Yung Wei Chiu

Baicalein inhibits the migration and invasive properties of human hepatoma cells

Flavonoids have been demonstrated to exert health benefits in humans. We investigated whether the flavonoid baicalein would inhibit the adhesion, migration, invasion, and growth of human hepatoma cell lines, and we also investigated its mechanism of action. The separate effects of baicalein and baicalin on the viability of HA22T/VGH and SK-Hep1 cells were investigated for 24h. To evaluate their invasive properties, cells were incubated on matrigel-coated transwell membranes in the presence or absence of baicalein. We examined the effect of baicalein on the adhesion of cells, on the activation of matrix metalloproteinases (MMPs), protein kinase C (PKC), and p38 mitogen-activated protein kinase (MAPK), and on tumor growth in vivo. We observed that baicalein suppresses hepatoma cell growth by 55%, baicalein-treated cells showed lower levels of migration than untreated cells, and cell invasion was significantly reduced to 28%. Incubation of hepatoma cells with baicalein also significantly inhibited cell adhesion to matrigel, collagen I, and gelatin-coated substrate. Baicalein also decreased the gelatinolytic activities of the matrix metalloproteinases MMP-2, MMP-9, and uPA, decreased p50 and p65 nuclear translocation, and decreased phosphorylated I-kappa-B (IKB)-β. In addition, baicalein reduced the phosphorylation levels of PKCα and p38 proteins, which regulate invasion in poorly differentiated hepatoma cells. Finally, when SK-Hep1 cells were grown as xenografts in nude mice, intraperitoneal (i.p.) injection of baicalein induced a significant dose-dependent decrease in tumor growth. These results demonstrate the anticancer properties of baicalein, which include the inhibition of adhesion, invasion, migration, and proliferation of human hepatoma cells in vivo.

Herbal Supplement Ameliorates Cardiac Hypertrophy in Rats with CCl 4 -Induced Liver Cirrhosis

We used the carbon tetrachloride (CCl4) induced liver cirrhosis model to test the molecular mechanism of action involved in cirrhosis-associated cardiac hypertrophy and the effectiveness of Ocimum gratissimum extract (OGE) and silymarin against cardiac hypertrophy. We treated male wistar rats with CCl4 and either OGE (0.02 g/kg B.W. or 0.04 g/kg B.W.) or silymarin (0.2 g/kg B.W.). Cardiac eccentric hypertrophy was induced by CCl4 along with cirrhosis and increased expression of cardiac hypertrophy related genes NFAT, TAGA4, and NBP, and the interleukin-6 (IL-6) signaling pathway related genes MEK5, ERK5, JAK, and STAT3. OGE or silymarin co-treatment attenuated CCl4-induced cardiac abnormalities, and lowered expression of genes which were elevated by this hepatotoxin. Our results suggest that the IL-6 signaling pathway may be related to CCl4-induced cardiac hypertrophy. OGE and silymarin were able to lower liver fibrosis, which reduces the chance of cardiac hypertrophy perhaps by lowering the expressions of IL-6 signaling pathway related genes. We conclude that treatment of cirrhosis using herbal supplements is a viable option for protecting cardiac tissues against cirrhosis-related cardiac hypertrophy.

Expression of Protein Kinase C α and the MZF-1 and Elk-1 Transcription Factors in Human Breast Cancer Cells

Recently, our research into hepatocellular carcinoma (HCC) has shown that the transcription factors Myeloid Zinc Finger-1 (MZF-1) and Ets-like-protein 1 are related to protein kinase C alpha (PKCα) expression. The purpose of this study was to determine the correlation of the expression of PKCαwith the expressions of Elk-1 and MZF-1 in various differentiated breast cancer cell lines: MDA- MB-231, Hs57BT, SKBR3, MDA-MB-468 and MCF-7. The malignant potential in the five lines of breast cancer cells was examined by using a cell proliferation/migration/invasion assay and the protein and mRNA levels of PKCα, ElK-1 and MZF-1 were examined by Western blot and RT-PCR analysis, re- spectively. The results showed that there were obvious signs of migration and invasion of cells in MDA- MB-231 and Hs57BT cells, little signs of cell migration and invasion in MDA-MB-468 cells, and no sign in SKBR3 and MCF-7 cells. Moreover, the highest expression levels of PKCα, Elk-1 and MZF-1 were also observed in MDA-MB-231 and Hs57BT cells when compared to the other breast cancer cell lines. These findings confirm that elevated expression of PKCαin breast cancer cells may be correlated with the potential of cell migration and invasion, and suggest an association between the expression of PKCα and the expression of the transcription factors Elk-1 and MZF-1.

