Myung-Joo Choi

Suppression by fucoidan of liver fibrogenesis via the TGF-β/Smad pathway in protecting against oxidative stress.

Fucoidan, a sulfated polysaccharide extracted from various types of brown seaweed, possesses a wide range of pharmacological properties. We investigated the protective effect of fucoidan on dimethylnitrosamine-induced liver fibrogenesis in rats and its mechanism. Liver fibrosis was induced by injecting DMN (10 mg/kg, 3 times per week, I.P.) for 4 weeks, and fucoidan was simultaneously administered (100 mg/kg, 3 times per week, P.O.). The anti-oxidative and anti-inflammatory effects of fucoidan were observed by relative mediators. Fucoidan improved liver fibrosis by inhibiting the expression of transforming growth factor beta 1 (TGF-β1)/Smad3 and the tissue inhibitor of metalloproteinase 1 (TIMP-1), and increasing the expression of metalloproteinase-9 (MMP-9). Fucoidan also significantly decreased the accumulation of the extracellular matrix (ECM) and collagen. These results suggest that fucoidan had an anti-fibrotic effect, which was exerted by inhibiting the TGF-β/Smad pathway, as well as anti-oxidative and anti-inflammatory effects.

SB365 inhibits angiogenesis and induces apoptosis of hepatocellular carcinoma through modulation of PI3K/Akt/mTOR signaling pathway

Identification of small molecules that safely inhibit cancer progression is critical for cancer therapeutics. Saponins exhibit cytostatic and cytotoxic activity against various cancer cells, but the mechanism is not well understood. Here, we investigated whether saponin D (designated SB365), an active component isolated from Pulsatilla koreana, could inhibit the progression of hepatocellular carcinoma (HCC) and considered its mechanism. SB365 strongly suppressed the growth of HCC cells in a dose‐dependent manner and induced apoptosis by increasing the proportion of sub G1 apoptotic cells from 8% to 21% through induction of expression of Bax and cleaved caspase‐3. In addition, SB365 exhibited potent anti‐angiogenic activity and decreased the expression of hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor, a key molecule for angiogenesis. Furthermore, SB365 suppressed the tube formation and migration of HUVEC, as well as in vivo neovascularization in a mouse Matrigel plug assay. In vivo study showed that SB365 significantly inhibited tumor growth in an HCC xenograft model, inducing apoptosis by increasing the expression of the cleaved caspase‐3 and DNA fragmentation. The expressions of vascular endothelial growth factor and CD34 in the tumor tissue were decreased by SB365 treatment. In examining its mechanism, SB365 was found to effectively suppress the phosphorylation of PI3K downstream factors, such as Akt, mTOR and p70S6K both in vitro and in vivo. Our study demonstrates that SB365 not only induces apoptosis but also inhibits cell growth and angiogenesis through modulation of the PI3K/Akt/mTOR pathway in human HCC. We suggest that SB365 may be a new chemotherapeutic candidate against HCC.

HS-116, a novel phosphatidylinositol 3-kinase inhibitor induces apoptosis and suppresses angiogenesis of hepatocellular carcinoma through inhibition of the PI3K/AKT/mTOR pathway

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival of human cancers. As PI3K is active in many cancer patients, resulting in cancer development and progression, we developed an azaindole derivative, HS-116 as a novel PI3K inhibitor. This study aimed to clarify the anticancer effect of HS-116 in human hepatocellular carcinoma (HCC). To identify the effect of HS-116 on HCC cells, a PI3K assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blotting were conducted. IC(50) of HS-116 for PI3Kα was 31nM, and it effectively suppressed the phosphorylation of PI3K downstream factors such as AKT, mTOR, p70S6K, and 4EBP1. Also, HS-116 induced apoptosis by increasing the proportion of sub-G1 apoptotic cells from 1.8% to 35% and increasing the expressions of Bax, cleaved-caspase-3, and cleaved-PARP as well as decreasing the expression of Bcl-2. In addition, chromatin condensation and apoptotic bodies were detected in HS-116-treated HCC cells. Furthermore, HS-116 decreased protein expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), and inhibited the tube formation and migration of human umbilical vein endothelial cells (HUVECs). In vivo, the ability of mice to vascularize subcutaneously implanted Matrigel plugs was diminished when the mice were treated with HS-116. These results show that HS-116 inhibits the PI3K/AKT/mTOR pathway via apoptosis and anti-angiogenesis in HCC cells. We suggest that HS-116 may be an effective novel therapeutic candidate against HCC.

