Parecoxib and 5-Fluorouracil Synergistically Inhibit EMT and Subsequent Metastasis in Colorectal Cancer by Targeting PI3K/Akt/NF-κB Signaling.

Colorectal cancer is one of the most common causes of cancer mortality worldwide, and innovative drugs for the treatment of colorectal cancer are continually being developed. 5-Fluorouracil (5-FU) is a common clinical chemotherapeutic drug. Acquired resistance to 5-FU is a clinical challenge in colorectal cancer treatment. Parecoxib is a selective COX-2-specific inhibitor that was demonstrated to inhibit metastasis in colorectal cancers in our previous study. This study aimed to investigate the synergistic antimetastatic activities of parecoxib to 5-FU in human colorectal cancer cells and determine the underlying mechanisms. Parecoxib and 5-FU synergistically suppressed metastasis in colorectal cancer cells. Treatment with the parecoxib/5-FU combination induced an increase in E-cadherin and decrease in ß-catenin expression. The parecoxib/5-FU combination inhibited MMP-9 activity, and the NF-κB pathway was suppressed as well. Mechanistic analysis denoted that the parecoxib/5-FU combination hindered the essential molecules of the PI3K/Akt route to obstruct metastatic colorectal cancer. Furthermore, the parecoxib/5-FU combination could inhibit reactive oxygen species. Our work showed the antimetastatic capacity of the parecoxib/5-FU combination for treating colorectal cancers via the targeting of the PI3K/Akt/NF-κB pathway.

Parecoxib expresses anti-metastasis effect through inhibition of epithelial-mesenchymal transition and the Wnt/ß-catenin signaling pathway in human colon cancer DLD-1 cell line.

Colorectal cancer is the third leading cause of cancer death in Taiwan. Current treatments involve combination of surgical resection, radiation, and chemotherapy. These treatments have demonstrated to increased five-year survival of a patient with colorectal cancer. However, metastasis is a major capability of cancer cells that causes poor prognosis, recurrence, and even death. Epidemiological and clinical studies have suggested the use of non-steroidal anti-inflammatory drugs (NSAIDs) as an effective class of compounds to prevent colon cancer. Parecoxib is an NSAID and the only parenterally administered selective cyclooxygenase (COX)-2 inhibitor. In this study, we evaluated whether parecoxib inhibits the metastasis of DLD-1 human colon cancer cells, a COX-2 null cell line, and the underlying mechanism. Cell migration of the DLD-1 cells was significantly inhibited by parecoxib treatment as shown by the Transwell migration assay. This enhanced anti-migration effect was correlated with the attenuated phosphorylation of Akt, expression of vimentin (a mesenchymal marker), and ß-catenin, and corresponded with the upregulated GSK3ß and E-cadherin (an epithelial marker). These findings suggested that parecoxib could inhibit the epithelial-mesenchymal transition (EMT) and metastasis in human colon cancer cells by downregulating ß-catenin. Thus, parecoxib could provide a novel prospective strategy for a combination treatment with chemotherapeutic drugs against metastasis of human colon cancer.

Yellow and green pigments from Calophyllum inophyllum L. seed oil induce cell death in colon and lung cancer cells.

Natural compounds have been candidates for anticancer medicine over the last 20 years. During the process of isolating seed oil from Calophyllum inophyllum L., yellow and green pigments containing multiple compounds with an aromatic structure were identified. High-performance liquid chromatography and nuclear magnetic resonance analysis of these pigments revealed that the compounds present were identical, but the concentration of the compounds was different. Treatment with the pigments was able to induce the death of DLD-1 human colon cancer cells and increase the percentage of the cells in the sub-G1 and sub-G2/M phases in a dose-dependent manner. Additionally, the pigments were able to exhibit cytotoxic activity on A549 and H1975 human non-small cell lung cancer (NSCLC) cell lines at 24 h, with half-maximal inhibitory concentrations (IC50) values of 0.1206 and 0.0676%, respectively for green pigments, and 0.0434 and 0.0501%, respectively for yellow pigments. Furthermore, a decrease in IC50 value was associated with an increase in the duration of treatment. However, a sharp decrease in IC50 value of the yellow pigment was observed for H1975 cells at 48 h and for A549 cells at 72 h compared with no change in IC50 value for the green pigment with time, suggesting that the pigments function and induce cell death differently in the two cell lines. An investigation was performed into the synergistic effect of the green pigment and gefitinib (Iressa®, ZD1839), which is a selective epidermal growth factor receptor-tyrosine kinase inhibitor to block growth factor-mediated cell proliferation. The combination of the green pigment and gefitinib resulted in an enhancement of the decrease in viability of A549 and H1975 cells compared with treatment with gefitinib alone, which suggested that treatment with the green pigments was able to enhance the sensitivity of NSCLC cells to gefitinib. In conclusion, these pigments may be considered for development as anti-colon cancer agents.

