Ji Young Sul

Fatty Acid Synthase Inhibition by Amentoflavone Suppresses HER2/neu (erbB2) Oncogene in SKBR3 Human Breast Cancer Cells

Fatty acid synthase (FASN) is a potential therapeutic target for treatment of cancer and obesity, and is highly elevated in 30% of HER2‐overexpressing breast cancers. Considerable interest has developed in searching for novel FASN inhibitors as therapeutic agents in treatment of HER2‐overexpressing breast cancers. Amentoflavone was found to be effective in suppressing FASN expression in HER2‐positive SKBR3 cells. Pharmacological inhibition of FASN by amentoflavone specifically down‐regulated HER2 protein and mRNA, and caused an up‐regulation of PEA3, a transcriptional repressor of HER2. In addition, pharmacological blockade of FASN by amentoflavone preferentially decreased cell viability and induced cell death in SKBR3 cells. Palmitate reduced the cytotoxic effect of amentoflavone, as the percentage of viable cells was increased after the addition of exogenous palmitate. Amentoflavone‐induced FASN inhibition inhibited the translocation of SREBP‐1 in SKBR3 cells. Amentoflavone inhibited phosphorylation of AKT, mTOR, and JNK. The use of pharmacological inhibitors revealed that the modulation of AKT, mTOR, and JNK phosphorylation required synergistic amentoflavone‐induced FASN inhibition and HER2 activation in SKBR3 cells. These results suggest that amentoflavone modulated FASN expression by regulation of HER2‐pathways, and induced cell death to enhance chemopreventive or chemotherapeutic activity in HER2‐positive breast cancers. Copyright

Laparoscopy Assisted versus Open Distal Gastrectomy with D2 Lymph Node Dissection for Advanced Gastric Cancer: Design and Rationale of a Phase II Randomized Controlled Multicenter Trial (COACT 1001)

Purpose Laparoscopy-assisted distal gastrectomy for early gastric cancer has gained acceptance and popularity worldwide. However, laparoscopy-assisted distal gastrectomy for advanced gastric cancer is still controversial. Therefore, we propose this prospective randomized controlled multi-center trial in order to evaluate the safety and feasibility of laparoscopy assisted D2-gastrectomy for advanced stage gastric cancer. Materials and Methods Patients undergoing distal gastrectomy for advanced gastric cancer staged cT2/3/4 cN0/1/2/3a cM0 by endoscopy and computed tomography are eligible for enrollment after giving their informed consent. Patients will be randomized either to laparoscopy-assisted distal gastrectomy or open distal gastrectomy. Sample size calculation revealed that 102 patients are to be included per treatment arm. The primary endpoint is the non-compliance rate of D2 dissection; relevant secondary endpoints are three-year disease free survival, surgical and postoperative complications, hospital stay and unanimity rate of D2 dissection evaluated by reviewing the intraoperative video documentation. Discussion Oncologic safety is the major concern regarding laparoscopy-assisted distal gastrectomy for advanced gastric cancer. Therefore, the non-compliance rate of clearing the N2 area was chosen as the most important parameter for the technical feasibility of the laparoscopic procedure. Furthermore, surgical quality will be carefully reviewed, that is, three independent experts will review the video records and score with a check list. For a long-term result, disease free survival is considered a secondary endpoint for this trial. This study will offer promising evidence of the feasibility and safety of Laparoscopy-assisted distal gastrectomy for advanced gastric cancer.Trial Registration: NCT01088204 (international), NCCCTS-09-448 (Korea).

Activation of AMP-activated Protein Kinase on Human Gastric Cancer Cells by Apoptosis Induced by Corosolic Acid Isolated from Weigela subsessilis

Corosolic acid is one of the triterpenoids present in the leaves of Weigela subsessilis. The antidiabetic activity of corosolic acid has been reported previously, but to date, the anticancer effects on gastric cancer have been poorly studied. In this study, corosolic acid showed growth inhibition on SNU‐601 human gastric cancer cells, with an IC50 value of 16.9 ± 2.9 μm. Corosolic acid also triggered the activation of caspase‐3 and poly (ADP‐ribose) polymerase, while it was recovered by Z‐VAD‐FMK. Moreover, the cell growth/apoptosis activities of corosolic acid were regulated by the AMP‐activated protein kinase‐mammalian target of rapamycin (AMPK‐mTOR) signals. These results showed that corosolic acid‐mediated AMPK activation leads to inhibition of mTOR, thus providing a possible mechanism of action of corosolic acid in the inhibition of cancer cell growth and the induction of apoptosis. Copyright

Chrysophanic acid blocks proliferation of colon cancer cells by inhibiting EGFR/mTOR pathway.