SB365, Pulsatilla saponin D suppresses the proliferation of human colon cancer cells and induces apoptosis by modulating the AKT/mTOR signalling pathway

Pulsatilla koreana has been used as a traditional medicine for the treatment of several diseases. The purpose of this study was to determine if SB365, Pulsatilla saponin D isolated from the root of P. koreana inhibits the progression of colon cancer. We found that SB365 strongly suppressed the growth and proliferation of colon cancer cells and induced their apoptosis. Also, SB365 showed anti-angiogenic activity by decreasing the expression of HIF-1α and VEGF. These results were confirmed by an in vivo study showing that SB365 significantly inhibited tumor growth by the induction of apoptosis and inhibition of angiogenesis with stronger anticancer activity than 5-FU. When further examined for its anticancer mechanism, SB365 effectively suppressed the AKT/mTOR pathway both in vitro and in vivo. Taken together, our study demonstrated that SB365 inhibits the AKT/mTOR pathway, leading to the suppression of tumor growth and angiogenesis together with induction of apoptosis. Therefore, SB365 is a good candidate as a natural product for use in the treatment of colon cancer.

Anti-cancer effects of a novel compound HS-113 on cell growth, apoptosis, and angiogenesis in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common malignancies, yet there have been no significant advances in effective therapeutics. In this study, HS-113 was synthesized as a novel compound, N-(5-(2-bromobenzyl) thiazole-2-yl) benzofuran-2-carboxamide and its cytotoxic activity and anti-cancer effect were examined in human HCC cells. HS-113 strongly suppressed growth of HCC cells in a dose-dependent manner, induced apoptosis by increasing the proportion of sub-G1 apoptotic cells, and caused cell cycle arrest at G0/G1 phase. Also, HS-113 increased the expression of p27 and decreased that of cyclin D1 associated with cell cycle arrest. Apoptosis by HS-113 was confirmed by DAPI and TUNEL staining, and the increases of the cleaved PARP and caspase-3 were observed. Furthermore, HS-113 decreased protein expression of HIF-1α and secretion of VEGF, and inhibited the tube formation of HUVECs. These results showed that HS-113 not only inhibited cell growth and angiogenesis, but also induced apoptosis of human HCC cells. We suggest that HS-113 may be a potential candidate for cancer therapy against HCC.

KRC-408, a novel c-Met inhibitor, suppresses cell proliferation and angiogenesis of gastric cancer

Among many cancer therapeutic targets, c-Met receptor tyrosine kinase has recently given particular attention. This kinase and its ligand, hepatocyte growth factor (HGF), play a central role in cell proliferation and the survival of several human cancers. Thus, we developed KRC-408 as a novel c-Met inhibitor and investigated its anti-cancer effects on human gastric cancer. KRC-408 inhibited the phosphorylation of c-Met and its constitutive downstream effectors such as phosphatidylinositol 3-kinase (PI3K), Akt, Mek, and Erk. This compound was found to exert anti-cancer effects stronger than those of 5-fluorouracil (5-FU) on gastric cancer cells, especially cell lines that overexpressed c-Met. Interestingly, cytotoxicity of KRC-408 was lower than that of 5-FU in normal gastric cells. Apoptosis induced by KRC-408 was accompanied by increased levels of cleaved caspase-3 and PARP as well as DNA condensation and fragmentation. Flow cytometry analysis showed an accumulation of gastric cancer cells in the G2/M phase with concomitant loss of cells in the S phase following treatment with this drug. In the angiogenesis studies, KRC-408 inhibited tube formation and migration of human umbilical vein endothelial cells (HUVECs), and suppressed microvessel sprouting from rat aortic rings ex vivo along with blood vessel formation in a Matrigel plug assay in mice. Results of an in vivo mouse xenograft experiment showed that the administration of KRC-408 significantly delayed tumor growth in a dose-dependent manner, and suppressed Akt and Erk phosphorylation as well CD34 expression in tumor tissues. These findings indicate that KCR-408 may exert anti-tumor effects by directly affecting tumor cell growth or survival via the c-Met receptor tyrosine kinase pathway. We therefore suggest that KRC-408 is a novel therapeutic candidate effective against gastric cancers that overexpress c-Met.