Anti-angiogenic effect of hexahydrocurcumin in rat corneal neovascularization.

AIM: This study was to investigate the anti-angiogenic effect of hexahydrocurcumin (HHC) to evaluate gene (p-basic fibroblast growth factor (bFGF)-SAINT-18 & p-vascular endothelial growth factor (VEGF)-SAINT-18 complex)-induced corneal neovascularization (CorNV) in rats. METHODS: CorNV was induced in 24 eyes of 24 rats. Four groups (Group A: 0 µg, B: 0.01 µg, C: 0.1 µg, and D: 1 µg) of HHC were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus at temporal side. The 1 µg of p-bFGF-SAINT-18 & p-VEGF-SAINT-18 complex were prepared and implanted into the rat corneal stroma 1.5 mm from the limbus at the same side. Inhibition of CorNV was observed and quantified from day 1 to day 60. bFGF and VEGF protein expression were analyzed by biomicroscopic examination, western blot analysis, and immunohistochemistry. RESULTS: Subconjunctival injection by 1 µg HHC successfully inhibited gene-induced CorNV in rats. bFGF and VEGF protein expression were reduced after 6 days. Meanwhile, the reduction of HLA-DR expression was detected. CONCLUSIONS: Our study showed that the HHC might provide an important anti-angiogenesis factor to inhibit CorNV development at the corneal experimental angiogenesis model.

The interaction of arsenic and N-butyl-N-(4-hydroxybutyl)nitrosamine on urothelial carcinogenesis in mice.

The bladder is an important organ for the storage of excreted water and metabolites. If metabolites with carcinogenic characteristics are present in urine, the urothelial lining of the bladder could be damaged and genetically altered. In this study, we analyzed the interaction of arsenic and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) on mouse bladder carcinogenesis. Our previous study found that arsenic affects BBN-altered urothelial enzymatic activity, protein expression, DNA oxidation and global DNA CpG methylation levels. In this study, two mouse models were used. First, after administering a co-treatment of BBN and arsenic for 20 weeks, BBN alone led to a urothelial carcinoma formation of 20%, and arsenic promoted a BBN-induced urothelial carcinoma formation of 10%. The protein expression of GSTM1, GSTO1, NQO1, and p21 did not change by arsenic along with the BBN co-treatment, but the Sp1 expression increased. In the second mouse model, BBN was a pretreatment promoter; arsenic dose-dependently deteriorated BBN-promoted dysplasia by 10% and 40% at 10 ppm and 100 ppm, respectively. Conversely, BBN pretreatment also accelerated arsenic-induced dysplasia by 30%. The urothelial carcinogenic effect reversed after ceasing BBN for a period of 20 weeks. In summary, three conclusions were drawn from this study. The first is the mutual promotion of arsenic and BBN in bladder carcinogenesis. Second, arsenic dosages without bladder carcinogenicity (10 ppm) or with slight carcinogenicity (100 ppm) promote BBN-induced mice bladder cancer progression. Finally, the dysplastic urothelium had reverted to near-normal morphology after ceasing BBN intake for 20 weeks, providing a good suggestion for people who want to quit smoking.

Propyl Gallate Exerts an Antimigration Effect on Temozolomide-Treated Malignant Glioma Cells through Inhibition of ROS and the NF-κB Pathway.

