Nitroprusside induces melanoma ferroptosis with serum supplementation and prolongs survival under serum depletion or hypoxia.

BACKGROUND: When proliferating tumor cells expand to areas distant from vascular sites, poor diffusion of oxygen and nutrients occur, generating a restrictive hypoxic gradient in which susceptible tumor cells die. The heterogeneous population surviving hypoxia and metabolic starvation include de-differentiated cancer stem cells (CSC), capable of self-renewing tumor-initiating cells (TICs), or those that divide asymmetrically to produce non-tumor-initiating differentiated (NTI-D) cell progeny. Under such restrictive conditions, both populations slowly proliferate, entering quiescence or senescence, when exiting from cell cycle progression. This may drive chemoresistance and tumor recurrence, since most anti-cancer treatments target rapidly proliferating cells. PURPOSE: Since persistent or additional stress may increase NTI-D cells conversion to TICs, we investigated whether nutrient depletion or hypoxia influence expression of tyrosinase, a crucial enzyme for melanin synthesis, and B16 melanoma survival, when exposed to iron-dependent cell death oxidative stress produced by the Fenton reaction, resembling ferroptosis. RESULTS: -a) proliferating B16 melanoma with 10% serum-supplementation (10%S) normoxically express hypoxia inducible factor 1α (HIF1α) but lose tyrosinase, in contrast to those transiently exposed to (SF) serum-free medium, in which both HIF1α and tyrosinase are co-expressed; b) in contrast to the resistance to SNP toxicity in (SF) cells with higher tyrosinase expression, those in (10%S) are killed by iron from nitroprusside/ferricyanide (SNP) irrespective of exogenous H2O2, in a reaction antagonized by the anti-oxidant and MEK inhibitor UO126; c) Moreover, under transient serum depletion, SNP cooperates with hypoxia (1.5% oxygen), prolonging B16 melanoma (SF) survival; d) the hypoxia mimetic CoCl2 inhibits proliferation-associated cyclin A, irrespective of SNP, in (10%S) cells or in transiently serum-depleted (SF) cells. However, only in the latter cells, CoCl2 but not SNP, induce loss of HIF1α and apoptosis-associated PARP cleavage; e) longer term adaptation to survive serum depletion, generates (SS) cells resistant to SNP toxicity, which aerobically co-express HIF1α and tyrosinase. In SS B16 melanoma, exogenous non-toxic 100 µM H2O2 super-induces the ratio of tyrosinase to HIF1α. However, co-treatment of SS-B16 cells with SNP plus exogenous H2O2, partly increases PARP cleavage by reciprocally decreasing tyrosinase expression. SIGNIFICANCE: - These results suggest that a phenotypic plasticity in response to depletion of nutrients and/or oxygen, helps decide whether melanoma cells undergo either death by ferroptosis, or resistance to it, when challenged by the same exogenous oxidative stress (iron ± H2O2).

Total saponins of Albiziae Cortex show anti-hepatoma carcinoma effects by inducing S phase arrest and mitochondrial apoptosis pathway activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Albiziae Cortex (AC) is a widely used traditional medicine in China. It is possess various properties to treat insomnia, traumatic injuries, diuresis, sthenia, and confusion. Total saponins of Albiziae Cortex (TSAC) are the most abundant bioactive components of AC, which were reported to show significant anti-tumor effects in vivo and in vitro. But the underlying mechanism of TSAC remained to be revealed. AIM OF STUDY: In this study, we investigated the anti-hepatoma carcinoma effects and the potential mechanism of TSAC in vivo and in vitro. MATERIALS AND METHODS: We first purified TSAC from crude extracts and characterized the major bioactive compounds by high performance liquid chromatography (HPLC). Effects of TSAC on viability of various hepatoma carcinoma cell lines were measured by MTT. Inhibition on cell proliferation was analysed using colony formation assay. Cell cycle distribution was revealed by flow cytometry. The apoptotic cells were observed by Hoechst 33258 staining and acridine orange (AO)/ethidium bromide (EB) double staining. Microstructures of apoptotic cells were examined by Transmission electron microscopy (TEM). The mitochondrial membrane potential were determined by JC-1 staining. Western blot was used to investigate the effects of TSAC on apoptosis-related proteins, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax), and S-phase related protein cyclin A, cyclin E and cyclin-dependent kinases 2 (CDK2). Effects on tumor growth was assessed by H22-bearing ICR mice. RESULTS: TSAC significantly decreased the hepatoma carcinoma cell viability and inhibited HepG2 cell colony formation in a concentration-dependent manner. We also found that TSAC inhibited HepG2 cell growth via induction of S phase arrest. Further study showed that TSAC significantly down-regulated the expressions of cyclin A, cyclin E and CDK2 in HepG2 cells. Meanwhile, TSAC could effectively induce mitochondria-dependent caspase apoptosis pathway activation. Furthermore, TSAC increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2. In vivo assay showed that the anti-tumor effects of TSAC were significantly augmented without increasing toxicity in H22-bearing ICR mice. CONCLUSION: TSAC could inhibit cell proliferation through inducing S phase arrest and activate cell apoptosis via mitochondria-dependent apoptosis pathway. Therefore, TSAC could be a promising agent in clinical trials for anti-hepatoma carcinoma treatment.

