Sheng-Teng Huang

Phyllanthus urinaria triggers the apoptosis and Bcl-2 down-regulation in Lewis lung carcinoma cells

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect in its water extract for the first time. The water extract of P. urinaria significantly decreased the number of Lewis lung carcinoma cells in a dose-and time-dependent manner as determined by MTT assay. However, the water extract of P. urinaria did not exert any cytotoxic effect on normal cells such as endothelial cells and liver cells. Result from flow cytometry revealed a dose-dependent increase of dead cells 24 hours after treating Lewis lung carcinoma cells with P. urinaria extract. The anticancer activity of P. urinaria extract was due to the apoptosis induced in Lewis lung carcinoma cells, which was demonstrated by DNA fragmentation analysis and increased caspase-3 activity. The apoptosis triggered by P. urinaria extract in Lewis lung carcinoma cells was associated with the down-regulation of Bcl-2 gene expression, but not with p53, p21 and Bax. Furthermore, the partial inhibition of P. urinaria-induced apoptosis in Lewis lung carcinoma cells by pretreatment with cyclosporin A, a mitochondria permeability transition pore inhibitor, suggesting that P. urinaria extract induced the apoptosis of Lewis lung carcinoma cells, at least in part, through a mitochondria-associated intrinsic pathway.

Wogonin, an active compound in Scutellaria baicalensis, induces apoptosis and reduces telomerase activity in the HL-60 leukemia cells.

Crude extract of Scutellaria baicalensis (S. baicalensis) has cytotoxic effect on human myelogenous leukemia cells (HL-60). We invesigated which compound from the crude extract is responsible for the cytotoxic effect on HL-60 cells. We identified 29 compounds from the crude extract using high performance liquid chromatography mass spectrometry (HPLC/MS). Two of the compounds, baicalin and wogonoside, are converted to baicalein and wogonin, respectively, after treatment with beta-glucuronidase. We observed a dose-dependent reduction in cell viability when cells with either wogonin or aqueous extract of S. baicalensis. Several of the apoptotic features including deoxyribonucleic acid (DNA) fragmentation and increased caspase-3 activity were found in cells treated with wogonin and aqueous extract. The changes were associated with down-regulation of Bcl-2, and not Bax. Furthermore, treatment of HL-60 cells with wogonin or S. baicalensis led to the inhibition of human telomerase reverse transcriptase (hTERT), human telomerase-associated protein 1 (hTP1) and c-myc messenger ribonucleic acid (m-RNA) expression. Wogonin and S. baicaleisis down-regulated the telomerase activity. Our findings suggest that wogonin may be the major compound in S. baicalensis responsible for HL-60 growth inhibition in vitro. The inhibition of HL-60 cell growth is mediated partly through the induction of Bax/Bcl-2 apoptosis and by telomerase inhibition through suppression of c-myc, which is a promoter of hTERT.

The anti-atherosclerotic effect of tanshinone IIA is associated with the inhibition of TNF-α-induced VCAM-1, ICAM-1 and CX3CL1 expression.

Tanshinone IIA is one of the major diterpenes in Salvia miltiorrhiza. The inhibitory effect of Tanshinone IIA on atherosclerosis has been reported, but the underlying mechanism is not fully understood. The present study aimed to study the anti-atherosclerosis effect of Tanshinone IIA on the adhesion of monocytes to vascular endothelial cells and related mechanism. Results showed that Tanshinone IIA, at the concentrations without cytotoxic effect, dose-dependently inhibited the adhesion of THP-1 monocytes to the TNF-α-stimulated human vascular endothelial cells. The expressions of cell adhesion molecules including VCAM-1, ICAM-1 and E-selectin were induced by TNF-α in HUVECs at both the mRNA and protein levels. The mRNA and protein expressions of VCAM-1 and ICAM-1, but not E-selectin, were both significantly suppressed by Tanshinone IIA in a dose dependent manner. In addition, the TNF-α-induced mRNA expression of fractalkine/CX3CL1 and the level of soluble fractalkine were both reduced by Tanshinone IIA. We also found that Tanshinone IIA significantly inhibited TNF-α-induced nuclear translocation of NF-κB which was resulted from the inhibitory effect of Tanshinone IIA on the TNF-α-activated phosphorylation of IKKα, IKKβ, IκB and NF-κB. As one of the major components of Salvia miltiorrhiza, Tanshinone IIA alone exerted more potent effect on inhibiting the adhesion of monocytes to vascular endothelial cells when compared with Salvia miltiorrhiza. All together, these results demonstrate a novel underlying mechanism for the anti-inflammatory effect of Tanshinone IIA by modulating TNF-α-induced expression of VCAM-1, ICAM-1 and fractalkine through inhibition of TNF-α-induced activation of IKK/NF-κB signaling pathway in human vascular endothelial cells.

