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Publications and source records attributed to Horng-Jyh Harn.
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Midbrain dopaminergic progenitors (mDAp) derived from human pluripotent stem cells have demonstrated promising safety and efficacy in Phase I clinical transplantation trials for Parkinson's disease (PD). To further improve the translational potential of this approach, strategies that accelerate the loss of pluripotency, increase the yield of in vitro mDA progenitors, and promote post-transplantation neurite outgrowth may be beneficial. Here, we developed an optimized protocol building upon our previously established neural induction method, with further refinements, to efficiently convert induced pluripotent stem cells (iPSCs) into mDA neurons. Additionally, we applied n-butylidenephthalide, which selectively reduced pluripotency-associated gene expression and enhanced neurite outgrowth during differentiation. With these improved techniques, 71% of iPSCs differentiated into mDA neurons, showing burst dopamine secretion and phasic electrophysiological activities with external stimuli. To evaluate the safety, cryopreserved mDAp were transplanted into immunodeficient mice. No teratoma or neural tumor was observed within 24 weeks post-implantation. In 6-OHDA PD rats, mDAp survived and differentiated into mDA neurons in the host striatum within eight weeks post-transplantation, leading to significant functional recovery. The current differentiation process, therefore, enables the generation of cryopreservable, off-the-shelf mDAp, with accelerated loss of the pluripotency marker OCT4 in vitro and early functional recovery following transplantation.
There was increasing evidence suggesting that angiotensin I-converting enzyme may play an important role in the pathogenesis of PD. Our former study has shown that angiotensin I-converting enzyme gene (ACE) may confer a susceptibility for the risk of Parkinson's disease (PD). Meanwhile, recent studies have emphasized that genetic factors may involve in the occurrence of the adverse effects of chronic L-dopa therapy in PD patients. This study was designed to assess whether genetic polymorphism of the ACE could be a predictor of L-dopa-induced adverse effects in PD. There were 251 patients included in this study and their mean age at onset of disease was 63.3+/-11.4 years. The duration of disease and the treatment with L-dopa was 6.3+/-5.1 and 5.0+/-4.3 years, respectively. The frequency of the homozygote ACE-II genotype of the ACE in PD patients with L-dopa-induced psychosis was significantly higher than that in PD patients without the adverse effect (63.3% vs 43.0%; chi(2)=6.347, OR=1.435, 95%CI=1.105-1.864, p=0.012). However, the ACE polymorphism was not associated with the risk to develop dyskinesia or motor fluctuation induced by L-dopa. Furthermore, a logistic regression analysis confirmed that the ACE-II genotype was an independent risk factor for L-dopa-induced psychosis in PD patients (OR=2.542, p=0.012). In conclusion, results of the study showed that ACE-II genotype might confer a primary predictor for the occurrence of psychosis in L-dopa-treated PD.
The naturally-occurring compound, n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis (AS-C), has been investigated with respect to the treatment of angina. In this study, we have examined the anti-tumor effects of n-butylidenephthalide on glioblastoma multiforme (GBM) brain tumors both in vitro and in vivo. In vitro, GBM cells were treated with BP, and the effects of proliferation, cell cycle and apoptosis were determined. In vivo, DBTRG-05MG, the human GBM tumor, and RG2, the rat GBM tumor, were injected subcutaneously or intracerebrally with BP. The effects on tumor growth were determined by tumor volumes, magnetic resonance imaging and survival rate. Here, we report on the potency of BP in suppressing growth of malignant brain tumor cells without simultaneous fibroblast cytotocixity. BP up-regulated the expression of Cyclin Kinase Inhibitor (CKI), including p21 and p27, to decrease phosphorylation of Rb proteins, and down-regulated the cell-cycle regulators, resulting in cell arrest at the G(0)/G(1) phase for DBTRG-05MG and RG2 cells, respectively. The apoptosis-associated proteins were dramatically increased and activated by BP in DBTRG-05MG cells and RG2 cells, but RG2 cells did not express p53 protein. In vitro results showed that BP triggered both p53-dependent and independent pathways for apoptosis. In vivo, BP not only suppressed growth of subcutaneous rat and human brain tumors but also, reduced the volume of GBM tumors in situ, significantly prolonging survival rate. These in vitro and in vivo anti-cancer effects indicate that BP could serve as a new anti-brain tumor drug.
