Chun-Ju Chang

Modulation of HER2 expression by ferulic acid on human breast cancer MCF7 cells

Background  The molecular mechanisms underlying the mitogenic effect of ferulic acid (FA), an active compound derived from Angelica sinensis, have never been elucidated. It was the aim of this study to investigate the proliferative effect of FA on human breast cancer cell lines and to elucidate its modulation mechanism on HER2 expression in MCF7 line.

Somatic mutations in mitochondrial genome and their potential roles in the progression of human gastric cancer

BACKGROUND Somatic mutation in mitochondrial DNA (mtDNA) has been proposed to contribute to initiation and progression of human cancer. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of gastric cancers. However, it is unclear whether somatic mutations occur in the coding region of mtDNA of gastric cancers. METHODS Using DNA sequencing, we studied 31 gastric cancer specimens and corresponding non-cancerous stomach tissues. Moreover, a human gastric cancer SC-M1 cell line was treated with oligomycin to induce mitochondrial dysfunction. Cisplatin sensitivity and cell migration were analyzed. RESULTS We identified eight somatic mutations in the coding region of mtDNAs of seven gastric cancer samples (7/31, 22.6%). Patients with somatic mutations in the entire mtDNA of gastric cancers did not show significant association with their clinicopathologic features. Among the eight somatic mutations, five point mutations (G3697A, G4996A, G9986A, C12405T and T13015C) are homoplasmic and three mutations (5895delC, 7472insC and 12418insA) are heteroplasmic. Four (4/8, 50%) of these somatic mutations result in amino acid substitutions in the highly conserved regions of mtDNA, which potentially lead to mitochondrial dysfunction. In addition, in vitro experiments in SC-M1 cells revealed that oligomycin-induced mitochondrial dysfunction promoted resistance to cisplatin and enhanced cell migration. N-acetyl cysteine was effective in the prevention of the oligomycin-enhanced migration, which suggests that reactive oxygen species generated by defective mitochondria may be involved in the enhanced migration of SC-M1 cells. GENERAL SIGNIFICANCE Our results suggest that somatic mtDNA mutations and mitochondrial dysfunction may play an important role in the malignant progression of gastric cancer.

Mitochondrial dysfunction represses HIF-1α protein synthesis through AMPK activation in human hepatoma HepG2 cells

BACKGROUND Hypoxia-inducible factor-1α (HIF-1α) is an important transcription factor that modulates cellular responses to hypoxia and also plays critical roles in cancer progression. Recently, somatic mutations and decreased copy number of mitochondrial DNA (mtDNA) were detected in hepatocellular carcinoma (HCC). These mutations were shown to have the potential to cause mitochondrial dysfunction. However, the effects and mechanisms of mitochondrial dysfunction on HIF-1α function are not fully understood. This study aims to explore the underlying mechanism by which mitochondrial dysfunction regulates HIF-1α expression. METHODS Human hepatoma HepG2 cells were treated with various mitochondrial respiration inhibitors and an uncoupler, respectively, and the mRNA and protein expressions as well as transactivation activity of HIF-1α were determined. The role of AMP-activated protein kinase (AMPK) was further analyzed by compound C and AMPK knock-down. RESULTS Treatments of mitochondrial inhibitors and an uncoupler respectively reduced both the protein level and transactivation activity of HIF-1α in HepG2 cells under normoxia or hypoxia. The mitochondrial dysfunction-repressed HIF-1α protein synthesis was associated with decreased phosphorylations of p70(S6K) and 4E-BP-1. Moreover, mitochondrial dysfunction decreased intracellular ATP content and elevated the phosphorylation of AMPK. Treatments with compound C, an AMPK inhibitor, and knock-down of AMPK partially rescued the mitochondrial dysfunction-repressed HIF-1α expression. CONCLUSIONS Mitochondrial dysfunctions resulted in reduced HIF-1α protein synthesis through AMPK-dependent manner in HepG2 cells. GENERAL SIGNIFICANCE Our results provided a mechanism for communication from mitochondria to the nucleus through AMPK-HIF-1α. Mitochondrial function is important for HIF-1α expression in cancer progression.

Expression of hedgehog signaling molecules as a prognostic indicator of oral squamous cell carcinoma.

Recent studies have indicated hedgehog pathway plays a role in carcinogenesis of certain cancers. We investigated the clinical significance of its signaling components, including Sonic hedgehog (Shh), Patched (Ptch), and Gli‐1, in oral squamous cell carcinoma (OSCC).

Effect of human milk and epidermal growth factor on growth of human intestinal Caco-2 cells.