Mechanism of p27 Unfolding for CDK2 Reactivation.

Cell-cycle regulatory protein, CDK2 is active when bound to its complementary partner protein, CyclinA or E. Recent discovery of the Kip/Cip family of proteins has indicated that the activity of CDK2 is also regulated by a member protein, p27. Although, the mechanism of CDK2 inhibition by p27 binding is known from crystal structure, little is known about the mechanism of CDK2 reactivation. Here, we execute classical and accelerated molecular dynamics simulations of unphosphorylated- and phosphorylated-p27 bound CDK2/CyclinA to unravel the CDK2 reactivation mechanism at molecular-to-atomic detail. Results suggest that the phosphorylation of p27 Y88 residue (pY88-p27) first disrupts the p27/CDK2 hybrid ß-sheet and subsequently ejects the p27 310 helix from CDK2 catalytic cleft. The unbinding of p27 from CDK2/CyclinA complex, thus, follows a two-step unfolding mechanism, where the 310 helix ejection constitutes the rate-limiting step. Interestingly, the unfolding of p27 leaves CDK2/CyclinA in an active state, where the prerequisite CDK2-CyclinA interfacial contacts were regained and ATP achieved its native position for smooth transfer of phosphate. Our findings match very well with NMR chemical shift data that indicated the flip-out of p27 310 helix from CDK2 pocket and kinetic experiments that exhibited significant kinase activity of CDK2 when saturated with pY88-p27.

Cratoxylum formosum Extracts Inhibit Growth and Metastasis of Cholangiocarcinoma Cells by Modulating the NF-κB and STAT3 Pathways.

Cratoxylum formosum Dyer has been used in Southeast Asian countries both for food and folk medicine. In this study, the leaf extracts of C. formosum were evaluated for anticancer effects on human cholangiocarcinoma (CCA) KKU-M156 cells. The results showed that the plant extracts possessed potent cytotoxicity against CCA cells. The cytotoxic activity was associated with an induction of cell apoptosis. Moreover, the colony forming ability of CCA cells was also inhibited by C. formosum extracts. Consistent with growth inhibitory effects, the plant extracts induced cell cycle arrest at the G2/M phase and downregulated cyclin A and Cdc25A protein expression. The extracts potently suppressed the migration and invasion properties of CCA cells. The effects were associated with the suppression of NF-κB and STAT3 nuclear translocation and transcriptional activity, and downregulation of genes involving in cancer progression and metastasis. Furthermore, the possible bioactive compounds in the extracts were analyzed by HPLC. Taken together, the potent anticancer activity of C. formosum against CCA indicates the plant promising use for CCA prevention and therapy.

Discovery of novel nonpeptide allosteric inhibitors interrupting the interaction of CDK2/cyclin A3 by virtual screening and bioassays.