N-acetylcysteine (NAC) is an antioxidant and cytoprotective agent with scavenging action against reactive oxygen species and inhibitory effects on pro-inflammatory cytokines. In a previous study, we found that pretreatment with NAC attenuated organ dysfunction and damage, reduced the production of free radicals, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) following endotoxemia elicited by administration of lipopolysaccharide (LPS). In the present study, we tested the effects of post-treatment with NAC on the sepsis-induced change. Post-treatment imitates clinical therapeutic regimen with administration of drug after endotoxemia. Endotoxin shock was induced by intravenous injection of Klebsiella pneumoniae LPS (10 mg/kg) in conscious rats. Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored for 48 h after LPS administration. NAC was given 20 min after LPS. Measurements of biochemical substances were taken to reflect organ functions. Biochemical factors included blood urea nitrogen (BUN), creatinine (Cre), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate transferase (GOT), alanine transferase (GPT), TNF-alpha, interleukin-6 (IL-6), and interleukin-10 (IL-10). LPS significantly increased blood BUN, Cre, LDH, CPK, GOT, GPT, TNF-alpha, IL-6, IL-10 levels and HR, and decreased MAP. Post-treatment with NAC diminished the decrease in MAP, increased the HR, and decreased the markers of organ injury (BUN, Cre, LDH, CPK, GOT, GPT) and inflammatory biomarkers (TNF-alpha, IL-6, IL-10) after LPS. We conclude that post-treatment with NAC suppresses the release of plasma TNF-alpha, IL-6, and IL-10 in endotoxin shock, and decreases the markers of organ injury. These beneficial effects protect against LPS-induced kidney, heart and liver damage in conscious rats. The beneficial effects may suggest a potential chemopreventive effect of this compound after sepsis.
We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (BS-AE) 60 microg/ml has anti-proliferation activity and apoptotic effects on A549 non-small cell lung cancer (NSCLC). A novel lignan, isochaihulactone (4-benzo[1,3]dioxol-5-ylmethyl-3(3,4,5-trimethoxyl-benzylidene)-dihydro-furan-2-one), was isolated from BS-AE and identified from spectral evidence ((1)H NMR, (13)C NMR, IR, and MS) and by comparison with authentic synthetic standards. Isochaihulactone was cytotoxic (IC(50)=10-50 microM) in a variety of human tumor cell lines. In in vitro and in vivo microtubule assembly assays, it inhibited tubulin polymerization in a concentration-dependent manner. As determined by flow cytometry, isochaihulactone caused G2/M phase arrest and apoptosis in a time- and concentration-dependent manner. G2/M arrest was correlated with increased p21/WAF1 levels, downregulation of the checkpoint proteins cyclin B1/cdc2 and mobility shift of cdc25C. Moreover, isochaihulactone (30 and 50 mg/kg) inhibited the growth of non-small cell lung carcinoma A549 xenograft in nude mice. These findings indicate isochaihulactone is a promising new antimitotic anticancer compound with potential for clinical application in the future.
AIM: To study the role of CDH1/E-cadherin (E-cad) gene alteration profiles including mutation, loss of heterozygosity (LOH), promoter polymorphism and hypermethylation in mechanisms of CDH1 inactivation in gastric carcinoma (GC). METHODS: Specimens were collected surgically from 70 patients with GC. Allelotyping PCR and detection of LOH, denaturing high pressure liquid chromatography and DNA sequencing, restriction fragment length polymorphism analysis, methylation specific PCR, and immunohistochemical staining were used. RESULTS: Promoter polymorphism was not a major mechanism of E-cad inactivation. Only one truncating mutation was found in a diffuse type tumor (3%). Both LOH and promoter hypermethylation were major mechanisms of E-cad inactivation, but interestingly, there was a negative association between the fraction of allelic loss (LOH) in tumors and hypermethylation of CDH1. Therefore LOH and hypermethylation were two different tumorigenic pathways involved in GC. CONCLUSION: Given the findings that somatic mutation was extremely low and the relationship between LOH and hypermethylation was inverse, any two combinations of these three factors cannot fulfill the classical two-hit hypothesis of CDH1 inactivation. Thus, other mechanisms operating at the transcriptional level or at the post-translational level might be required to induce E-cadherin inactivation.
BACKGROUND: Colorectal cancer (CRC), which has become especially prevalent in developed countries, is currently the third highest cause of cancer mortality in Taiwan. Mutation of the adenomatous polyposis coli (APC) gene, a tumour suppressor, is thought to be an early event in colorectal tumourigenesis. To date, however, no large-scale screening for APC gene variants in Chinese subjects has been performed. The present study was undertaken to identify APC gene variants that are significantly associated with the occurrence of CRC in Taiwanese subjects. METHODS: In order to compare the genotype distribution of variant sites, the full-length APC genes of 74 healthy individuals and 80 CRC patients were sequenced. RESULTS: Among the 154 Taiwanese subjects examined in this study, three new mutations, but no previously reported mutations, were found. One deletion at codon 460 leading to a frameshift and two missense mutations resulting in p.V1125A and p.S1126R substitutions were identified. Additionally, three high risk genotypes associated with three single nucleotide polymorphisms and one low risk genotype at codon 1822 were identified. CONCLUSION: The findings of this case-control study are consistent with the proposal that Taiwanese subjects differ from other subjects with respect to phenotypic presentation of APC and CRC risk.
