-six patients with bronchiectasis were randomly divided into inspiratory muscle training and control groups. In the inspiratory muscle training group (n = 13), the training programme started with an intensity of 30% maximal inspiratory pressure (MIP), which was increased by 2 cmH2O each week, for 30 minutes daily, 5 days a week for eight weeks. The control group (n = 13) did not receive inspiratory muscle training. Main outcome measures included spirometry, resting oxyhaemoglobin saturation by pulse oximetry (SpO2), lowest SpO2 and Borg Scale during 6-minute walking tests, 6-minute walking distance (6MWD), 6-minute walking work (6Mwork), MIP, maximal expiratory pressure (MEP) and St George’s Respiratory Questionnaire. Results: There were significant differences in change from baseline in 6MWD (411.9 (133.5) vs. 473.2 (117.2) m, P = 0.021), 6Mwork (21 051.0 (8286.7) vs. 23 915.5 (8343.0) kg-m, P = 0.022), MIP (60.8 (21.8) vs. 84.6 (29.0) cmH2O, P = 0.004), and MEP (72.3 (31.1) vs. 104.2 (35.7) cmH2O, P = 0.004) in the inspiratory muscle training group. Significant improvements in both MIP (23.8 (25.3) vs. 2.3 (16.4) cmH2O, adjusted P-value = 0.005) and MEP (31.9 (30.8) vs. 11.5 (20.8) cmH2O, adjusted P-value = 0.038) levels after adjusting for age by linear regression analysis were observed between groups. Conclusions: An eight-week home-based inspiratory muscle training is feasible and effective in improving both inspiratory and expiratory muscle strength, but has no effect on respiratory function and quality of life in patients with bronchiectasis.

Grp78 as a therapeutic target for refractory head–neck cancer with CD24 − CD44 + stemness phenotype

Cancer stem cells are refractory to conventional therapy, which result to cancer metastasis and chemo-radioresistance. Grp78 is known to have important roles in cytoprotection and tumorigenesis in several cancers. We therefore examined whether Grp78 can serve as a therapeutic target for refractory stemness phenotype of head and neck cancer (HNC). Six HNC cell lines were used. Fluorescence-activated cell sorting (FACS) analysis was used to sort CD24−CD44+ and Grp78+ cells. The small interfering RNA (siRNA) knockdown and cDNA transfection were applied to examine the effects of Grp78 on cellular function. Western blot and confocol microscopy were used to determine the effects of downstream protein expressions. Xenografted mouse tumors and immunohistochemistry were used to validate the results. We found that Grp78 regulated the conversion of CD24−CD44+ cells, a characteristic of HNC stem cells. The CD24−CD44+Grp78+ cells showed superior chemo-radioresistance and invasion ability compared with CD24−CD44+, Grp78+ or the parental cells. Silencing Grp78 increased chemo-radiosensitivity, inhibited cell invasion, reverse epithelial–mesenchymal transition, suppressed cancer stemness, withdrew CD24−CD44+ cell conversion and induced differentiated phenotype. Study in xenografted mice further showed that CD24−CD44+Grp78+ cells exhibited highest tumorigenesis, compared with CD24−CD44+ CD24+CD44+ or the parental cells. Grp78 knockdown dramatically restrained tumor growth along with the inhibition of stem cell regulatory proteins Oct-4 and Slug. Grp78 may serve as a molecular target that can be further developed for eradication of refractory HNC with stemness phenotype.

Aberrant FHIT transcripts in hepatocellular carcinomas

To study abnormalities of the FHIT gene in human hepatocellular carcinoma (HCC), eight liver cancer cell lines, 18 matched tumorous and non-tumorous tissues from patients with HCC and three normal liver tissues were analysed by microsatellite polymorphism analysis and reverse transcription of FHIT mRNA followed by polymerase chain reaction (PCR) amplification and sequencing of the products. No loss of heterozygosity at chromosome 3p14.2 as defined by markers D3S1300 and D3S1312 was detected in any of the specimens. In addition, a normal transcript of the gene without any sequence change was found to be expressed in all the cell lines, 17 of the 18 tumorous and all 21 non-tumorous liver tissues tested. Although five out of eight liver cancer cell lines (62.5%), 12 out of 18 HCC tissues (66.7%) and 8 out of 18 paired non-tumorous liver tissues (44.4%) displayed abnormal faint bands of smaller size, sequence analysis revealed that they were aberrant FHIT transcripts lacking three or more exons and might represent alternatively spliced transcripts only. In conclusion, these studies indicate that abnormalities of the FHIT gene transcripts occur in a fairly high frequency of tumorous and non-tumorous liver tissues. However, it might not be causally related to the hepatocarcinogenesis.