Background Epidermal growth factor (EGF) in human milk has been thought to be mitogenic for cell growth. This study investigated the effects of human milk and EGF on the growth of human intestinal Caco-2 cells to determine whether the action occurred through regulation of the cell cycle or through c-jun expression. Methods Cells were incubated with 5% human milk, 0.375 nmol/L EGF (relevant to EGF concentration in 5% human milk, 0.05 × EGF), 7.5 nmol/L EGF (1 × EGF), or 75 nmol/L EGF (10 × EGF). Cell numbers; cellular RNA, DNA, and protein concentrations; DNA content in the cell cycle, and expressions of c-Jun protein and mRNA were analyzed. Results Cell numbers increased in the 1 × and 10 × EGF groups at 48 hours. Cellular RNA increased in the 5% human milk and 10 × EGF groups. DNA and protein contents increased in the 1 × and 10 × EGF groups. The 1 × and 10 × EGF groups increased DNA content in the G1 phase compared with the 5% human milk group at 24 hours. The greatest c-jun protein expression was 2.6, 1.4, 1.8, and 1.9 times the control, and the c-jun mRNA increased by 202%, 14%, 150%, and 181%, respectively, in the 5% human milk, 0.05 ×, 1 ×, and 10 × EGF groups. Conclusions In a dose-dependent manner, EGF stimulated intestinal growth in vitro, by increasing DNA content in the G1 phase and c-jun mRNA expression. However, low concentrations of human milk (5%) and its equivalent EGF did not affect cell growth.

Si-Wu-Tang and its constituents promote mammary duct cell proliferation by up-regulation of HER-2 signaling

Objective: The consumption of over-the-counter natural products by perimenopausal women remains a challenging problem. It is our aim to investigate the proliferative effect of Si-Wu-Tang (SWT) and its constituents on MCF7 breast cancer cells as well as the SWT-modulated cell signaling and HER-2 gene expression. Design: By using the MCF7 (ER+, HER-2 low), BT474 (ER+, HER-2 high), MDAMB231 (ER−, HER-2 low), and SKBR3 (ER−, HER-2 high) mammary duct cell lines as our in vitro model, the mitogenic effects of SWT and its constituents were assessed by trypan blue dye exclusion assay and DNA flow cytometry. SWT-modulated cell signaling and HER-2 gene expression were evaluated in the MCF7 line by Western blot and reverse transcriptase-polymerase chain reaction analyses. Results: The results showed that SWT and some of its constituents dose-dependently stimulated cell proliferation of MCF7 cells. The activation of HER-2, its downstream signaling molecules AKT and ERK1/2, as well as HER-2 gene up-regulation were involved in SWT-stimulated cell proliferation. The addition of neutralizing antibody against HER-2 abrogated the SWT-up-regulated HER-2 expression, indicating a positive feedback control for the action of HER-2 in this setting. Ferulic acid, one of the major compounds in SWT, not only promoted cell proliferation of MCF7, BT474, MDAMB231, and SKBR3 cells, but also increased the phosphorylation of HER-2, AKT, and ERK1/2, as well as overexpression of HER-2, on MCF7 cells. Conclusions: We conclude that SWT and its active constituents stimulate mammary duct cell proliferation by modulating HER-2, PI3K/AKT, and MAPK signaling and the positive feedback of HER-2 gene expression. This provides important information for perimenopausal women who are at risk of or have breast cancer or other growths in breast tissue.

Screening to Identify Commonly Used Chinese Herbs That Affect ERBB2 and ESR1 Gene Expression Using the Human Breast Cancer MCF-7 Cell Line

Aim. Our aim the was to screen the commonly used Chinese herbs in order to detect changes in ERBB2 and ESR1 gene expression using MCF-7 cells. Methods. Using the MCF-7 human breast cancer cell line, cell cytotoxicity and proliferation were evaluated by MTT and trypan blue exclusion assays, respectively. A luciferase reporter assay was established by transient transfecting MCF-7 cells with plasmids containing either the ERBB2 or the ESR1 promoter region linked to the luciferase gene. Chinese herbal extracts were used to treat the cells at 24 h after transfection, followed by measurement of their luciferase activity. The screening results were verified by Western blotting to measure HER2 and ERα protein expression. Results. At concentrations that induced little cytotoxicity, thirteen single herbal extracts and five compound recipes were found to increase either ERBB2 or ESR1 luciferase activity. By Western blotting, Si-Wu-Tang, Kuan-Shin-Yin, and Suan-Tsao-Ren-Tang were found to increase either HER2 or ERα protein expression. In addition, Ligusticum chuanxiong was shown to have a great effect on ERBB2 gene expression and synergistically with estrogen to stimulate MCF-7 cell growth. Conclusion. Our results provide important information that should affect clinical treatment strategies among breast cancer patients who are receiving hormonal or targeted therapies.