Hemorrhagic shock is a common cause of death in emergency rooms. Current animal models of hemorrhage encounter a major problem that the volume and the rate of blood loss cannot be controlled. In addition, the use of anesthesia obscures physiological responses. Our experiments were designed to establish an animal model based on the clinical situation for studying hemorrhagic shock. Hemorrhagic shock was induced by withdrawing blood from a femoral arterial catheter. The blood volume withdrawn was 40% of the total blood volume for group 1 and 30% for group 2 and 3. Group 3 was anesthetized with sodium pentobarbital (25 mg/kg, i.v.) at the beginning of blood withdrawal. Our data showed that the survival rate was 87.5% at 48 h in the conscious group and 0% at 9 h in anesthetic group after hemorrhage. The levels of mean arterial pressure, heart rate, white blood count, TNF-alpha, IL1-beta, CPK, and LDH after blood withdrawal in the anesthetic group were generally lower than those in conscious groups. These results indicated that anesthetics significantly affected the physiology of experimental animals. The conscious, unrestrained and cumulative volume-controlled hemorrhagic shock model was a good experimental model to investigate the physical phenomenon without anesthetic interfernce.
AIM: To study Helicobacter pylori (H. pylori) infection in relation to E-cadherin (E-cad) promoter polymorphism and hypermethylation in GCs. METHODS: Specimens were taken from representative cancerous lesions and adjacent non-cancerous epithelia of 67 resected GCs. H. pylori was detected by real-time PCR of the cagA gene from non-neoplastic epithelium. E-cad promoter polymorphism and hypermethylation were determined by restriction fragment length polymorphism analysis and methylation-specific PCR, respectively. Expression of E-cad protein was determined by immunohistochemistry. RESULTS: H. pylori was found in 57% of patients with GC. H. pylori infection was more frequently found in tumors with the -160C/C genotype than those with the -160C/A and -160A/A genotypes (74% vs 47%, P = 0.02). H. pylori infection was associated with E-cad methylation in non-neoplastic epithelium; however, no significant difference in H. pylori was observed between methylated and unmethylated cancerous lesions. CONCLUSION: Patients with the -160C/C genotype might require H. pylori infection to promote the inactivation of CDH1, suggesting that H. pylori infection might affect GC in an initial stage because polymorphism is germ line. Mechanism of hypermethylation of CDH1 promoter in GC is complex, and H. pylori infection might affect it in an initial stage.
PURPOSE: In this study, we have examined the antitumor effects of chloroform extract of Angelica sinensis (AS-C), a traditional Chinese medicine, on glioblastoma multiforme (GBM) brain tumors in vitro and in vivo. EXPERIMENTAL DESIGN: In vitro, GBM cells were treated with AS-C, and the cell proliferation, changes in distributions of cell cycle, and apoptosis were determined. In vivo, human DBTRG-05MG and rat RG2 GBM tumor cells were injected s.c. or i.c. and were treated with AS-C. Effects on tumor growth were determined by tumor volume, magnetic resonance imaging, survival, and histology analysis. RESULTS: The AS-C displays potency in suppressing growth of malignant brain tumor cells without cytotoxicity to fibroblasts. Growth suppression of malignant brain tumor cells by AS-C results from cell cycle arrest and apoptosis. AS-C can up-regulate expression of cdk inhibitors, including p21, to decrease phosphorylation of Rb proteins resulting in cell arrest at the G0-G1 phase for DBTRG-05MG and RG2 cells. The apoptosis-associated proteins are dramatically increased and activated in DBTRG-05MG cells and RG2 cells by AS-C but RG2 cells without p53 protein expression. In vitro results showed AS-C triggered both p53-dependent and p53-independent pathways for apoptosis. In in vivo studies, AS-C not only can suppress growths of malignant brain tumors of rat and human origin but also shrink the volumes of in situ GBM, significantly prolonging survivals. CONCLUSIONS: The in vitro and in vivo anticancer effects of AS-C indicate that it has sufficient potential to warrant further investigation and development as a new anti-brain tumor agent.