Anti-angiogenic effect of Tanshinone IIA involves inhibition of matrix invasion and modification of MMP-2/TIMP-2 secretion in vascular endothelial cells

Tanshinone IIA (Tan IIA) is one of the major lipophilic components of Salvia miltiorrhiza Bunge reported to exhibit anti-carcinogenic effect. In the present study, we further evaluated the anti-angiogenic effect of Tan IIA using the chorioallantoic membrane (CAM) in chicken embryos and human umbilical vascular endothelial cells (HUVECs). Tan IIA was confirmed to inhibit in vivo angiogenesis by CAM assay. Tan IIA also exhibited in vitro anti-angiogenic effects as demonstrated by tube formation assay, transwell migration assay and TNF-α-induced matrix invasion assay. The mRNA expressions of matrix metalloproteinase-2, -3, -9, -14 (MMP-2, -3, -9, -14), tissue inhibitor of metalloproteinase-2 (TIMP-2) and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were not affected by Tan IIA as analyzed by reverse transcription polymerase chain reaction (RT-PCR). However, the extracellular matrix metalloproteinase-2 (MMP-2) activity was found to be reduced dose-dependently by Tan IIA as determined by gelatin zymography. Results from western blot analysis and ELISA further demonstrated the dose-dependent decrease of MMP-2 and increase of TIMP-2 secretion from cytosol of vascular endothelial cells simultaneously after Tan IIA treatment. Together, the present study confirmed the anti-angiogenic effects of Tan IIA both in vivo and in vitro. Our results also demonstrated that Tan IIA could modulate the secretion of MMP-2 and TIMP-2 in an opposite way and resulted in the decreased MMP-2 activity of vascular endothelial cells.

Ellagic Acid, the Active Compound of Phyllanthus urinaria, Exerts In Vivo Anti-Angiogenic Effect and Inhibits MMP-2 Activity

This study aimed to assess the potential anti-angiogenic mechanism of Phyllanthus urinaria (P. urinaria) and characterize the major compound in P. urinaria that exerts anti-angiogenic effect. The water extract of P. urinaria and Ellagic Acid were used to evaluate the anti-angiogenic effect in chorioallantoic membrane (CAM) in chicken embryo and human vascular endothelial cells (HUVECs). The matrix metalloproteinase-2 (MMP-2) activity was determined by gelatin zymography. The mRNA expressions of MMP-2, MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Level of MMP-2 proteins in conditioned medium or cytosol was determined by western blot analysis. We confirmed that P. urinarias in vivo anti-angiogenic effect was associated with a reduction in MMP-2 activity. Ellagic acid, one of the major polyphenolic components as identified in P. urinaria by high performance liquid chromatography mass spectrometry (HPLC/MS), exhibited the same anti-angiogenic effect in vivo. Both P. urinaria and Ellagic Acid inhibited MMP-2 activity in HUVECs with unchanged mRNA level. The mRNA expression levels of MMP-14 and TIMP-2 were not altered either. Results from comparing the change of MMP-2 protein levels in conditioned medium and cytosol of HUVECs after the P. urinaria or Ellagic Acid treatment revealed an inhibitory effect on the secretion of MMP-2 protein. This study concluded that Ellagic Acid is the active compound in P. urinaria to exhibit anti-angiogenic activity and to inhibit the secretion of MMP-2 protein from HUVECs.