In this study, we demonstrated that temozolomide (TMZ) and propyl gallate (PG) combination enhanced the inhibition of migration in human U87MG glioma cells. PG inhibited the TMZ-induced reactive oxygen species (ROS) generation. The mitochondrial complex III and NADPH oxidase are two critical sites that can be considered to regulate antimigration in TMZ-treated U87MG cells. PG can enhance the antimigration effect of TMZ through suppression of metalloproteinase-2 and metalloproteinase-9 activities, ROS generation, and the NF-κB pathway and possibly provide a novel prospective strategy for treating malignant glioma.

Anti-oxidative Responses on Hepatic Tissue of Zebrafish (Danio rerio) in a Short Duration of Sub-lethal Concentrations of Cadmium Exposure.

The aim of the present study was to identify whether the responses of oxidative stress in zebrafish liver are similar to those in mammalians upon low doses of Cd2+ exposure in short durations. Fish were exposed to 1.78 µM Cd2+ (treatment) and 0.0 µM Cd2+ (control) for 0, 1, 3, and 6 h. The reactive oxygen species (ROS) and lipid peroxidation (LPO) of hepatic tissues significantly increased after 3 and 6 h of Cd2+ exposure, respectively. Antioxidants glutathione peroxidase (gpx1a), superoxide dismutase (sod), and catalase (cat) were up regulated after 1-3 h, and metallothionein isoforms (smtB and mt2) increased after 3-6 h of Cd2+ exposure. The caspase-3 and p53 mRNA expressions significantly increased threefolds after 1 h of Cd2+ exposure. Results confirmed that oxidative stress in the hepatic tissue was induced by Cd2+ within 3 h. However, anti-oxidative functions immediately up regulated, causing cell apoptosis levels to decrease after 6 h of Cd2+ exposure.

An oxidative stress mechanism of shikonin in human glioma cells.

Shikonin is a quinone-containing natural product that induces the apoptotic death of some cancer cell lines in culture through increasing intracellular reactive oxygen species (ROS). Quinone-based drugs have shown potential in the clinic, making shikonin an interesting compound to study. Our previous study found that shikonin induces apoptosis in neuroblastoma by induction of ROS, but its mechanism of action and scope of activity are unknown. In this study, we investigated the mode of oxidative stress of shikonin in human glioma cells. ROS induction by shikonin was of mitochondrial origin, as demonstrated by detection of superoxide with MitoSOX Red. Pre-incubation of shikonin with inhibitors of different complexes of the respiratory chain suggested that shikonin-induced ROS production occurred via complex II. In addition, NADPH oxidase and lipooxygenase are two other main ROS-generated sites in shikonin treatment. ROS production by shikonin resulted in the inhibition of nuclear translocation of Nrf2. Stable overexpression of Nrf2 in glioma cells inhibited ROS generation by shikonin. ROS generation from mitochondrial complex II, NADPH oxidase and lipooxygenase is likely the primary mechanism by which shikonin induces apoptosis in glioma cells. These findings also have relevance to the development of certain ROS producers as anti-cancer agents. These, along with shikonin have potential as novel chemotherapeutic agents on human glioma.

Folate deficiency triggers an oxidative-nitrosative stress-mediated apoptotic cell death and impedes insulin biosynthesis in RINm5F pancreatic islet ß-cells: relevant to the pathogenesis of diabetes.

It has been postulated that folic acid (folate) deficiency (FD) may be a risk factor for the pathogenesis of a variety of oxidative stress-triggered chronic degenerative diseases including diabetes, however, the direct evidence to lend support to this hypothesis is scanty. For this reason, we set out to study if FD can trigger the apoptotic events in an insulin-producing pancreatic RINm5F islet ß cells. When these cells were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature proceeding through a mitochondria-dependent pathway. In addition to evoke oxidative stress, FD condition could also trigger nitrosative stress through a NF-κB-dependent iNOS-mediated overproduction of nitric oxide (NO). The latter compound could then trigger depletion of endoplasmic reticulum (ER) calcium (Ca(2+)) store leading to cytosolic Ca(2+) overload and caused ER stress as evidence by the activation of CHOP expression. Furthermore, FD-induced apoptosis of RINm5F cells was found to be correlated with a time-dependent depletion of intracellular glutathione (GSH) and a severe down-regulation of Bcl-2 expression. Along the same vein, we also demonstrated that FD could severely impede RINm5F cells to synthesize insulin and their abilities to secret insulin in response to glucose stimulation were appreciably hampered. Even more importantly, we found that folate replenishment could not restore the ability of RINm5F cells to resynthesize insulin. Taken together, our data provide strong evidence to support the hypothesis that FD is a legitimate risk factor for the pathogenesis of diabetes.