We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (AE-BS) 60 microg/ml has anti-proliferation activity and apoptosis effects to A549 human lung cancer cells. They can also cause tumor cell arrest in G2/M phase. To better understand its target protein in A549 cell, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry were applied. The modification of keratin 8 was identified. By immunoblot, the expression of phosphorylated keratin 8 at Ser-73 was increased from 2.0 to 3.0-fold after AE-BS treatment 24 to 48 hr respectively as compared with untreated A549 control cells. Furthermore, the A549 cells were pretreated with 50 microM PD98059, a specific inhibitor of the upstream regulator of ERK1/2, or with the p38 kinase inhibitor 20 microM SB203580 or JNK inhibitor 20 microM SP600125 for 30 min, followed by 24 h of incubation with AE-BS, PD98059 can inhibit K8-Ser-73 hyperphosphorylation and prevented cell apoptosis which was induced by AE-BS significantly. By immunoblot, AE-BS also can induce ERK 1/2 phosphorylation. In conclusion, our data indicate that the AE-BS induced tumor apoptosis in A549 cells was related to ERK 1/2 activation. The molecular mechanism of hyperphosphorylation of K8 on Ser-73 was associated with ERK 1/2 activation rather than JNK and p38 kinase. The apoptosis induced by AE-BS may be related to K8 phosphorylation.
Studies have shown that migraine may have a major genetic component. Meanwhile, angiotensin converting enzyme (ACE) gene has been implicated as a genetic factor associated with migraine. We designed a case-control study to investigate the association between ACE and migraine in 240 migraine patients and 200 healthy controls, matched by age and sex. There was no significant difference in allelic frequency (I and D) and genotype polymorphism (DD, DI and II) of the ACE gene in migraine patients and controls. Analysis of the difference in ACE polymorphism stratified by gender revealed that male migraine patients with the homozygote DD genotype (ACE-DD) were significantly fewer than that of male controls (OR = 0.331, p = 0.045). There was no existence of a difference among the frequency and duration of headache in each subgroup of migraine patients stratified by ACE genotype. Our findings indicate that ACE-DD may have a slight protective effect against migraine in male patients.
Nan-Chai-Hu, the root of Bupleurum scorzonerifolium, is a traditional Chinese herb used in treatment of liver diseases such as hepatitis and cirrhosis. We recently reported that the acetone extract of B. scorzonerifolium (BS-AE) could inhibit proliferation and induce apoptosis in A549 human lung cancer cells. We further examined its anti-proliferative mechanisms and in vivo anticancer effect. Our results showed that BS-AE had the ability to cause cell cycle arrest in G2/M phase, inducing tubulin polymerization, and activating caspase-3 and -9 in A549 cells. BS-AE-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk in majority. The result of in vivo study showed that BS-AE could suppress growth in A549 subcutaneous xenograft tumors. These results indicate that BS-AE exerts antiproliferative effects on A549 cells in vitro and in vivo, and prompted us to further evaluate and elucidate the chemical composition profile of BS-AE.
Angelica sinensis (Oliv.) Diels, a traditional Chinese medicine, has been widely prescribed in treatment of gynecological diseases. Bio-based assays for extracts of Angelica sinensis showed that the acetone extract (AE-AS) had dose-dependently antiproliferative effect on A549, HT29, DBTRG-05MG and J5 human cancer cells. The IC50 values of AE-AS on mentioned cancer cells ranged from 35 to 50 microg/ml after 24 h of treatment. After 72 h of exposure, AE-AS (40 microg/ml) significantly reduced A549 cell proliferation to 24 +/- 3.2% of control. In A549 cells, the cell cycle analysis showed that AE-AS induced a significant increase in the number of cells in G0/G1, with a concomitant decrease in the number of cells in S phase. AE-AS-induced chromatin changes and apoptosis of A549 cells were confirmed by Hoechst 33342 DNA staining and annexin V staining. A549 cells treated with AE-AS caused activation of caspase-9 and -3, and AE-AS-induced apoptosis could be inhibited by the broad-spectrum caspase inhibitor, z-VAD-fmk. The Western blot indicated the AE-AS-triggered apoptosis is mediated via suppression of Bcl-2 oncoprotein expression rather than p53 or Bax. Besides, AE-AS decreased the levels of cdk4 protein was observed. These results indicate that the AE-AS could induce G1/S arrest and activate the mechanism of apoptosis in human cancer cells. Extracts obtained from different methods of fractionation might possess distinct bioactivity. These results prompted us to further evaluate the in vivo anticancer effects and elucidate the chemical composition profile of AE-AS.
