Quantifying Cell Confluency by Plasmonic Nanodot Arrays to Achieve Cultivating Consistency.

The determination of cell confluency and subculture timing for cell culture consistency is crucial in the field of cell-based research, but there is no universal standard concerning optimal confluence. In this study, gold nanodot arrays on glass substrates were used as culture substrates, and their spectral shifts of localized surface plasmon resonance (LSPR) were employed to monitor cell growth and quantify cell confluency. Experiments including cell counting, metabolic activity, focal adhesion, and cell cycle were also performed to confirm the cell growth monitoring accuracy of the LSPR signals. The LSPR signal exhibited the same trends like the increase of cell numbers and cell metabolic activity and reached the maximum as the cell growth achieved confluency, suggesting its great capability as an effective indicator to predict suitable subculture timing. The proposed sensing approach is a noninterventional, nondestructive, real-time, and useful tool to help biologists quantify the optimal subculture timing, achieve cell culture consistency, and obtain reproducible experimental results efficiently.

Anti-Cancer Effects of Protein Extracts from Calvatia lilacina, Pleurotus ostreatus and Volvariella volvacea.

Calvatia lilacina (CL), Pleurotus ostreatus (PO) and Volvariella volvacea (VV) are widely distributed worldwide and commonly eaten as mushrooms. In this study, cell viabilities were evaluated for a human colorectal adenocarcinoma cell line (SW480 cells) and a human monocytic leukemia cell line (THP-1 cells). Apoptotic mechanisms induced by the protein extracts of PO and VV were evaluated for SW480 cells. The viabilities of THP-1 and SW480 cells decreased in a concentration-dependent manner after 24 h of treatment with the protein extracts of CL, PO or VV. Apoptosis analysis revealed that the percentage of SW480 cells in the SubG(1) phase (a marker of apoptosis) was increased upon PO and VV protein-extract treatments, indicating that oligonucleosomal DNA fragmentation existed concomitantly with cellular death. The PO and VV protein extracts induced reactive oxygen species (ROS) production, glutathione (GSH) depletion and mitochondrial transmembrane potential (ΔΨ(m)) loss in SW480 cells. Pretreatment with N-acetylcysteine, GSH or cyclosporine A partially prevented the apoptosis induced by PO protein extracts, but not that induced by VV extracts, in SW480 cells. The protein extracts of CL, PO and VV exhibited therapeutic efficacy against human colorectal adenocarcinoma cells and human monocytic leukemia cells. The PO protein extracts induced apoptosis in SW480 cells partially through ROS production, GSH depletion and mitochondrial dysfunction. Therefore, the protein extracts of these mushrooms could be considered an important source of new anti-cancer drugs.

Humic acid enhances the cytotoxic effects of arsenic trioxide on human cervical cancer cells.

Cervical cancer is the second leading cancer affecting women, and recent studies have demonstrated arsenic trioxide (As(2)O(3)) has therapeutic effects on cervical cancer by promoting apoptosis and inhibiting metastasis in vitro and in vivo. Humic acid (HA) possesses various pharmacologic properties, including anti-inflammatory, anti-neoplastic, and anti-proliferative effects by inducing apoptosis. We examined the growth inhibition properties and the combined effects of HA and As(2)O(3) in human cervical adenocarcinoma cell lines. Our results shown both As(2)O(3) and HA-induced inhibition of cell growth, most likely by ROS-mediated cell damage and activation of the apoptosis pathway, and HA enhanced the anti-proliferative action of As(2)O(3) in HeLa and SiHa cells, which reduced the LC(50) about 57.62 or 73.52% (300µg HA/mL) to 83.67 or 79.03% (500µg HA/mL), respectively. This study is relevant to the development of chemotherapeutic approaches using As(2)O(3) in treating human cervical cancer.

Complications and management of post-vitrectomy circumferential retinal detachment.

Four consecutive patients with chronic peripheral circumferential detachment developed vision-threatening complications requiring further treatment. All four cases had more than one previous vitreoretinal surgery; two cases had silicone oil intravitreally and three had crystalline lens removed. The main complications included chronic hypotony (1 case), rubeosis iridis (4 cases), intraocular hemorrhage (1 case), neovascular glaucoma (1 case), and cataract (1 case). Retinectomy with peripheral traction release and silicone oil infusion resulted in improvement or stabilization of visual function in all four cases.