Herbal Supplement Attenuation of Cardiac Fibrosis in Rats with CCl4-Induced Liver Cirrhosis

Previously we found carbon tetrachloride (CCl₄) induced cirrhosis associated cardiac hypertrophy and apoptosis. The purpose of this study is to determine whether further CCl₄ treatment would induce cardiac cell fibrosis. The cardiac tissues were analyzed by H&E. histological staining, Trichrome Masson staining and Western blotting. The results showed that the CCl₄-treated-only group exhibits more trichrome staining, meaning that more fibrosis is present. Moreover, CCl₄ could further induce cardiac-fibrosis via TGF-β-p-Smad2/3-CTGF pathway. However, our data showed that the CCl₄- indcued cardiac abnormalities were attenuated by Ocimum gratissimum extract (OGE) and silymarin co- treatments. In conclusion, our results indicated that the OGE and silymarin may be a potential traditional herb for the protection of cardiac tissues from the CCl4 induced cirrhosis associated cardiac fibrosis through modulating the TGF-β signaling pathway.

Expression of protein kinase Cα and the MZF-1 and elk-1 transcription factors in human bladder transitional cell carcinoma cells.

In a recent study on hepatocellular carcinoma (HCC), we have shown that the transcription factors Myeloid Zinc Finger-1 (MZF-1) and Ets-like-protein 1 (Elk-1) are significantly related to protein kinase C alpha (PKCα) expression. The purpose of this study was to determine the correlation of the expression of PKCα with the expression of Elk-1 and MZF-1 in various differentiated urinary bladder transitional cell carcinoma (TCC) cell lines: 5637, BFTC905, TSGH8301, HT1376 and HT1197 cells. The malignant potential in the five TCC cell lines was examined by using cell proliferation/migration/invasion assay and the protein and mRNA levels of PKCα, ElK-1 and MZF-1 were examined by Western blot and RT-PCR analysis. The results showed that the rate of cell proliferation in the TSGH8301 cell line was higher than that in other cell lines, while there were obvious signs of cell migration and invasion in 5637, BFTC905 and HT1376 cells, and no sign in TSGH8301 and HT1197 cells. The resulting expression levels of Elk-1 and PKCα were the highest in 5637 cells, but the MZF-1 expression observed in all five cell lines showed no significant difference. To determine whether a correlation exists between PKCα and Elk-1, a shRNA knockout assay was performed and the results showed that the reduction of Elk-1 expression in 5637 cells did not result in the decreased PKCα expression. Therefore, although the findings showed elevated expression of Elk-1 and PKCα in 5637 cells, the regulator of PKCα in bladder cancer cells is yet to be determined.

Ocimum gratissimum is Effective in Prevention against Liver Fibrosis in Vivo and in Vitro

Ocimum gratissimum is a traditional herb commonly found in tropical regions, which prevents free radical damage and protects the liver from oxidative stress. In this study, we tested in vivo and in vitro the effectiveness of O. gratissimum extracts (OGEs) in anti-hepatic fibrosis in rats. Male Wistar rats were administered with carbon tetrachloride (CCl4) by intraperitoneal injection and varying amounts of oral injection of OGE doses (0-40 mg/kg body weight) for 8 weeks. Our experiments showed that OGE significantly reduced liver damage, including steatosis and fibrosis, in a dose-dependent manner, as well as significantly decreased the elevation in plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT). It also inhibited the formation of lipid peroxidative products during CCl4 treatment. Moreover, OGE-inhibited CCl4-induced liver collagen accumulation and promoted the expression of catalase, an anti-oxidative enzyme. The inhibition of fibrosis factors α-SMA expression was also observed. In primary cultures, OGE significantly inhibited the serum-induced activation of hepatic stellate cells (HSCs), and the expression of α-SMA and collagen α (I). These data suggest that O. gratissimum possesses anti-hepatic fibrosis properties via its anti-oxidative components.

MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells

Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160–72 or Elk-1145–157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial–mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain.

Ocimum gratissmum aqueous extract reduces plasma lipid in hypercholesterol-fed hamsters

Objectives: Hyperlipidemia is a significant risk factor in the development of atherosclerosis and related diseases which are major health problem in many developed and developing countries that can lead to fatality due to the changes in lifestyle and dietary habits in this modern age. Methods: In the present study, the Ocimum gratissimum aqueous extract (OGE) was tested for the lowering effect on the serum lipid level of male hamsters on a high-fat (12%) and high-cholesterol (0.2%) diet (HFCD). Results: The results showed that the levels of serum high-density-lipoprotein-cholesterol (HDL-C) low-density-lipoprotein-cholesterol (LDL-C), total cholesterol (TC), and triglycerols (TG) were increased in the HFCD group (113±11, 259±87, 629±175 and 625±262, respectively), as compared to the control normal diet group (51±8, 19±5, 77±16 and 101±44, respectively). When co-treated with various doses (10 and 20 mg/kg) of the OGE or rosuvastatin, the rats exhibited the restoration of normal serum LDL-C, TC, and TG levels. Conclusion: Therefore, we suggest that the Ocimum gratissimum aqueous extract may have the potential function of lowering serum lipid in rats.

Protective Effects of Ocimum gratissimum Polyphenol Extract on Carbon Tetrachloride-Induced Liver Fibrosis in Rats

Ocimum gratissimum found in tropical regions is a traditional herb commonly which prevents free radical damage and protects liver from oxidative stress and fibrosis. Ocimum gratissimum polyphenol extract (OGPE) was purified by resin tube to 33.24% polyphenol and 8.2% flavonoid, which were three-fold higher compared with the pre-purification concentrations. The abstract was used to determine if the antioxidant components in the O. gratissimum extract (OGE) were responsible for protective effects on liver fibrosis. High-performance liquid chromatography analysis revealed that the content levels of catechin, caffeic acid and epicatechin in OGPE also increased three-fold. Male Wistar rats were administered with carbon tetrachloride (CCl₄) and varying amounts of OGPE doses [0-12 mg/kg body weight (BW)] or OGE dose (40 mg/kg BW) for 8 weeks. Results showed that OGPE at 12 mg/kg BW, similar to OGE at 40 mg/kg BW, maintained the liver weight, significantly ameliorated CCl₄-induced steatosis, and mitigated other pathological changes. OGPE (12 mg/kg BW) also maintained the levels of serum alanine aminotransferase and aspartate aminotransferase, as well as the levels of malondialdehyde, catalase and α-smooth muscle actin in liver tissues from CCl₄-induced changes. These findings suggest that antioxidant components in OGPE were the major factors that prevented liver fibrosis. Moreover, higher polyphenol concentrations were necessary for higher effectiveness.

Amelioration of estrogen-deficiency-induced obesity by Ocimum gratissimum

Objectives: Menopausal transition in women initiates with declining estrogen levels and is followed by significant changes in their physiological characteristics. These changes often lead to medical conditions, such as obesity, which is correlated with chronic low-grade/subclinical inflammation. Ocimum gratissimum L. is a food spice or traditional herb in many countries; the plant is rich in antioxidants, which possess anti-inflammation activities and multitude of other therapeutic functions. Methods: In this study, we evaluated effects of O. gratissimum extract (OGE) in preventing obesity by using ovariectomized (OVX) animal models to mimic menopausal women. Methods: OVX rats showed increase in body weight and in adipocyte size in perigonadal adipose tissue (p <0.05) and decrease in uterus weight. By contrast, OGE (0.2 mg/ml) significantly reduced body weight gain and adipocyte in OVX rats and showed insignificant changes in uterus weight. Further investigation indicated that OGE exerted no influence on levels of dorsal fat, serum total cholesterol, and serum triacylglycerol and on serum biochemical factors, calcium, phosphorus, and glucose. Conclusion: These findings suggested that OGE dietary supplements may be useful in controlling body weight of menopausal women.