Inactivation of epidermal growth factor receptor (EGFR) is a prime method used in colon cancer therapy. Here it is shown that chrysophanic acid, a natural anthraquinone, has anticancer activity in EGFR‐overexpressing SNU‐C5 human colon cancer cells. Chrysophanic acid preferentially blocked proliferation in SNU‐C5 cells but not in other cell lines (HT7, HT29, KM12C, SW480, HCT116 and SNU‐C4) with low levels of EGFR expression. Chrysophanic acid treatment in SNU‐C5 cells inhibited EGF‐induced phosphorylation of EGFR and suppressed activation of downstream signaling molecules, such as AKT, extracellular signal‐regulated kinase (ERK) and the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K). Chrysophanic acid (80 and 120 µm) significantly blocked cell proliferation when combined with the mTOR inhibitor, rapamycin. These findings offer the first evidence of anticancer activity for chrysophanic acid via EGFR/mTOR mediated signaling transduction pathway. Copyright

Sentinel Node Mapping Using a Fluorescent Dye and Visible Light During Laparoscopic Gastrectomy for Early Gastric Cancer: Result of a Prospective Study From a Single Institute

Objective: The aim of this study was to investigate the feasibility of sentinel node mapping using a fluorescent dye and visible light in patients with gastric cancer. Background: Recently, fluorescent imaging technology offers improved visibility with the possibility of better sensitivity or accuracy in sentinel node mapping. Methods: Twenty patients with early gastric cancer, for whom laparoscopic distal gastrectomy with standard lymphadenectomy had been planned, were enrolled in this study. Before lymphadenectomy, the patients received a gastrofiberoscopic peritumoral injection of fluorescein solution. The sentinel basin was investigated via laparoscopic fluorescent imaging under blue light (wavelength of 440–490 nm) emitted from an LED curing light. The detection rate and lymph node status were analyzed in the enrolled patients. In addition, short-term clinical outcomes were also investigated. Results: No hypersensitivity to the dye was identified in any enrolled patients. Sentinel nodes were detected in 19 of 20 enrolled patients (95.0%), and metastatic lymph nodes were found in 2 patients. The latter lymph nodes belonged to the sentinel basin of each patient. Meanwhile, 1 patient (5.0%) experienced a postoperative complication that was unrelated to sentinel node mapping. No mortality was recorded among enrolled cases. Conclusions: Sentinel node mapping with visible light fluorescence was a feasible method for visualizing sentinel nodes in patients with early gastric cancer. In addition, this method is advantageous in terms of visualizing the concrete relationship between the sentinel nodes and surrounding structures.

Inhibitory effect of emodin on fatty acid synthase, colon cancer proliferation and apoptosis