Serine/threonine-specific cyclin-dependent kinases (CDKs) are key regulatory elements in eukaryotic cell cycle progression, and the dysregulation of CDKs has been implicated in cancers. Therefore, CDKs have been identified as anti-cancer targets for the development of small-molecule drugs. In this Letter, virtual screening and biological evaluation were performed to identify novel lead structures that allosterically disrupt the interaction between CDK2 and cyclin A3, which are directed toward a noncatalytic binding pocket of CDK2. Ultimately, B2 was identified as exhibiting superior CDK2/cyclin A3 inhibition activity. In addition, our results indicated that B2 exhibited antiproliferative activities against a broad spectrum of human cancer cell lines. Significantly, B2 certainly interrupted the interaction between CDK2 and cyclin A3 and exhibited a concentration-dependent trend. In summary, our results suggest that B2 is the first effective allosteric chemical small-molecule CDK2 inhibitor to be discovered, and further lead optimization may result in a series of novel anti-CDK2 agents.

Secreted protein acidic and rich in cysteine antagonizes bufalin-induced apoptosis in gastric cancer cells.

Bufalin is an active compound in the traditional Chinese medicine Chan Su, which has been shown to induce apoptosis in a range of cancer cell types. However, certain gastric cancer cells are known to be resistant to bufalin. Intracellular secreted protein acidic and rich in cysteine (SPARC) regulates proliferation and apoptosis. This study aimed to evaluate the role of SPARC in bufalin-induced apoptosis in SGC7901 and MGC803 gastric cancer cells. SGC7901 cells with high SPARC expression were more resistant to bufalin than MGC803 cells with low SPARC expression. This resistance was significantly reversed by small interfering (si)RNA-mediated knockdown of SPARC. Furthermore, it was shown that SPARC negatively regulated bufalin-induced intrinsic apoptosis by protecting mitochondrial integrity, decreasing the release of cytoplasmic cytochrome c and increasing the ratio of Bcl-2/Bax. In addition, SPARC overcame bufalin-induced G2/M phase arrest by increasing levels of Cyclin B1 and Cyclin A protein expression. SPARC also activated cellular survival signals, including Src and Akt, but not extracellular signal-regulated kinase. This study demonstrated that SPARC antagonizes bufalin-induced apoptosis via inhibition of the intrinsic apoptosis pathway, inhibition of cell cycle arrest and activation of certain pathways involved in proliferation. This provides novel evidence for SPARC as a potential target by which to sensitize gastric cancer cells to bufalin.

The ellagic acid-derived gut microbiota metabolite, urolithin A, potentiates the anticancer effects of 5-fluorouracil chemotherapy on human colon cancer cells.

Chemotherapy increases the overall survival in colorectal cancer (CRC) patients. 5-Fluorouracil (5-FU) remains as a drug of first choice in CRC therapy over the last four decades. However, only 10-15% of patients with advanced CRC respond positively to 5-FU monotherapy. Therefore, new strategies to enhance the 5-FU effectiveness, overcome the tumor cell resistance and decrease the unspecific toxicity are critically needed. Urolithin A (Uro-A) is the main metabolite produced by the human gut microbiota from the dietary polyphenol ellagic acid. Uro-A targets the colonic mucosa of CRC patients, and preclinical studies have shown the anti-inflammatory and cancer chemopreventive activities of this metabolite. We evaluated here whether Uro-A, at concentrations achievable in the human colorectum, could sensitize colon cancer cells to 5-FU and 5'DFUR (a pro-drug intermediate of 5-FU). We found that both 5-FU and 5'DFUR arrested the cell cycle at the S phase by regulating cyclins A and B1 in the human colon cancer cells Caco-2, SW-480 and HT-29, and also triggered apoptosis through the activation of caspases 8 and 9. Co-treatments with Uro-A decreased IC50 values for both 5-FU and 5'DFUR and additionally arrested the cell cycle at the G2/M phase together with a slight increase in caspases 8 and 9 activation. Overall, we show that Uro-A potentiated the effects of both 5-FU and 5'DFUR on colon cancer cells. This suggests the need for lower 5-FU doses to achieve similar effects, which could reduce possible adverse effects. Further in vivo investigations are warranted to explore the possible role of Uro-A as a chemotherapy adjuvant.