An Effective Assessment of Simvastatin-Induced Toxicity with NMR-Based Metabonomics Approach

Background Simvastatin, which is used to control elevated cholesterol levels, is one of the most widely prescribed drugs. However, a daily excessive dose can induce drug-toxicity, especially in muscle and liver. Current markers for toxicity reflect mostly the late stages of tissue damage; thus, more efficient methods of toxicity evaluation are desired. Methodology/Principal Findings As a new way to evaluate toxicity, we performed NMR-based metabonomics analysis of urine samples. Compared to conventional markers, such as AST, ALT, and CK, the urine metabolic profile provided clearer distinction between the pre- and post-treatment groups treated with toxic levels of simvastatin. Through multivariate statistical analysis, we identified marker metabolites associated with the toxicity. Importantly, we observed that the treatment group could be further categorized into two subgroups based on the NMR profiles: weak toxicity (WT) and high toxicity (HT). The distinction between these two groups was confirmed by the enzyme values and histopathological exams. Time-dependent studies showed that the toxicity at 10 days could be reliably predicted from the metabolic profiles at 6 days. Conclusions/Significance This metabonomics approach may provide a non-invasive and effective way to evaluate the simvastatin-induced toxicity in a manner that can complement current measures. The approach is expected to find broader application in other drug-induced toxicity assessments.

Suppression of tumor proliferation and angiogenesis of hepatocellular carcinoma by HS-104, a novel phosphoinositide 3-kinase inhibitor

Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway frequently instigates tumorigenesis leading to hepatocellular carcinoma (HCC). We synthesized N-(5-(3-(3-methyl-1,2,4-oxadiazol-3-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (HS-104), a novel PI3K inhibitor, and investigated its in vitro anticancer effect and in vivo capacity in an animal xenograft model. The inhibition of cell growth by HS-104 revealed that it was effective against HCC cell lines. Also, the activation of the AKT/mTOR signal cascade was inhibited by HS-104 treatment in a dose dependent manner. Flow cytometry analysis showed an accumulation of HCC cells in the G2/M phase with concomitant loss of cells in the S phase. The apoptotic effect of HS-104 was accompanied by increased evidence of cleaved caspase-3 and PARP, as well as DNA fragmentation. In angiogenesis studies, HS-104 inhibited the tube formation of vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs), and suppressed microvessel sprouting from a rat aortic ring, ex vivo, and blood vessel formation in the Matrigel plug assay in mice. HS-104 inhibited the expression of the downstream proteins of PI3K including p-AKT, p-mTOR and p-p70S6K in VEGF-induced HUVECs. In the xenograft animal model, HS-104 significantly delayed tumor growth in a dose dependent manner and suppressed the expression of PCNA, CD34 and cleaved caspase-3 in tumor tissue. These studies show that HS-104 inhibited the PI3K/AKT/mTOR signaling pathway resulting in cell growth/angiogenesis inhibition and apoptosis induction. Therefore, HS-104 is considered as a novel drug candidate for the treatment of HCC.