In Vitro and In Vivo Effects of Jia-Wei-Xiao-Yao-San in Human Breast Cancer MCF-7 Cells Treated With Tamoxifen

Introduction. There is epidemiological evidence that Jia-Wei-Xiao-Yao-San (JWXYS) is the most common Chinese medicine decoction coprescribed with tamoxifen (Tam) when breast cancer is treated by hormonal therapy. However, whether there is interaction between JWXYS and Tam remains to be clarified. The aim of this study was to investigate the in vitro and in vivo effects of JWXYS on human breast cancer MCF-7 cells treated with Tam. Methods. In vitro cultured MCF-7 cells were cotreated with JWXYS and Tam. This was followed by MTT ([4,5-cimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide) assays and cell cycle analysis to assess cell proliferation; Western blot analysis was used to analyze the expression of various proteins involved in growth-related signal pathways. In addition, immunohistochemistry was used to detect autophagy among the cancer cells. In vivo analysis used female athymic nude mice implanted with MCF-7 cells; these mice were randomly assigned to 6 groups. All mice were killed humanely after 21 days of treatment; body weight, tumor volume, and tumor weight were then measured. Results. JWXYS was not cytotoxic to MCF-7 cells, based on the fact that there were no statistically significant changes between the JWXYS + Tam groups and the Tam-alone group in cell numbers, cell cycle progression, and cell proliferation signals, the latter including the expression levels of AKT, ERK, P38, p27(Kip1), and light chain (LC3)-I, II. Furthermore, using the MCF-7 xenograft mouse model, there were no significant changes between the JWXYS (1.3-3.9 gm/kg) + Tam groups and the Tam-alone group in terms of tumor weight and the protein expression levels of AKT, ERK, P38, and p27 (Kip1). However, there was a significant decrease in LC3-II protein expression with the low-dose JWXYS + Tam group but not with the middle- or high-dose JWXYS + Tam groups compared with the Tam-alone group. Conclusion. Based on in vitro studies and in vivo functional studies, there is no obvious interaction between JWXYS and Tam. However, the presence of interference at the molecular level in relation to LC3-II expression provides important information and may affect treatment strategies when physicians have patients with estrogen receptor-α(+) or progesterone receptor(+) breast cancers.

Flow-cytometric DNA content analysis of oesophageal carcinoma. Comparison between tumour and sequential non-tumour mucosae

The DNA content in oesophageal carcinoma and in sequential non-tumour mucosa was evaluated in 35 patients with oesophageal carcinoma, to explore the hypotheses that DNA distribution pattern and S-phase fraction can reflect malignant potential and that DNA aneuploidy can provide an early-warning signal of developing cancer. DNA flow cytometry was performed on 129 specimens from the tumours and on 119 specimens from non-tumour mucosa. Control specimens from gastric fundus had normal diploid DNA content and low S-phase fraction. Aneuploidy was found in 94.3% of the carcinoma specimens and intratumoral heterogeneity in 54.3%. Of the non-tumour specimens, 43.7% showed aneuploidy and none multiple aneuploidy. Pattern III distribution was present in 8.6% of the tumour specimens but not in non-tumour mucosa, where the incidence of aneuploidy rose with closeness to the tumour (p < 0.001). S-phase fraction was smaller in non-tumour than in tumour specimens (p < 0.0001). The study indicated that histologically tumour-free oesophageal mucosa may have a high malignant potential in patients with oesophageal carcinoma. The relative instability of such mucosa, with aneuploid cells and low S-phase fraction, may facilitate transition to abnormally proliferating cells in response to environmental signals. Cigarette smoking and alcohol may increase the risk of multicentric cancer development.

A novel rat model simulating biliary atresia after a Kasai operation.

ABSTRACT Purpose: The mechanisms of liver fibrosis in biliary atresia (BA) after a Kasai operation deserve studying to improve the clinical outcomes. This study aimed to create a rat model simulating BA after a Kasai operation. Methods: We inserted a polyethylene tube (PE10) into the common hepatic duct (CHD) and ligated the common bile duct (CBD) in 30 newborn rats and injected 95% ethanol into IHD at postoperative week-one (POW-1). The PE10 was removed at POW-3. The rats were sacrificed at POW-5. The CBD cystojejunostomy was performed on another 10 rats at POW-5. Results: The IHD obliteration and CBD dilatation were noted at POW-3 cholangiography before removal of the PE tube. The gross findings at sacrifice in the rats without cystojejunostomy included biliary fibrosis, CBD cyst, and IHD obliteration. The microscopic findings of the liver were like BA. Seven of the 10 rats with CBD cystojejunostomy were jaundice-free at POW-8. The fibrosis grade at POW-8 of the rats with CBD cystojejunostomy was significantly lower in the jaundice-free rats (Ishak fibrosis score, 3.4 ± 0.9 and 1.5 ± 0.3 in the jaundiced rats and jaundice-free rats, respectively, p < .05). Conclusion: Based on our study, CBD cystojejunostomy five weeks after CBD ligation with ethanol injection into the IHD in newborn rats can provide a model for investigating mechanisms and treatments of liver fibrosis in BA after a Kasai operation.