Synergistic anti-cancer effect of baicalein and silymarin on human hepatoma HepG2 Cells.

This study investigated the effect of baicalein, silymarin, and their combination, on two human liver-derived cell lines, HepG2 (hepatocellular carcinoma) and Chang liver (non-tumor liver cells). It was found that 6.75 microg/ml baicalein or 100 microg/ml silymarin alone significantly inhibited the growth of HepG2. When baicalein was used in combination with silymarin on HepG2, an additive effect at 24 h and a synergistic effect at 48 h were observed. The viability at 48 h was 85.62% from 6.75 microg/ml baicalein treatment; but the viability reduced to 49.67%, 38.56%, and 19.61% when 25, 50, and 100 microg/ml silymarin respectively, was added to the treatment. By contrast, each treatment had little or no effect on Chang liver. Compared to treatment of baicalein or silymarin alone on HepG2, combination of both drugs synergistically increased the percentages of cells in G0/G1 phase and decreased those in S-phase, which were associated with up-regulation of Rb, p53, p21(Cip1) and p27(Kip1) and down-regulation of cyclin D1, cyclin E, CDK4 and phospho-Rb. The results indicate that the combination of baicalein and silymarin eradicates tumor cells efficiently, has minimal deleterious effects to the surrounding normal cells, and offers mechanistic insight for further exploitation of HCC treatment.

Antioxidant activities of Toona Sinensis leaves extracts using different antioxidant models.

The aim of the present study was to investigate the antioxidant activity of aqueous extracts of Toona sinensis (TS; 0-100 microg/mL) and gallic acid (0-50 microg/mL), with the purified natural phenolic components evaluated using different antioxidant models. It was found that the TS extracts and gallic acid possess effective antioxidant activity against various oxidative systems in vitro, including the scavenging of free and superoxide anion radicals, reducing power, and metal chelation. However, antioxidant activity in terms of metal chelation was not observed for the gallic acid. Moreover, TS extracts and gallic acid appear to possess powerful antioxidant properties with respect to oxidative modification of human LDL induced by CuSO4, AAPH or sodium nitroprusside, as assessed by the relative electrophoretic mobility, TBARS formation, and cholesterol degradation of oxidized LDL. Furthermore, AAPH-induced oxidative hemolysis, lipid peroxidation, and decline in superoxide dismutase (SOD) activity in human erythrocytes were prevented by both the TS extracts and the gallic acid. Our findings suggest that T. sinensis may act as a chemopreventative agent, providing antioxidant properties and offering effective protection from atherogenesis.

Toona sinensis extracts induces apoptosis via reactive oxygen species in human premyelocytic leukemia cells.

Toona sinensis (T. sinensis), well known in Taiwan as a traditional Chinese medicine, has been shown to exhibit antioxidant effects. In this study, therefore, the ability of T. sinensis to induce apoptosis was studied in cultured human premyelocytic leukemia HL-60 cells. Treatment of the HL-60 cells with a variety of concentrations of the aqueous extracts of T. sinensis (TS extracts) (10-75 microg/ml) and gallic acid (5-10 microg/ml), the natural phenolic components purified from TS extracts, resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by loss of cell viability and internucleosomal DNA fragmentation. Furthermore, apoptosis in the HL-60 cells was accompanied by the release of cytochrome c, caspase 3 activation and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). This increase in TS extracts- and gallic acid-induced apoptosis was also associated with a reduction in the levels of Bcl-2, a potent cell-death inhibitor, and an increase in those of the Bax protein, which heterodimerizes with and thereby inhibits Bcl-2. Interestingly, TS extracts- and gallic acid-induced dose-dependent reactive oxygen species (ROS) generation in HL-60 cells. We found that catalase significantly decreased TS extracts- or gallic acid-induced cytotoxicity, DNA fragmentation, and ROS production, however, slight reduction was observed with vitamins C and E. Our results indicate that TS extracts- or gallic acid-induced HL-60 apoptotic cell death could be due to the generation of ROS, especially H(2)O(2). The data suggest that T. sinensis exerts antiproliferative action and growth inhibition on HL-60 cells through apoptosis induction, and, therefore, that it may have anticancer properties valuable for application in food and drug products.