Flavonoids from Gynostemma pentaphyllum exhibit differential induction of cell cycle arrest in H460 and A549 cancer cells.

Flavonoids, containing mainly kaempferol rhamnohexoside derivatives, were extracted from Gynostemma pentaphyllum (G. pentaphyllum) and their potential growth inhibition effects against H460 non-small cell lung cancer cells was explored and compared to that on A549 cells. The extracted flavonoids were found to exhibit antiproliferation effects against H460 cells (IC50 = 50.2 µg/mL), although the IC50 of H460 is 2.5-fold that of A549 cells (IC50 = 19.8 µg/mL). Further investigation revealed that H460 cells are more susceptible to kaempferol than A549, whereas A549 cell growth is better inhibited by kaempferol rhamnohexoside derivatives as compared with H460. In addition, flavonoids from G. pentaphyllum induced cell cycle arrest at both S and G2/M phases with concurrent modulated expression of the cellular proteins cyclin A, B, p53 and p21 in A549 cells, but not H460. On the contrary, apoptosis and concomitant alteration in balance of BCL-2 and BAX expression as well as activation of caspase-3 were equally affected between both cells by flavonoid treatment. These observations strongly suggest the growth inhibition discrepancy between H460 and A549 following flavonoid treatment can be attributed to the lack of cell cycle arrest in H460 cells and the differences between H460 and A549 cells may serve as contrasting models for further mechanistic investigations.

Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 µM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

Cucurbitacin-I, a natural cell-permeable triterpenoid isolated from Cucurbitaceae, exerts potent anticancer effect in colon cancer.

Cucurbitacin-I is a triterpenoids found in medicinal plants and have diverse pharmacological and biological activities. In this study, the antitumor effects of cucurbitacin-I on colon cancer and possible roles in apoptosis and cell cycle arrest were investigated. Treatment of SW480 cells, a human colon cancer cells, with cucurbitacin-I decreased cell viability and cell proliferation in a concentration-dependent manner. Also, cucurbitacin-I induced G2/M phase cell cycle arrest in SW480 cells with a decreased expression of cell cycle proteins including cyclin B1, cyclin A, CDK1, and CDC25C. Moreover, cucurbitacin-I induced increased cleavage of caspase-3, -7, -8, -9, and poly ADP ribose polymerase. When we examined the inhibitory effect of cucurbitacin-I on tumor growth in vivo, cucurbitacin-I effectively inhibited the tumorigenicity and growth of CT-26 cells in syngenic BALB/c mice. In summary, the present study showed that cucurbitacin-I reduced colon cancer cell proliferation by enhancing apoptosis and causing cell cycle arrest at the G2/M phase.

[Protective effect and mechanism of astragaloside IV on oxidative stress injury of mesangial cells].

OBJECTIVE: To study the protective effect of astragaloside IV (AS IV) on H2O2 induced human mesangial cells (HMC), and further explore its molecular mechanism. METHOD: The cultured mesangial cells were divided into 5 groups: the normal control group, the H2O2 model group, the AS IV (12.5, 100 nmol x L(-1)) group and the Tempol (1 x 10(5) nmol x L(-1)) group. The MTT method was used to observe cell viability. Hoechst 33258 staining was used to observe the HMC apoptosis and DHE staining was used to detect the generation of reactive oxygen species (ROS). The flow cytometry was used to detect the changes in cell cycle. Western blot was used to detect the expression of Cyclin D1, CyclinA, p38, and T-p38. RESULT: H2O2 (1 x 10(5), 2 x 10(5), 3 x 10(5), and 4 x 10(5) nmol x L(-1)) could induce HMC oxidative stress injury, with significant decrease in the cell survival rate. AS IV (100 nmol x L(-1)) could significantly inhibit HMC oxidative stress injury induced by H2O2 (3 x 10(5) nmol x L(-1)), increase the survival rate of HMC cells, inhibit cell apoptosis, and decrease intracellular ROS production. AS IV could also increase the expression of Cyclin D1, recover normal cell proliferation, and decrease the expression of p38. CONCLUSION: AS IV can protect H2O2 induced oxidative stress injury in mesangial cells. Its mechanisms may be related to inhibiting the p38/MAPK signaling pathway, increasing the expression of Cyclin D1 and decreasing the intracellular ROS oxidative stress injury.