Shikonin suppresses the migratory ability of hepatocellular carcinoma cells.

Shikonin is a traditional Oriental medical herb extracted from Lithospermum erythrorhizon. Many studies have shown that shikonin possesses anticancer ability against many different cancers, including hepatocellular carcinoma (HCC). Recently, tumor metastasis has been become an important clinical obstacle. However, the effect of shikonin on metastasis by HCC is unknown. The 50% inhibitory concentration (IC50) of shikonin on HCC cells was determined by an MTT assay and the xCELLigence biosensor system. The migratory ability of HCC cells was detected by a transwell migration assay and the xCELLigence biosensor system. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) expression levels were determined by Western blotting, and the activities of MMP-2 and -9 were determined by gelatin zymography. We found that IC50 values of HepJ5 and Mahlavu cells to shikonin treatment were around 2 µM. Exposure to a low dose of shikonin (0-0.4 µM) did not influence the survival of HCC cells. Interestingly, exposure to a low dose of shikonin inhibited the migratory ability on HepJ5 and Mahlavu cells. To further dissect the mechanism, we found that treatment with a low dose of shikonin reduced the activities and expression levels of MMP-2 and -9, which were correlated with the decreased cell migratory ability of HCC cells. In addition, we found a decrease of vimnetin expression, but no influence on the expression levels of N-cadherin, TWIST, or GRP78. In mechanism dissecting, we found that shikonin treatment may suppress the phosphorylation of AKT and then reduce the NF-κB (NF = nuclear factor) levels, but has no influence on the levels of c-Fos and c-Jun. Furthermore, we also found that shikonin may also reduce the phosphorylation of IκB. We concluded that a low dose of shikonin can suppress the migratory ability of HCC cells through downregulation of expression levels of vimentin and MMP-2 and -9. Our findings suggest that shikonin may be a new compound to prevent the migration of HCC cells.

Inhibition of corneal neovascularization with the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18 (p-PEDF-SAINT-18) vector in a rat corneal experimental angiogenesis model.

Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonal antibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeability properties. In this study, we demonstrated that the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18(p-PEDF-SAINT-18) has a favorable antiangiogenic effect on corneal NV. Four groups(Group A: 0 µg + 0 µg, B: 0.1 µg + 0.1 µg, C: 1 µg + 1 µg, and D: 10 µg + 10 µg) of bevacizumab + p-PEDF-SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus on the temporal side. Then, 1 µgof p-bFGF-SAINT-18 was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. The inhibition of NV was observed and quantified from days 1 to 60. Biomicroscopic examination, western blot analysis and immunohistochemistry were used to analyze the 18-kDa bFGF, 50-kDa PEDF and VEGF protein expression. No inhibition activity for normal limbal vessels was noted. Subconjunctival injection with the combination of bevacizumab and p-PEDF-SAINT-18 successfully inhibited corneal NV.The bFGF and PEDF genes were successfully expressed as shown by western blot analysis,and a mild immune response to HLA-DR was shown by immunohistochemistry. We concluded that the combination of bevacizumab and p-PEDF-SAINT-18 may have more potent and prolonged antiangiogenic effects, making it possible to reduce the frequency of subconjunctival bevacizumab administration combined with a relatively safe profile and low toxicity.

Cell cycle regulation by bevacizumab in ARPE-19 human retinal pigment epithelial cells.

Bevacizumab, a recombinant humanized monoclonal antibody, binds vascular endothelial growth factor (VEGF) and inhibits its interaction with receptors found on endothelial cells. Bevacizumab has been increasingly used as an off-label treatment for exudative age-related macular degeneration (AMD). Whether or not bevacizumab is capable of arresting the growth of human retinal pigment epithelial cells remains to be clarified. In this study, flow cytometry was used to evaluate whether bevacizumab markedly induced the G1/S phase arrest. The G1/S phase cycle-related protein analysis demonstrated that the expression of cyclin-dependent kinase (CDK)2, 4 and 6 and of cyclin D and E, as well as the phosphorylation of retinoblastoma tumor suppressor protein (ppRB) production were found to be markedly reduced by bevacizumab. By contrast, the protein levels of p53, p16, p21 and p27 were increased in bevacizumab-treated ARPE-19 cells (a human retinal pigment epithelial cell line). These events of G1/S arrest induced by bevacizumab in ARPE-19 cells suggest that a preventive effect of bevacizumab exists in AMD.