Prognostic values of serum IP-10 and IL-17 in Patients with Pulmonary Tuberculosis

Objective: To identify patients at high risk of relapse after anti-tuberculosis (TB) therapy or with poor long-term outcomes. Methods: Fifty-one patients with pulmonary TB: 7 were classified as high association with both cavitations on initial chest radiography and positive sputum smear/cultures after two months of anti-TB treatment (HA group); 19 medium association (MA, one risk alone); and 25 low association (LA, neither risk). Serum interferon (IFN)-γ-inducible protein 10 (IP-10), interleukin-17 (IL-17), and C-reactive protein levels were investigated. Results: There was a trend towards higher serum IP-10 levels (p = 0.042) for HA patients throughout the 6-month treatment period. Month-2 IP-10 levels were higher in the HA than in the MA/LA group (656.2 ± 234.4 vs. 307.6 ± 258.5 pg/ml, adjusted p = 0.005). Receiver operating characteristic curves showed that the risk of relapse was well-captured by month-2 IP-10 levels at a cut-off value of 431 pg/ml (AUC=0.857, 95% CI 0.75–0.97, p = 0.003). Month-2 serum IL-17 levels were lower in non-survivors than survivors (15.7 ± 2.9 pg/ml vs. 24.6 ± 8.2 pg/ml, p = 0.001). Multivariate analysis demonstrated that a month-2 serum IL-17 level of ≤ 17 pg/ml (p = 0.026) was independently associated with all-cause mortality. Conclusions: Serum IP-10 and IL-17 levels after 2 months of anti-TB treatment may be biomarkers for estimating risk of both cavitation and delayed sputum conversion, and for predicting long-term mortality, respectively.

Leukocyte Mitochondrial DNA Copy Number Is Associated with Chronic Obstructive Pulmonary Disease.

Background Oxidative stress is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Evidence suggests that leukocytes mitochondria DNA (mtDNA) is susceptible to undergo mutations, insertions, or depletion in response to reactive oxidative stress (ROS). We hypothesize that mtDNA copy number is associated with the development of COPD. Methodology/Principal Findings Relative mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral leukocytes. MtDNA copy number of peripheral leukocytes in the COPD group (n = 86) is significantly decreased compared with non-smoker group (n = 77) (250.3± 21.5 VS. 464.2± 49.9, P<0.001). MtDNA copy number in the COPD group was less than that in the healthy smoking group, but P value nearly achieved significance (250.3± 21.5 VS. 404.0± 76.7, P = 0.08) MtDNA copy number has no significance with age, gender, body mass index, current smoking, and pack-years in COPD group, healthy smoker group and no smoker group, respectively. Serum glutathione level in the COPD group is significantly decreased compared with healthy smoker and non-smoker groups (4.5± 1.3 VS. 6.2± 1.9 and 4.5± 1.3 VS. 7.1±1.1 mU/mL; P<0.001 respectively). Pearson correlation test shows a significant liner correlation between mtDNA copy number and serum glutathione level (R = 0.2, P = 0.009). Conclusions/Significance COPD is associated with decreased leukocyte mtDNA copy number and serum glutathione. COPD is a regulatory disorder of leukocytes mitochondria. However, further studies are needed to determine the real mechanisms about the gene and the function of mitochondria.

Association of HPV infections with second primary tumors in early-staged oral cavity cancer

OBJECTIVE The infection of human papilloma virus (HPV) has been reported in head and neck cancer; however, the clinical significance of HPV infection on the pathogenesis of oral cavity squamous cell carcinoma (OSCC) is still uncertain. MATERIALS AND METHODS The study recruited 103 patients with pathological early-stage OSCC between March 1997 and December 2003 from Chang Gung Memorial Hospital, Taiwan. Tumor specimens were HPV-genotyped by the EasychipVR HPV Blot method. Clinical association study was performed by using chi-square, Kaplan-Meier, and logrank tests. RESULTS Thirty-one patients (30.1%) were positive for HPV infection. The most frequent HPV types were types 16 (16 patients, 51.6%) and 18 (seven patients, 22.6%). HPV infection was not associated with tumor aggressiveness (pathological tumor stage or differentiation status), risk exposure (alcohol, cigarette, or areca quid chewing habit), or the treatment outcome (disease-free survival or overall survival). However, infection with HPV-18 was associated with the occurrence of a second primary cancers (P = 0.033), indicating the infection of HPV in OSCC enhances the susceptibility of developing secondary malignancy. CONCLUSIONS There are 30% of the patients with OSCC infected with HPV, with most high-risk types. HPV-18 infection may enhance the susceptibility of second primary tumors. Large scale of validation study will be needed to confirm this result.