Maple polyphenols, ginnalins A-C, induce S- and G2/M-cell cycle arrest in colon and breast cancer cells mediated by decreasing cyclins A and D1 levels.

Polyphenols are bioactive compounds found in plant foods. Ginnalins A-C are polyphenols present in the sap and other parts of the sugar and red maple species which are used to produce maple syrup. Here we evaluated the antiproliferative effects of ginnalins A-C on colon (HCT-116) and breast (MCF-7) tumourigenic and non-tumourigenic colon (CCD-18Co) cells and investigated whether these effects were mediated through cell cycle arrest and/or apoptosis. Ginnalins A-C were twofold more effective against the tumourigenic than non-tumourigenic cells. Among the polyphenols, ginnalin A (84%, HCT-116; 49%, MCF-7) was more effective than ginnalins B and C (50%, HCT-116; 30%, MCF-7) at 50 µM concentrations. Ginnalin A did not induce apoptosis of the cancer cells but arrested cell cycle (in the S- and G(2)/M-phases) and decreased cyclins A and D1 protein levels. These results suggest that maple polyphenols may have potential cancer chemopreventive effects mediated through cell cycle arrest.

An inhibitory effect of resveratrol in the mitotic clonal expansion and insulin signaling pathway in the early phase of adipogenesis.

Resveratrol is known as a potent antiobesity compound that acts partly through inhibition of adipogenesis. However, the direct targets responsible for its antiadipogenic action are unclear. Our hypothesis is that resveratrol inhibits adipogenesis through modulation of mitotic clonal expansion (MCE) and cell signaling pathways in the early phase of differentiation. To test this, we examined the effects of resveratrol on MCE and insulin signaling pathway in the early phase of adipogenesis in murine preadipocytes. We observed that the antiadipogenic action of resveratrol is largely limited to the early phase of adipogenesis. Specifically, the presence of resveratrol in the first 24 hours of adipogenesis was required for its antiadipogenic effect. During the first 24 hours of adipogenesis, resveratrol impaired the progression of MCE by suppressing the cell cycle entry of preadipocytes to G2/M phase, and expression of cell cycle regulators cyclin A and cyclin-dependent kinase 2. Concomitantly, resveratrol inhibited insulin signaling pathway in the early phase of adipogenesis. Furthermore, we revealed an inhibitory effect of resveratrol on insulin receptor (IR) activity, and this is likely through a direct physical interaction between resveratrol and IR. The antiadipogenic effect of resveratrol is through inhibition of the MCE and IR-dependent insulin signaling pathway in the early phase of adipogenesis.

Induction of apoptosis and cell cycle arrest by pericarp polyphenol-rich extract of Baneh in human colon carcinoma HT29 cells.

Plants as important source of natural active components with anticancer effects commonly are different in structure and biological properties. The pericarp of Pistacia atlantica sub kurdica with local name of Baneh, a rich source of active phytochemicals, contains noticeable amounts of polyphenolic compounds, flavonoids and anthocyanins. Therefore, the antiproliferative, apoptosis induction and cell cycle alterations of Baneh were evaluated in human colon carcinoma HT29 cells. The Baneh extract (0.7 mg/ml) resulted in 50% growth inhibition similar to 500 nM of Doxorubicin (Dox) in HT29 cells after 72 h. The down-regulation of cyclin A protein by Baneh extract induced S phase delay in cell cycle progression of HT29 cells. Unlike the Baneh extract, Dox showed G2/M accumulation of HT29 cells which was associated with an increase in cyclin A and cyclin B1 protein expression. Furthermore, the induction of apoptosis following Baneh extract and Dox treatment in HT29 cells was confirmed by DNA fragmentation and translocation of phosphatidylserine. The morphological characteristics of apoptosis were also observed in HT29 cells exposed to the Baneh extract and Dox. These results suggest that due to the existence of bioactive components, methanolic extract of the Baneh has significant cytotoxic effects against human colon carcinoma HT29 cells.