Aberrant expression of Wnt family contributes to the pathogenesis of diabetes‐induced erectile dysfunction

Diabetic erectile dysfunction (ED) has multiple causative factors, such as endothelial and smooth muscle dysfunction and cavernous fibrosis. Wnt signalling is essential for normal embryonic development and for tissue homeostasis in adults. Aberrant activation of Wnt family members has been implicated in tissue fibrosis and in angiogenesis. In this study, we investigated the differential expression of Wnts in the penises of mice with streptozotocin‐induced diabetic ED. We also examined the effect of transforming growth factor‐β1 (TGF‐β1) on the expression of Wnts in primary cultured fibroblasts isolated from human tunica albuginea. Among the mouse and human Wnts tested, 16 mouse Wnts and 14 human Wnts were detected in the corpus cavernosum tissue of normal mice and in fibroblasts derived from human tunica albuginea respectively. We observed up‐regulation of Wnt10b (known to be involved in tissue fibrosis) and down‐regulation of Wnt16 (known to be involved in vasculogenesis and hematopoiesis), both in the diabetic condition in vivo and with treatment of fibroblasts with TGF‐β1 in vitro. Wnt10b was mainly expressed in fibroblasts and Wnt16 was colocalized with smooth muscle cells in the corpus cavernosum tissue. Cavernous TGF‐β1 protein expression and the degree of cavernous fibrosis determined by the ratio of collagen to smooth muscle content were significantly higher in diabetic mice than in controls. Cavernous endothelial content was significantly decreased by the diabetic condition. Overexpression of Wnt16 with plasmid vector accelerated tube formation in primary cultured mouse cavernous endothelial cells. However, down‐regulation of Wnt10b with small interfering RNA did not decrease the production of extracellular matrix protein in human fibroblasts. This is the first report demonstrating the differential expression of Wnts in diabetic mouse penis. Aberrant Wnt expression might contribute to the pathogenesis of ED.

A guanidinylated bioreducible polymer as a novel gene carrier to the corpus cavernosum of mice with high-cholesterol diet-induced erectile dysfunction

A prerequisite for the successful clinical application of gene therapy in erectile dysfunction (ED) is the availability of safe and efficient gene delivery systems. The aim of this study was to examine the effectiveness of guanidinylated bioreducible polymer (GBP) polyplexes for gene delivery systems, which take advantage of the biodegradability of reducible disulfide bonds and the cell‐penetrating ability of guanidine groups. For in vitro transfection experiments, we used mouse cavernous endothelial cells and A7r5 rat vascular smooth muscle cells. For in vivo experiments, we used a mouse model of hypercholesterolaemic ED in which 2‐month‐old male C57BL/6 mice were fed a diet containing 4% cholesterol and 1% cholic acid for 3 months. Animals or cells were treated with pCMV‐Luc, poly(ethyleneimine) (PEI)25k/pCMV‐Luc polyplex (weight ratio: 1) and GBP/pCMV‐Luc polyplexes (weight ratio: 20, 40, 60 and 80). Gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. GBP had greater transfection efficiency as the weight ratio increased. GBP had sevenfold higher gene delivery efficiency in A7r5 cells at a weight ratio of 80 than did PEI25k. Moreover, the gene expression was more profoundly induced by GBP/pCMV‐Luc than by pCMV‐Luc in both the corpus cavernosum tissue of hypercholesterolaemic mice and in mouse cavernous endothelial cells, although the expression levels induced by the GBP gene delivery system were lower than those induced by the PEI25k gene delivery system. GBP revealed no considerable cytotoxicity to A7r5 cells and mouse cavernous endothelial cells (relative cell viability: 95 and 88% respectively), whereas PEI25k resulted in high cytotoxicity. Interestingly, immunofluorescent double staining revealed that luciferase expression induced by the GBP polyplex mainly overlapped with cavernous endothelial cells, but rarely with smooth muscle cells. The GBP‐based non‐viral gene expression system may be useful for the development of gene therapy in vasculogenic ED.