Fatty acid synthase (FASN) is a key anabolic enzyme for de novo fatty acid synthesis, which is important in the development of colon carcinoma. The high expression of FASN is considered a promising molecular target for colon cancer therapy. Emodin, a naturally occurring anthraquinone, exhibits an anticancer effect in various types of human cancer, including colon cancer; however, the molecular mechanisms remain to be fully elucidated. Cell viability was evaluated using a Cell Counting Kit-8 assay. The apoptosis rate of cells was quantified via flow cytometry following Annexin V/propidium iodide staining. FASN activity was measured by monitoring oxidation of nicotinamide adenine dinucleotide phosphate at a wavelength of 340 nm, and intracellular free fatty acid levels were detected using a Free Fatty Acid Quantification kit. Western blot analysis and reverse transcription-polymerase chain reaction were used to detect target gene and protein expression. The present study was performed to investigate whether the gene expression of FASN and its enzymatic activity are regulated by emodin in a human colon cancer cell line. Emodin markedly inhibited the proliferation of HCT116 cells and a higher protein level of FASN was expressed, compared with that in SW480, SNU-C2A or SNU-C5 cells. Emodin significantly downregulated the protein expression of FASN in HCT116 cells, which was caused by protein degradation due to elevated protein ubiquitination. Emodin also inhibited intracellular FASN enzymatic activity and reduced the levels of intracellular free fatty acids. Emodin enhanced antiproliferation and apoptosis in a dose- and time-dependent manner. The combined treatment of emodin and cerulenin, a commercial FASN inhibitor, had an additive effect on these activities. Palmitate, the final product of the FASN reaction, rescued emodin-induced viability and apoptosis. In addition, emodin altered FASN-involved signaling pathways, including phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinases/extracellular signal-regulated kinases 1/2. These results suggested that emodin-regulated cell growth and apoptosis were mediated by inhibiting FASN and provide a molecular basis for colon cancer therapy.

Corosolic acid enhances 5‑fluorouracil‑induced apoptosis against SNU‑620 human gastric carcinoma cells by inhibition of mammalian target of rapamycin

5‑Fluorouracil (5‑FU), one of the oldest anticancer therapeutic agents, is increasingly being administered in cancer chemotherapy. In the present study, the anticancer effects of 5‑FU combined with corosolic acid (CRA) were determined in SNU‑620 human gastric carcinoma cells and the underlying mechanisms were examined. A combination treatment of 5‑FU and CRA inhibited the viability of cells additively. Furthermore, apoptotic activity following combination treatment was found to be stronger than that of the single treatments, as observed using an Annexin V/propidium iodide assay. The protein level of Bcl‑2 was decreased significantly by the combination treatment, whereas the protein level of Bim was increased. The release of mitochondrial cytochrome c was increased as a result of the combination treatment, however, the combination treatment additively increased caspase‑3 and poly‑(ADP‑ribose) polymerase cleavages. Additionally, the mammalian target of rapamycin (mTOR) signaling pathway, which is highly activated in gastric cancer, was regulated by 5‑FU and CRA, and additive mTOR/eukaryotic translation initiation factor 4E‑binding protein 1 (4‑EBP1) inhibition was observed with the combination treatment. Additional rapamycin treatment along with the combination treatment of 5‑FU and CRA showed a more marked inhibition of mTOR/4‑EBP1 in the cells, as well as increased apoptosis and antiproliferation. Thus, these data indicate that CRA enhances the anticancer activities of 5‑FU via mTOR inhibition in SNU‑620 human gastric carcinoma cells.

Combination of pristimerin and paclitaxel additively induces autophagy in human breast cancer cells via ERK1/2 regulation

Pristimerin, a quinonemethide triterpenoid, has demonstrated anticancer activity against a number of types of cancer, including breast cancer. However, its mechanism of action remains unclear. The present study investigated the autophagy-induced anticancer efficacy of pristimerin on MDA-MB-231 human breast cancer cells. Pristimerin inhibited the growth of these cells in a concentration-dependent manner. Treatment with pristimerin dose-dependently induced an increase of light chain 3B (LC3-II), whereas autophagy inhibitor 3-methyladenine (3-MA) inhibited pristimerin-induced LC3-II accumulation and cytotoxic effects. Autophagy was also activated by paclitaxel as observed by an elevated LC3-II level. Although 24 µM paclitaxel induced autophagy without cytotoxicity, combined with pristimerin it additively induced cell growth inhibition and autophagy induction. Autophagy induction was measured with an autophagy detection kit and LC3-II levels were monitored by western blot analysis. Treatment with 3-MA inhibited LC3-II accumulation and cell death induced by a combination of paclitaxel and pristimerin. Pristimerin and paclitaxel inhibited extracellular signal-regulated kinase (ERK)1/2/p90RSK signaling, consistent with autophagy indicators, namely p62 degradation and beclin 1 expression. In addition, ERK activator ceramide C6 treatment suppressed the LC3-II levels induced by a combination of paclitaxel and pristimerin. These results suggested that exposure to pristimerin induced autophagic cell death, whereas a combination treatment of pristimerin and paclitaxel resulted in an additive effect on ERK-dependent autophagic cell death.