Cambogin is preferentially cytotoxic to cells expressing PDGFR.

Platelet-derived growth factor receptors (PDGFRs) have been implicated in a wide array of human malignancies, including medulloblastoma (MB), the most common brain tumor of childhood. Although significant progress in MB biology and therapeutics has been achieved during the past decades, MB remains a horrible challenge to the physicians and researchers. Therefore, novel inhibitors targeting PDGFR signaling pathway may offer great promise for the treatment of MB. In the present study, we investigated the cytotoxicity and mechanisms of cambogin in Daoy MB cells. Our results show that cambogin triggers significant S phase cell cycle arrest and apoptosis via down regulation of cyclin A and E, and activation of caspases. More importantly, further mechanistic studies demonstrated that cambogin inhibits PDGFR signaling in Daoy and genetically defined mouse embryo fibroblast (MEF) cell lines. These results suggest that cambogin is preferentially cytotoxic to cells expressing PDGFR. Our findings may provide a novel approach by targeting PDGFR signaling against MB.

Molecular mechanisms of celery seed extract induced apoptosis via s phase cell cycle arrest in the BGC-823 human stomach cancer cell line.

BACKGROUND: Mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Loss of cell cycle control, leading to uncontrolled proliferation, is common in cancer. Therefore, the identification of potent and selective cyclin dependent kinase inhibitors is a priority for anti-cancer drug discovery. There are at least two major apoptotic pathways, initiated by caspase-8 and caspase-9, respectively, which can activate caspase cascades. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. This is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane. Anti-tumor effects of celery seed extract (CSE) and related mechanisms regarding apoptosis were here investigated in human gastric cancer BGC-823 cells. METHODS: CSE was produced by supercritical fluid extraction. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay and apoptosis by flow cytometry using Annexin/PI staining and DAPI staining and a laser scanning confocal microscope (LSCM). Cell cycling was evaluated using PI staining with flow cytometry and expression of cell cycle and apoptosis-related proteins cyclin A, CDK2, bcl-2 and bax was assessed by immunohistochemical staining. RESULTS: CSE had an anti-proliferation effect on human gastric cancer BGC-823 cells in a dose- and time-dependent manner. After treatment, the apoptotic rate significantly increased, with morphological changes typical of apoptosis observed with LSCM by DAPI staining. Cell cycle and apoptosis related proteins, such as cyclin A, CDK2 and bcl-2 were all down-regulated, whereas bax was up-regulated. CONCLUSIONS: The molecular determinants of inhibition of cell proliferation as well as apoptosis of CSE may be associated with cycle arrest in the S phase.

Peptides from water buffalo cheese whey induced senescence cell death via ceramide secretion in human colon adenocarcinoma cell line.

SCOPE: Milk proteins are a source of bioactive peptides. Recent studies have indicated that protein-derived peptides released in buffalo cheese acid whey exert a cytomodulatory effect in human epithelial colon cancer (CaCo2) cells. The aim of the present study was to explain the molecular mechanism involved in the response of CaCo2 cells to oxidative stress in the presence of peptide fractions of buffalo cheese whey, purified and characterized by mass spectrometry. METHODS AND RESULTS: We demonstrated that treatment of CaCo2 treated with H2O2 (H-CaCo2) cells with a partially purified peptide sub-fraction (f3) from buffalo cheese acid whey induced a reduction of mitochondrial superoxide anion with subsequent decrease in heat shock protein 70 and 90 expression. Moreover, we observed a 5-fold decrease in cyclin A expression and cell cycle arrest in G1/G0 phases. These responses were associated with increased activity of alkaline phosphatase and beta-galactosidase, markers of differentiation and senescence respectively. CONCLUSIONS: The structural characterization of the active peptide fraction and the elucidation of the effects induced by its treatment on H-CaCo2 cells in vitro demonstrated an activity of this peptide sub-fraction in the modulation of cell cycle, thus suggesting potential application for the development of nutraceuticals as well as health-promoting functional foods.

Inhibition of adipocyte differentiation and adipogenesis by the traditional Chinese herb Sibiraea angustata.