Mechanisms of apoptosis induction and cell cycle regulation in irradiated leukemia U937 cells and enhancement by arsenic trioxide.

Apoptosis is a common mode of cell death after exposure of tumor cells to radiation and/or chemotherapy. The factors that determine the rate of induction of apoptosis are generally related to the functioning of cell cycle checkpoints. In the present study, we investigated the involvement of several genes in cell cycle redistribution and induction of apoptosis in U937 cells after low and high doses of radiation. Activation of CDC2 was observed after both low and high doses of radiation in U937 cells that underwent apoptosis. Expression of CDK2, CDC2 and cyclin A was induced rapidly in the process of radiation-induced apoptosis. In addition, we investigated the use of a clinically relevant dose of radiation to promote As2O3-induced apoptosis in U937 cells. We found that combining radiation and As2O3 may be a new and more effective means of cancer treatment.

Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata.

Antrodia camphorata (A. camphorata) is well known in Taiwan as a traditional Chinese medicine, and it has been shown to exhibit antioxidant and anticancer effects. In this study, therefore, its ability to induce apoptosis in cultured MCF-7 breast cancer cells was studied. Treatment of the MCF-7 cells with a variety of concentrations of the fermented culture broth of A. camphorata (25-150 microg/ml) resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation, and sub-G1 phase accumulation. Furthermore, apoptosis in the MCF-7 cells was accompanied by the release of cytochrome c, activation of caspase 3, and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). Although, the A. camphorata-induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Interestingly, A. camphorata induced dose-dependent reactive oxygen species (ROS) generation in MCF-7 cells. Analysis of the data suggests that A. camphorata exerts antiproliferative action and growth inhibition on MCF-7 cells through apoptosis induction, and that it may have anticancer properties valuable for application in drug products.

Anti-inflammatory potential of Antrodia Camphorata through inhibition of iNOS, COX-2 and cytokines via the NF-kappaB pathway.

Antrodia camphorata (A. camphorata), well known in Taiwan as a traditional Chinese medicine, has been shown to exhibit antioxidant and anticancer effects. In the present study, therefore, we have examined the effects of the fermented culture broth of A. camphorata (25-100 microg/ml) in terms of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in RAW 264.7 macrophages. Our results indicate concentration-dependent A. camphorata inhibition of LPS-induced NO and PGE2 production, without appreciable cytotoxicity on the RAW 264.7 cells. A. camphorata also attenuates the production of LPS-induced tumor necrosis factor (TNF-alpha) and interleukin (IL)-1beta. Furthermore, A. camphorata blocks the IkappaB-alpha degradation induced by LPS. These results indicate that A. camphorata inhibits LPS induction of cytokine, iNOS and COX-2 expression by blocking NF-kappaB activation. Therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.

The PKC delta inhibitor, rottlerin, induces apoptosis of haematopoietic cell lines through mitochondrial membrane depolarization and caspases' cascade.

Rottlerin is a widely selective protein kinase C delta (PKCdelta) inhibitor isolated from Mallotus philippinensis. It shown to be effective against several human tumor cell lines and in potentiating chemotherapy-induced cytotoxcicity. Using the trypan blue exclusion assay, we demonstrated that rottlerin reduced the viability in a dose- and time-dependent manner of human leukemia HL60 cells, human acute T cell leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. Rottlerin caused apoptosis and the apaptotic processing was inhibited by a caspase inhibitor, z-VAD-fmk, in these haematopoietic cells. The apoptosis-inducing activities were determined by nuclear condensation, sub-G1 appearance, DNA fragmentation, loss of mitochondrial membrane potential (Deltapsim), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Expression of PKCdelta and Bcl-2 protein inhibited Deltapsim change and repressed cell death. These studies suggest that the cytotoxic effects of rottlerin through inhibition of PKCdelta cause mitochondrial dysfunction, cytochrome c release from mitochondria into cytoplasm and the activation of caspases' cascade.

Antitumor effects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action.

