Inhibitory effects of (-)-epigallocatechin-3-gallate and pterostilbene on pancreatic cancer growth in vitro.

BACKGROUND: It has been previously shown that the naturally occurring antioxidant (-)-epigallocatechin-3-gallate (EGCG), found in green tea, and pterostilbene, a stilbenoid derived from blueberries, inhibit pancreatic cancer in vitro when used individually. We hypothesized that the combination of EGCG and pterostilbene would reveal additive effects in vitro. METHODS: Using the pancreatic cancer cell lines MIA PaCa-2 and PANC-1, efficacy and synergism were evaluated for cell proliferation and viability (3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays, cell cycle analysis) and mitochondrial apoptosis (mitochondrial depolarization, cytochrome C release, caspase-3/7 activity, cell death detection using enzyme-linked immunosorbent assay). RESULTS: Cell proliferation assays revealed significant additive antiproliferative effects with pterostilbene and EGCG in both cell lines at the later, 72-h, point (P < 0.05). MIA underwent S-phase arrest with the combination (10-12% increase); however, cell cycle arrest was not observed in PANC. The combination induced mitochondrial depolarization and upregulated cytochrome C (P < 0.05) in MIA, but these effects were not observed in PANC. EGCG increased caspase-3/7 in MIA; however, the combination did not significantly increase the activity in either cell line (P < 0.05). Apoptosis was only observed in PANC (P < 0.05). The reduction in proliferation in MIA in the 3-(4,5-dimethyltiazol-2-y1)-2,5-diphenltetrazolium bromide assays with the combination indicated that cell death occurs, possibly through another mechanism. CONCLUSIONS: Our results are encouraging regarding the future use of EGCG and pterostilbene to improve traditional pancreatic cancer therapies. In conclusion, EGCG and pterostilbene have additive, antiproliferative effects in vitro and alter the apoptotic mechanisms in both cell lines by modulation at different points in the mechanism.

Pterostilbene inhibits pancreatic cancer in vitro.

INTRODUCTION: Stilbenes are phenolic compounds present in grapes and blueberries. Resveratrol, a naturally occurring compound present in grapes, has been shown to have potent antioxidant properties as well as an ability to induce apoptosis. Resveratrol has also been reported to have significant inhibitory effects against a variety of primary tumors including breast, colon, and prostate. Pterostilbene, a naturally occurring analogue of resveratrol found in blueberries, also has antioxidant and antiproliferative properties. It is also substantially more bioavailable orally than resveratrol. These effects have not been studied in pancreatic cancer. We hypothesized that pterostilbene would inhibit pancreatic cancer cell growth in vitro. MATERIALS AND METHODS: Two pancreatic cancer cell lines (MIA PaCa and PANC-1) were cultured using standard techniques. Cells were treated with graduated doses of pterostilbene ranging from 10 to 100 microM. Cell viability was measured by MTT at 24, 48, and 72 h. RESULTS: Pterostilbene decreases cell viability in both cancer cell lines in a concentration- and time-dependent manner. Higher doses (75-100 microM) caused a significant reduction in cell viability at 24 and 48 h. However, by 72 h, all tested concentrations of pterostilbene (10 to 100 microM) resulted in significantly reduced cell viability in both pancreatic cancer cell lines in a dose-dependent fashion. Pterostilbene caused a dose-dependent 10-63% inhibition in MIA PaCa-2 cells and 10-75% inhibition in PANC-1 cells. DISCUSSION: Treatment of pancreatic cancer cells in vitro with Pterostilbene leads to inhibition of cell proliferation and/or cell death, cell cycle arrrest, mitochondrial membrane depolarization, and activation of effector caspases. This naturally occurring agent may have a role in treating pancreatic cancer. CONCLUSIONS: Pterostilbene inhibits the growth of pancreatic cancer in vitro. Further, in vitro mechanistic studies and in vivo experiments are warranted to determine its potential for the treatment of pancreatic cancer.