GRP78 knockdown enhances apoptosis via the down-regulation of oxidative stress and Akt pathway after epirubicin treatment in colon cancer DLD-1 cells.

INTRODUCTION: The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3ß, as well as downstream targets of ß-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway. CONCLUSIONS: Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells.

Novel multiple apoptotic mechanism of shikonin in human glioma cells.

BACKGROUND: Shikonin is the main naphthoquinone compound of the root of Lithospermum erythrorhizon. Our previous study demonstrated that shikonin possesses anticancer activity in human hepatoma cells. However, the anticancer mechanism of shikonin in human glioma cells is unclear at present. In the present study, we demonstrated that shikonin induces apoptosis in three human glioma cell lines: U87MG, Hs683, and M059K cells. METHODS: Cell cycle, generation of reactive oxygen species (ROS), depletion of glutathione (GSH), and disruption of mitochondrial transmembrane potential in shikonin-treated cells were determined by flow cytometry. Apoptosis-related proteins, catalase, and superoxide dismutase-1 (SOD-1) were determined by Western blot testing. N-acetylcysteine (NAC), pifithrin-α (PFT-α), or cyclosporin A were applied to evaluate the molecular mechanism of shikonin in apoptosis. RESULTS: Shikonin induces the generation of ROS, depletion of GSH, disruption of mitochondrial transmembrane potential, upregulation of p53, and cleavage of PARP [poly(ADP-ribose) polymerase] in U87MG glioma cells. Moreover, shikonin causes catalase downregulation and SOD-1 upregulation as well as decreased Bcl-2 and increased Bax expression. Pretreatment with NAC, PFT-α, or cyclosporin A causes the recovery of shikonin-induced apoptosis. The ROS generation and GSH depletion induced by shikonin trigger mitochondrial transmembrane potential disruption. ROS production was partially dependent on the upregulation of p53 upon shikonin treatment. CONCLUSIONS: These studies are the first to show that shikonin-induced apoptosis occurs through multiple pathways in human glioma cells. We conclude that shikonin may be used as a potential chemotherapeutic agent against human glioma.

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.

Enhancement of temozolomide-induced apoptosis by valproic acid in human glioma cell lines through redox regulation.

Temozolomide (TMZ) is an oral alkylating agent that has been widely used in the treatment of refractory glioma, although inherent and acquired resistance to this drug is common. The clinical use of valproic acid (VPA) as an anticonvulsant and mood-stabilizing drug has been reported primarily for the treatment of epilepsy and bipolar disorder and less commonly for major depression. VPA is also used in the treatment of glioma-associated seizures with or without intracranial operation. In this study, we evaluated the potential synergistic effect of TMZ and VPA in human glioma cell lines. Compared with the use of TMZ or VPA alone, concurrent treatment with both drugs synergistically induced apoptosis in U87MG cells as evidenced by p53 and Bax expression, mitochondrial transmembrane potential loss, reactive oxygen species production, and glutathione depletion. This synergistic effect correlated with a decrease in nuclear translocation of the nuclear factor-erythroid 2 p45-related factor and corresponded with reduced heme oxygenase-1 and γ-glutamylcysteine synthetase expression. Pretreatment with N-acetylcysteine partially recovered the apoptotic effect of the TMZ/VPA combination treatment. The same degree of synergism is also seen in p53-mutant Hs683 cells, which indicates that p53 may not play a major role in the increased proapoptotic effect of the TMZ/VPA combination. In conclusion, VPA enhanced the apoptotic effect of TMZ, possibly through a redox regulation mechanism. The TMZ/VPA combination may be effective for treating glioma cancer and may be a powerful agent against malignant glioma. This drug combination should be further explored in the clinical setting.