Antrodia camphorata is a popular folk medicine that has attracted great attention due to its fame for antitumor activity against cancer. However, there is little information available about its action. In the present study, we purified a unique polysaccharide component from A. camphorata mycelia (AC-PS) and found that it has pronounced anti-tumor effects on both in vitro and in vivo model. Our results showed that AC-PS alone did not show any direct cytotoxic effect to human leukemic U937 cells, even at high concentration (200 microg/ml). However, it could inhibit the proliferation of U937 cells via activation of mononuclear cells (MNCs). Treatment of U937 cells with AC-PS-stimulated-MNC-CM could significantly inhibit its proliferation with 55.3% growth inhibition rate. The in vitro antitumor activity was substantiated by the in vivo therapeutical study of AC-PS in sarcoma 180-bearing mice. Intraperitoneal and oral administration of AC-PS, 100 and 200 mg/kg significantly suppressed the tumor growth with the inhibition rate of 69.1% and 58.8%, respectively. In vivo studies also showed that several immunoparameters, such as the spontaneous proliferation of spleen cells, after AC-PS administration, were two-fold higher than in control mice. Furthermore, the cytolytic activity of spleen cells also increased from 9.8 +/- 1.1% in control mice to 34.2 +/- 5.5% and 48.2 +/- 2.5%, after oral and intraperitoneal treatment, respectively. Besides, the mice serum interleukin-12 levels increased significantly by AC-PS treatment. Considering all these results, it is suggested that AC-PS elicit its anti-tumor effect by promoting a Th1-dominant state and killer activities.

Effect of baicalein on apoptosis of the human Hep G2 cell line was induced by mitochondrial dysfunction.

The effects of baicalein on the human hepatoblastoma G2 (Hep G2) cell line were investigated in this study. By an SRB viability assay, we demonstrated that baicalein reduced the viability in a dose- and time-dependent manner. The apoptotic features such as chromatin condensation and DNA fragmentation were observed in the baicalein-treated cells. During the process of apoptosis, we noticed a sequential dissipation of mitochondrial membrane potential (DeltaPsim) and an apparent redistribution of cytochrome c from the mitochondria to the cytosol in baicalein-treated cells. Furthermore, the mitochondrial Bcl-2 protein represented a dramatic change in response to baicalein treatment. Altogether, our data suggested that the effect of baicalein on apoptosis of the human Hep G2 cell line was induced by mitochondrial dysfunction and Bcl-2 regulation.

Different effects of baicalein, baicalin and wogonin on mitochondrial function, glutathione content and cell cycle progression in human hepatoma cell lines.

The effects of the flavonoids from Scutellaria baicalensis Georgi (baicalein, baicalin and wogonin) in cultured human hepatoma cells (Hep G2, Hep 3B and SK-Hep1) were compared by MTT assay and flow cytometry. All three flavonoids dose-dependently decreased the cell viabilities accompanying the collapse of mitochondrial membrane potential and the depletion of glutathione content. However, the influence of baicalein, baicalin or wogonin on cell cycle progression was different. All three flavonoids resulted in prominent increase of G2/M population in Hep G2 cells, whereas an accumulation of sub G1 (hypoploid) peak in Hep 3B cells was observed. In SK-Hep1 cells, baicalein and baicalin resulted in dramatic boost in hypoploid peak, but wogonin made mainly in G1 phase accumulation. These data, together with the previous findings in other hepatoma cell lines, suggest that baicalein, baicalin and wogonin might be effective candidates for inducing apoptosis or inhibiting proliferation in various human hepatoma cell lines.

Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by fermented soy milk.

The effect of a fermented soy milk product (FSP) on various human breast carcinoma cell lines was investigated, and it was shown to have a growth-inhibitory effect, especially on MCF-7 cells. Thus the MCF-7 cell line was used to study the mechanism of action. In female severe combined immune deficiency mice implanted with MCF-7 cells, pretreatment with FSP significantly inhibited tumor growth. The inhibitory effect of FSP on MCF-7 cells seemed to be caused by the additive effects of a wide variety of constituents. The active components of FSP are mainly in the water phase, and the lipid-soluble fraction, which includes the soy isoflavones such as genistein and daidzein, is relatively ineffective. A variety of methods were used to demonstrate that FSP caused apoptotic cell death in MCF-7 cells. FSP induced generation of reactive oxygen species (ROS). Growth inhibition and ROS generation induced by FSP could be inhibited by catalase and deferoxamine, indicating that the ROS production probably was the cause of this apoptotic cell death. This study suggests that FSP retards tumor growth in vivo and can trigger apoptosis in vitro. It may, therefore, be a potential nutritional supplement in chemotherapy.