Obesity has become a major health concern due to its strong association with the metabolic syndrome. Inhibition of adipocyte differentiation represents a key strategy to inhibit obesity. Sibiraea angustata (SA), a traditional Chinese herb, has a wide range of pharmacological effects, such as improving digestive functions. Here, we report a novel antiadipogenic effect of SA. By using the SA water extract (SAW), SA acetic ether extract (SAA) and the 3T3-L1 model of adipocyte differentiation and adipogenesis, we showed that both SAW and SAA impaired the proliferation and adipo-differentiation of 3T3-L1 in a dose- and time-dependent manner. At the molecular level, treatment of 3T3-L1 cells with SAW or SAA inhibited the expression of the key adipocyte differentiation regulator CCAAT enhancer binding protein ß (C/EBPß), as well as peroxisome proliferator activated receptor γ, adipocyte protein-2, lipoprotein lipase and glucose transporter 4. Cell cycle analysis showed that both SAW and SAA blocked cell cycle at the G1-S transition phase, causing cells to remain in the preadipocyte state. The expression of CyclinA and cyclin-dependent kinase 2 was also inhibited by SAW and SAA. Treatment with SAW also prevented the localization of C/EBPß to the centromeres. Taken together, our results show that SA has a potent antiadipogenic effect in 3T3-L1 cells due to the inhibition of adipocyte differentiation and adipogenesis. We propose that SA may be used as a safe and effective neutraceutical to manage obesity.

The effect of Longan seed polyphenols on colorectal carcinoma cells.

BACKGROUND: Polyphenol-rich longan seed extract (LSP) is a free radical scavenger and antioxidant. However, the effect of LSP on the growth of human colorectal carcinoma cells (CRC) has not yet been evaluated. MATERIALS AND METHODS: Polyphenols of longan seeds were extracted and measured by colorimetry. Four CRC cell lines (Colo 320DM, SW480, HT-29 and LoVo) were treated with LSP and assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for apoptosis by annexin V labelling and for changes in the levels of proteins involved in cell cycle control or apoptosis by immunoblotting. RESULTS: Total phenol content of LSP was 695 mg g(-1) and total flavonoids were 150 mg g(-1). LSP inhibited the proliferation (25 microg mL(-1)-200 microg mL(-1)) of Colo 320DM, SW480 and HT-29, but not LoVo. LSP inhibited the proliferation by blocking cell cycle progression during the DNA synthesis phase and inducing apoptotic death. Western blotting indicated that LSP blocks the S phase, reducing the expression of cyclin A and cyclin D1. Colo 320DM and SW480 treated with LSP also showed the activation of caspase 3 and increased Bax : Bcl-2 ratio. CONCLUSION: LSP induces S phase arrest of the cell cycle and apoptotic death in three CRC cell lines. The results indicate that LSP is a potential novel chemoprevention and treatment agent for colorectal cancer.

High performance liquid chromatographic analysis and anticancer potential of Oplopanax horridus: comparison of stem and berry extracts.

Oplopanax horridus or devil's club is a herbal medicine distributed in North America. The constituents and pharmacological activities of O. horridus (OPH) are largely unknown. In this study, we assayed OPH stem and berry extracts using high performance liquid chromatography (HPLC). The anticancer potentials of extracts on different human cancer cell lines (SW-480, HCT-116, HT-29, MCF-7 and NSCLC) were determined by MTS method. The effect of stem extract on cancer cell cycle, expression of cyclin A, and apoptosis were assayed using flow cytometry. HPLC data showed that the composition of OPH stem extract is more complicated than the berry extract. The wavelength of maximum absorption of the major constituent in stem and berry is 196.0 nm and 201.9 nm, respectively. Compared to the berry extract, the stem extract showed significant potent antiproliferative effect on all the studied cell lines. The stem extract at 0.1 mg/ml arrested cancer cells in S- and G2/M-phases, and significantly induced expression of cyclin A. After treatment with 0.1 mg/ml of stem extract for 72 h, apoptotic cells were increased to 45.2%, while control was 9.6%. The cell cycle arrest and induction of apoptosis may play a critical role in cancer chemoprevention by Oplopanax horridus stem extract.