Zinc-Chelation Contributes to the Anti-Angiogenic Effect of Ellagic Acid on Inhibiting MMP-2 Activity, Cell Migration and Tube Formation

Background Ellagic acid (EA), a dietary polyphenolic compound, has been demonstrated to exert anti-angiogenic effect but the detailed mechanism is not yet fully understood. The aim of this study was to investigate whether the zinc chelating activity of EA contributed to its anti-angiogenic effect. Methods and Principal Findings The matrix metalloproteinases-2 (MMP-2) activity, a zinc-required reaction, was directly inhibited by EA as examined by gelatin zymography, which was reversed dose-dependently by adding zinc chloride. In addition, EA was demonstrated to inhibit the secretion of MMP-2 from human umbilical vein endothelial cells (HUVECs) as analyzed by Western blot method, which was also reversed by the addition of zinc chloride. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), known to down-regulate the MMP-2 activity, was induced by EA at both the mRNA and protein levels which was correlated well with the inhibition of MMP-2 activity. Interestingly, zinc chloride could also abolish the increase of EA-induced RECK expression. The anti-angiogenic effect of EA was further confirmed to inhibit matrix-induced tube formation of endothelial cells. The migration of endothelial cells as analyzed by transwell filter assay was suppressed markedly by EA dose-dependently as well. Zinc chloride could reverse these two effects of EA also in a dose-dependent manner. Since magnesium chloride or calcium chloride could not reverse the inhibitory effect of EA, zinc was found to be involved in tube formation and migration of vascular endothelial cells. Conclusions/Significance Together these results demonstrated that the zinc chelation of EA is involved in its anti-angiogenic effects by inhibiting MMP-2 activity, tube formation and cell migration of vascular endothelial cells. The role of zinc was confirmed to be important in the process of angiogenesis.

Phyllanthus urinaria Increases Apoptosis and Reduces Telomerase Activity in Human Nasopharyngeal Carcinoma Cells

Background: This study was designed to obtain the chemical fingerprint and to investigate the effect of Phyllanthus urinaria on telomerase activity and apoptotic pathways in the human nasopharyngeal carcinoma cell line (NPC-BM1). Materials and Methods: The polyphenol compounds in P. urinaria were investigated by HPLC/MS. Cell viability with the treatment of P. urinaria, gallic acid, ellagic acid, quercetin and cisplatin was detected by MTT assay. TUNEL assay, DNA fragmentation analysis and caspase3 activity were used to confirm apoptotic changes. Telomerase activity was determined using the TRAP assay. RNA isolation and RT-PCR were used to analyze the related genes expression. All experiments on treatments with P. urinaria from 0–3 mg/ml were carried out for 24 h. Results: 5 major compounds including gallic acid, brevifolin carboxylic acid, corilagin, phyllanthusiin C and ellagic acid were identified as a plant fingerprint by HPLC/MS. With the MTT assay, we demonstrated that P. urinaria, gallic acid and ellagic acid reduce cell viability. The apoptosis features showed DNA fragmentation and increased caspase-3 activity associated with the down-regulation of Bcl-2, but not of Bax, p53, and PCNA (proliferating cell nuclear antigen) in P. urinaria-treated NPC-BM1 cells. Furthermore, treatment of NPC-BM1 cells led to an inhibition of hTERT (human telomerase reverse transcriptase), hTP1 (human telomerase-associated protein 1) and c-myc mRNA expression and to decreased telomerase activity. Conclusion: This study suggests that P. urinaria induces the death of NPC-BM1 cells in vitro through the induction of apoptosis and inhibited telomerase activity.