AIM: Most cancer cells acquire immortal capability by telomerase activation. The human telomerase reverse transcriptase gene (hTERT) is considered to be the major determinant of the enzymatic activity of human telomerase, and the hTERT promoter contains several c-Myc binding sites that mediate hTERT transcriptional activation. Few studies have examined the role of hTERT in hepatocarcinogenesis, and the relationship between c-Myc and telomerase in human hepatocellular carcinoma tissue is unknown. METHODS: We measured hTERT mRNA levels and c-Myc oncoprotein expression in 57 patients with hepatocellular carcinoma using in situ hybridization and immunohistochemistry, respectively. The transcription regulation of hTERT was evaluated by transient transfection of pGL3-1375 into the human hepatocellular carcinoma cell line J5. To determine the relationship between c-Myc and the hTERT promoter, a 1375-bp DNA fragment encompassing the promoter was placed upstream of the luciferase reporter gene and transiently transfected into the cell line. Two additional hTERT promoter constructs (-776 and -100 bp region) and an hTERT promoter-LUC construct containing 2 c-Myc mutations (pGL3-181 MycMT) were also used for luciferase assays. RESULTS: In 30 of 57 cases (52%), hTERT mRNA expression was associated with c-Myc protein expression. However, 16 of 57 cases (28%) showed strong hTERT mRNA detection without c-Myc protein expression, and 11 cases (19%) showed weak hTERT mRNA expression and strong c-Myc expression. Although luciferase activity was decreased between upstream 1 375 bp and 776 bp, there was no significant difference between upstream 776 bp and 100 bp. Finally, there was no significant decrease in activity after transfection of the hTERT promoter-LUC construct. CONCLUSION: The results indicate that c-Myc does not play a major role in gene regulation of the catalytic subunit of telomerase (hTERT) in human hepatocellular carcinoma. Other regulatory elements or epigenetic phenomena should be further investigated to understand hTERT gene regulation in human hepatocellular carcinoma.
BACKGROUND: Telomerase is activated in the majority of cancer tissues and immortalized cell lines. The hTERT (human telomerase reverse transcriptase-major component of telomerase) gene promoter has been cloned and contains many c-myc binding sites that mediate hTERT transcriptional activation. Thus far, the role of hTERT in tumorigenesis in hepatocellular carcinoma (HCC) has been little studied using RNA in situ hybridization. The relationship between c-myc and telomerase in human HCC tissue is undetermined. MATERIALS AND METHODS: The telomerase activity was assayed using TRAP in specimens from 23 HCC patients, hTERTmRNA was detected using in situ hybridization from 57 HCC patients. The immunohistochemistry for c-myc and DNA sequence for hTERT promoter, and tumour differentiation in relation to hTERT and c-myc expression were determined in 57 specimens. RESULT: hTERTmRNA was found in 47/57 (82.5%) HCC specimens using in situ hybridization. The hTERT expression paralleled telomerase activity, but hTERTmRNA regulation was not significantly associated with c-myc level ( P<0.954) The DNA sequence analysis of the hTERT promoter in specimens from 17 HCC revealed 15 cases of nucleotide transition (T-->C) over 5'-end of distal E-box and one case of nucleotide transversion (G-->C) over 5'-end of proximal E-box. Neither the hTERT expression (P< 0.890) nor c-myc level (P < 0.348) were related to HCC differentiation. CONCLUSIONS: The hTERT expression paralleled telomerase activity. The telomerase activity in HCC was not only regulated by c-myc. Another pathways might contribute to hTERT and telomerase activity regulation. The lack of telomerase activity in specimens from 17.4% of HCC cases might indicate an alternative pathway for maintaining telomere length. Furthermore, both the telomerase activity and c-myc had no significant role in HCC differentiation.
Bupleuri radix, a traditional Chinese herb, has been widely used to treat liver diseases such as hepatitis and cirrhosis. The acetone extract of Bupleurum scorzonerifolium (AE-BS) showed a dose-dependently antiproliferative effect on the proliferation of A549 human lung cancer cells. The IC(50) of AE-BS, i.e., the concentration required to inhibit proliferation of A549 cells, was 59 +/- 4.5 microg/ml on day 1. The IC(50) of AE-BS for WI38 human normal lung fibroblast cells, however, was significant higher than that for A549 cells (150 +/- 16 microg/ml, p< 0.01). After 72 hours of exposure, AE-BS (60 microg/ml) significantly reduced A549 cell proliferation to 33 +/- 3.2% of control. In TUNEL assay, A549 cells treated with AE-BS showed typical morphologic features of apoptosis, and the percentage of apoptotic cells was approximately 38 % on day 1. In the TRAP assay, AE-BS-treated cells demonstrated significantly lower telomerase activity on day 3. This result indicates that the AE-BS could suppress the proliferation of lung cancer cells via inhibition of telomerase activity and activation of apoptosis.