Role of redox signaling regulation in propyl gallate-induced apoptosis of human leukemia cells.

Propyl gallate (PG) is a synthetic antioxidant that has been used in processed food and medicinal preparations. The anti-cancer effect of PG in leukemia is unclear. In the present study, we demonstrate that PG reduced cell viability in THP-1, Jurkat, and HL-60 leukemia cells and induced apoptosis in THP-1 cells. PG activated caspases 3, 8, and 9 and increased the levels of p53, Bax, Fas, and Fas ligand. PG activated mitogen-activated protein kinases (MAPKs), inhibited nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf-2) and induced intracellular glutathione (GSH) depletion. In addition, PG increased superoxide dismutase-1 expression and decreased intracellular levels of reactive oxygen species. Our data show for the first time that an early event of PG-induced apoptosis is MAPKs/Nrf-2-mediated GSH depletion and that PG induced apoptosis via multiple pathways in human leukemia. PG might serve as a potential chemotherapeutic agent or food supplement for human leukemia patients.

4'-Chloro-3,5-dihydroxystilbene, a resveratrol derivative, induces lung cancer cell death.

AIM: To examine the antitumor effect of 4'-chloro-3,5-dihydroxystilbene, a resveratrol derivative, on lung adenocarcinoma A549 cells. METHODS: The cytotoxic IC(50) was determined by direct cell counting. Flow cytometry, monodansylcadaverine (MDC) staining, transfection, Western blot and a proteasome activity assay were used to study the cellular mechanism of 4'-chloro-3,5-dihydroxystilbene. A xenograft nude mouse model was used to analyze the antitumor effect in vivo. RESULTS: 4'-Chloro-3,5-dihydroxystilbene induced a rapid and persistent increase in the intracellular reactive oxygen species in the cells, but the cell death could not be inhibited by two antioxidant agents. The derivative caused sub-G(1) formation, a decrease in the mitochondria membrane potential and poly (ADP-ribose) polymerase degradation, and the caspase inhibitor Z-VAD-FMK could partially prevent cell death. It also induced a significant increase in intracellular acidic vacuoles, LC3-II formation and intracellular GFP-LC3 aggregation. An autophagic inhibitor partially reversed cell death. Additionally, 4'-chloro-3,5-dihydroxystilbene induced the accumulation of ubiquitinated conjugates and inhibited proteasome activity in cells. In an in vivo study, 4'-chloro-3,5-dihydroxystilbene retarded tumor growth in nude mice. CONCLUSION: These data suggest that the resveratrol derivative 4'-chloro-3,5-dihydroxystilbene could be developed as an anti-tumor compound.

Calvatia lilacina protein-extract induces apoptosis through glutathione depletion in human colorectal carcinoma cells.

This paper reports that a novel protein extract isolated from Calvatia lilacina (CL) can induce cell death against four types of human colorectal cancer cells. Importantly, CL was shown to be free of apoptotic effects against normal rat liver cells. We have also identified that CL-induced glutathione (GSH) depletion is the major contributor responsible for the apoptotic cell death induction of SW 480 cells, as evidenced by the observation that exogenously added N-acetylcysteine (NAC), or GSH, but not vitamin C, could offer a near complete protection of CL-treated cells against apoptotic cell death. Furthermore, the participation of reactive oxygen species (ROS) evoked a drop in the transmembrane potential (Delta Psi(m)) in the CL-induced apoptotic cell death. This observation can only be deemed as a minor pathway due to the fact that cyclosporine A (CyA) could only partially rescue the CL-treated cells from apoptotic cell death. Likewise, despite the fact that CL could induce the upregulation of Bax, its knockdown via siRNA (48 h) failed to completely mitigate apoptotic cell death, indicating that its role in this apoptotic process was insignificant. To further explore the possible underlying mechanism associated with CL-induced GSH depletion, we proceeded to determine the effect of CL on the cellular gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme responsible for GSH biosynthesis, and demonstrated that indeed gamma-GCS could be repressed by CL. Taken together, we report here for the first time that the anticancer effect of CL on human colorectal cancer cells is mediated through GSH depletion mechanism rather than a ROS-mediated killing process. This functional attribute of CL can thus provide the basis for the strategic design of a treatment of colorectal cancer.

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.