Davallia bilabiata Inhibits TNF-α-Induced Adhesion Molecules and Chemokines by Suppressing IKK/NF-kappa B Pathway in Vascular Endothelial Cells

Davallia bilabiata (D. bilabiata) is also called GuSuiBu in Taiwan and is used as a substitute for Drynaria fortunei J. Sm. It is often used for trauma and bone repair. The inhibitory effect of D. bilabiata on inflammatory activity has not been reported. In the present study, we aimed to study the mechanism of anti-inflammation of D. bilabiata on the adhesion of leukocytes to vascular endothelial cells. The results showed that D. bilabiata, at concentrations without cytotoxic effect, inhibited the adhesion of monocytes (THP-1) to the TNF-α-stimulated human umbilical vascular endothelial cells (HUVECs). D. bilabiata suppressed the expression of the adhesion molecules ICAM, VCAM, and E-selectin at both the mRNA and protein level. In addition, both of the TNF-α-induced mRNA and protein expression of chemokines including fractalkine/CX3CL1, MCP-1 and RANTES as well as the level of secreted soluble fractalkine were decreased by D. bilabiata. We also verified that D. bilabiata inhibited the TNF-α-induced nuclear translocation of NF-κB through the inhibitory process on the TNF-α-activated phosphorylation of IKKα, IKKβ, IκB and NF-κB. All together, we concluded that the D. bilabiata affected the canonical pathway of TNF-α-induced NF-κB activation and down-regulated cell adhesion molecules and chemokine expression through inhibition of the NF-κB/IκBα/IKK signaling pathway. These findings strongly indicated that D. bilabiata might be a promising alternative/adjunct treatment for inflammatory diseases, such as rheumatoid arthritis and osteoarthritis.

Inhibition of Melanogenesis in Murine B16/F10 Melanoma Cells by Ligusticum sinensis Oliv.

Ligusticum sinensis Oliv. (LSO) is an herbal drug commonly used as a topical treatment of epidermal hyperdepigmentation in Chinese medicine. However, the mechanism underlying the depigmentation by LSO is still unclear. The purpose of this study was to investigate the effects of LSO on the process of melanogenesis and its possible underlying mechanism. Suppressed DOPA oxidase activity of mushroom tyrosinase was first noted when incubated with aqueous extracts of LSO, demonstrating the direct inhibitory effect of LSO on mushroom tyrosinase. Further experiments were carried out in murine B16/F10 melanoma cells and the effects of LSO extract on melanin formation, tyrosinase activity and tyrosinase gene expression were tested. Under conditions without affecting the viability of murine B16/F10 melanoma cells, LSO extract significantly reduced the cellular melanin content in a dose-dependent manner. The DOPA oxidase activity of tyrosinase in B16/F10 cells was dose-dependently inhibited by LSO treatment, possibly mediated by the suppressed tyrosinase mRNA expression in LSO-treated B16/F10 cells. In conclusion, the inhibitory effect of LSO on melanogenesis is likely associated with decreased DOPA oxidase activity of tyrosinase that is most likely the result of the down-regulation of tyrosinase mRNA expression.

Tanshinone IIA induces intrinsic apoptosis in osteosarcoma cells both in vivo and in vitro associated with mitochondrial dysfunction

Tanshinone IIA (Tan IIA), a phytochemical derived from the roots of Salvia miltiorrhiza, has been shown to inhibit growth and induce apoptosis in various cancer cells. The association of its inhibitory effect on the primary malignant bone tumor, osteosarcoma, with mitochondrial dysfunction remains unclear. This study aimed to investigate the anti-proliferative effects of Tan IIA on human osteosarcoma 143B cells both in vitro and in vivo. Administration of Tan IIA to NOD-SCID mice implanted with 143B cells led to significant inhibition of tumor development. The inhibition of proliferation, migration, and invasion was observed in 143B cells treated with Tan IIA. The tumor proliferation markers, Ki67 and PCNA, were suppressed and apoptosis by TUNEL assay was activated respectively. Apoptosis in the Tan IIA-treated 143B cells and xerograft mice was associated with the activation of caspase cascade via the modulation of Bcl-2 family. The CD31 was inhibited in Tan IIA-treated xenografts to indicate anti-neovasculization. Tan IIA administration resulted in a significant decrease in the mitochondrial fusion proteins, Mfn1/2 and Opa1, as well as an increase in the fission protein Drp1. We concluded that mitochondrial dysfunction associated with dynamic change was involved in apoptosis and anti-angiogenesis elicited by Tan IIA.