A natural ent‑kaurane diterpenoid induces antiproliferation in ovarian cancer cells via ERK1/2 regulation and inhibition of cellular migration and invasion

Ovarian cancer is one of the most common causes of female mortalities from gynecological tumors. An ent-kaurane diterpenoid compound CRT1 (ent−18-acetoxy-7β-hydroxy kaur-15-oxo-16-ene), mainly isolated from the Vietnamese herb Croton tonkinesis has been used in folk medicine in Vietnam for cancer treatment. However, the effect of this compound on human ovarian cancer cells has not yet been reported. The objective of the present study was to determine the effect of CRT1 on the cell viability, apoptosis and metastasis of SKOV3 human ovarian cancer cells using a Cell Counting Kit-8 assay, flow cytometric analysis of Annexin V-fluorescein isothiocyanate/propidium iodide staining, western blot analysis, soft agar colony forming assay, wound healing assay and Matrigel invasion assay. The results revealed that CRT1 possessed significant anti-proliferative effects on SKOV3 cells. CRT1 treatment at 25 and 50 µM induced apoptosis, enhanced the percentage of Annexin V-positive cells, increased the expression of pro-apoptotic protein B-cell lymphoma 2 (Bcl-2)-associated X protein, cytochrome c release from the mitochondria to the cytosol, cleaved caspase-3, caspase-7, caspase-9, and poly (adenosine diphosphate-ribose) polymerase. However, it decreased the expression of Bcl-2 in a dose-dependent manner. The percentage of necrotic cells increased following CRT1 treatment at <10 µM. CRT1 at 50 µM significantly induced the phosphorylation of extracellular signal-regulated kinase (ERK). Growth inhibition and the apoptotic effects of CRT1 could be reversed by PD98059, an ERK inhibitor. Additionally, CRT1 inhibited cell migration and invasion via ERK1/2 activation in SKOV3 cells. These results indicated that CRT1, an ent-kaurane diterpenoid, may be a potential inhibitor of ovarian cancer by the activating ERK1/2/p90 ribosomal S6 kinase signaling pathway.

Corosolic acid reduces 5‑FU chemoresistance in human gastric cancer cells by activating AMPK

5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutic agents for gastric cancer. Resistance to 5-FU-based chemotherapy remains the major obstacle in the treatment of gastric cancer. A growing body of evidence has suggested that adenosine monophosphate-activated protein kinase (AMPK) is pivotal for chemoresistance. However, the mechanism by which AMPK regulates the chemosensitivity of gastric cancer remains unclear. In the present study, how corosolic acid enhanced the chemosensitivity of gastric cancer cells to 5-FU via AMPK activation was investigated. A 5-FU-resistant gastric cancer cell line (SNU-620/5-FUR) was established, which had a marked increase in thymidine synthase (TS) expression but reduced AMPK phosphorylation when compared with the parental cell line, SNU-620. AMPK regulation by 5-aminoimidazole-4-carboxamide ribonucleotide or compound c was revealed to be markedly associated with TS expression and 5-FU-resistant cell viability. In addition, corosolic acid activated AMPK, and decreased TS expression and the phosphorylation of mammalian target of rapamycin/4E-binding protein 1 in a dose-dependent manner. Corosolic acid treatment significantly reduced cell viability while compound c reversed corosolic acid-induced cell growth inhibition. The 5-FU-resistance sensitization effect of corosolic acid was determined by the synergistic reduction of TS expression and inhibition of cell viability in the presence of 5-FU. The corosolic acid-induced AMPK activation was markedly increased by additional 5-FU treatment, while compound c reversed AMPK phosphorylation. In addition, compound c treatment reversed corosolic acid-induced apoptotic markers such as capase-3 and PARP cleavage, and cytochrome c translocation to cytosol, in the presence of 5-FU. Corosolic acid treatment in the presence of 5-FU induced an increase in the apoptotic cell population based on flow cytometry analysis. This increase was abolished by compound c. In conclusion, these results implied that corosolic acid may have therapeutic potential to sensitize the resistance of gastric cancer to 5-FU by activating AMPK.