Pterostilbene inhibits lung cancer through induction of apoptosis.

BACKGROUND: Lung cancer remains the leading cause of cancer mortality in the United States. Resveratrol is a potent antioxidant found in grapes that inhibits several types of cancer, including lung cancer. Herein, we investigated the effects of pterostilbene, an analog of resveratrol found in blueberries, on lung cancer, in vitro. We hypothesized that pterostilbene would inhibit lung cancer cell growth in vitro by a pro-apoptotic mechanism. METHODS: Two lung cancer cell lines (NCI-H460 and SK-MES-1) were cultured using standard techniques. Cells were treated with increasing doses of pterostilbene (10-100 microM). Cell viability was measured at 24, 48, and 72h using a MTT assay. Apo-ONE Caspase-3/7 assay was used to evaluate caspase activity. T-test and two-way ANOVA were used for statistical analysis. RESULTS: Pterostilbene significantly decreased cell viability in lung cancer cells in a concentration- and time-dependent manner (P<0.001). Concentrations greater than 20 microM of pterostilbene produced significant growth inhibition by 72h (P<0.001). Apoptosis and caspase-3/7 activity were significantly increased by pterostilbene treatment (P<0.05). CONCLUSIONS: Pterostilbene inhibits growth via apoptosis induction in vitro. Further in vitro mechanistic studies and in vivo experiments are warranted to determine the potential role for pterostilbene in lung cancer treatment or prevention.

Pterostilbene inhibits breast cancer in vitro through mitochondrial depolarization and induction of caspase-dependent apoptosis.

BACKGROUND: Epidemiologic studies suggest that diets high in fruits and vegetables reduce cancer risk. Resveratrol, a compound present in grapes, has been shown to inhibit a variety of primary tumors. Pterostilbene, an analogue of resveratrol found in blueberries, has both antioxidant and antiproliferative properties. We hypothesized that pterostilbene would induce apoptosis and inhibit breast cancer cell growth in vitro. METHODS: Breast cancer cells were treated with graduated doses of pterostilbene. Cell viability was measured by MTT assay. Apoptosis was evaluated via DNA fragmentation assay and TUNEL assay. Apo-ONE caspase-3/7 assay was used to evaluate caspase activity. Flow cytometry was used to evaluate mitochondrial depolarization, superoxide formation, and cell cycle. Student's t-test and two-way ANOVA with Bonferroni posttests were utilized for statistical analysis. RESULTS: Pterostilbene decreased breast cancer cell viability in a concentration- and time-dependent manner. Pterostilbene treatment increased caspase-3/7 activity and apoptosis in both cell lines. Caspase-3/7 inhibitors completely reversed pterostilbene's effects on cell viability. Pterostilbene treatment triggered mitochondrial depolarization, increased superoxide anion, and caused alteration in cell cycle. CONCLUSIONS: Pterostilbene treatment inhibits the growth of breast cancer in vitro through caspase-dependent apoptosis. Mitochondrial membrane depolarization and increased superoxide anion may contribute to the activation downstream effector caspases. Caspase inhibition leads to complete reversal of pterostilbene's effect on cell viability. Further in vitro mechanistic studies and in vivo experiments are warranted to determine its potential for the treatment of breast cancer.

Effects of pterostilbene on melanoma alone and in synergy with inositol hexaphosphate.

BACKGROUND: Pterostilbene and inositol-6-phosphate (IP6) have been shown to inhibit melanoma growth in vitro. However, pterostilbene's mechanism of action has not been clearly demonstrated. We aimed to further investigate the mechanism of action for pterostilbene and to determine whether combination treatment with IP6 produced synergistic growth inhibition. METHODS: Melanoma cells were treated with increasing doses of pterostilbene, IP6, or combinations thereof. Cell viability was measured at 24 hours, 48 hours, and 72 hours using a MTT assay. Caspase activity and vascular endothelial growth factor (VEGF) production were measured using enzyme-linked immunosorbent assay (ELISA). Analysis of variance (ANOVA) and t tests were used for statistical analysis. RESULTS: Pterostilbene inhibits melanoma growth in vitro in association with increased effector caspase activity. Combination treatment with inositol hexaphosphate produces synergistic growth inhibition, greater than either treatment alone. CONCLUSIONS: Pterostilbene produces caspase-dependent apoptosis in melanoma cell lines. Combination treatment with IP6 produces synergistic growth inhibition. Both compounds have significant potential for a therapeutic role in the treatment of melanoma.

Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway.

We recently reported that embryonic stem cells-conditioned medium (ES-CM) contains antiapoptotic factors that inhibit apoptosis in the cardiac myoblast H9c2 cells. However, the mechanisms of inhibited apoptosis remain elusive. In this report, we provide evidence for the novel mechanisms involved in the inhibition of apoptosis provided by ES-CM. ES-CM from mouse ES cells was generated. Apoptosis was induced after exposure with H(2)O(2) (400 mum) in H9c2 cells followed by the replacement with ES-CM or culture medium. H9c2 cells treated with H(2)O(2) were exposed to ES-CM, and ES-CM plus cell survival protein phosphatidylinositol 3-kinase/Akt inhibitor, LY-294002, or extracellular signal-regulated kinase (ERK1/2) inhibitor, PD-98050. After 24 h, H9c2 cells treated with ES-CM demonstrated a significant increase in cell survival. ES-CM significantly inhibited (P < 0.05) apoptosis determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, and caspase-3 activity. Importantly, enhanced cell survival and inhibited apoptosis with ES-CM was abolished with LY-294002. In contrast, PD-98050 shows no effect on ES-CM-increased cell survival. Furthermore, H(2)O(2)-induced apoptosis is associated with decreased levels of phosphorylated (p)Akt activity. Following treatment with ES-CM, we observed a decrease in apoptosis with an increase in pAkt, and the increased activity was attenuated with the Akt inhibitor, suggesting that the Akt pathway is involved in the decreased apoptosis and cell survival provided by ES-CM. In contrast, we observed no change in ES-CM-decreased apoptosis or pERK with PD-98050. In conclusion, we suggest that ES-CM inhibited apoptosis and is mediated by Akt but not the ERK pathway.

Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells.

Our recent study (Singla DK, Hacker TA, Ma L, Douglas PS, Sullivan R, Lyons GE, Kamp TJ, J Mol Cell Cardiol 40: 195-200, 2006) suggests that transplanted embryonic stem (ES) cells subsequent to myocardial infarction differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration is relatively meager compared with the observed functional improvement. The mechanisms underlying their improved function are completely unknown. In this report, we provide evidence using a cell culture model system for novel mechanisms that involve the release of cytoprotective, anti-apoptotic factor(s) from ES cells and inhibit H(2)O(2)-induced apoptosis in the rat cardiomyocyte-derived cell line H9c2. Conditioned medium (CM) from growing mouse ES cells treated with and without H(2)O(2) was generated. Apoptosis was induced after exposure to H(2)O(2) in H9c2 cells for 2 h followed by replacement with fresh cell culture or ES cell-CM. After 24 h, H9c2 cells treated with both ES cell-CMs demonstrated significantly decreased apoptosis, as determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, caspase-3 activity, and DNA ladder. Next, using Luminex technology, we examined the presence of antiapoptotic proteins cystatin c, osteopontin, and clusterin and anti-fibrotic, tissue inhibitor of metalloproteinase-1 (TIMP-1) in both ES cell-CMs. The levels of released factors were 2- to 170-fold higher than those in H9c2 cell-CM. Antiapoptotic effects of ES cell-CM were significantly inhibited with TIMP-1 antibody, suggesting that TIMP-1 is an important factor to inhibit apoptosis. Furthermore, we used CM from an TIMP-1-overexpressing cell line and demonstrated that H(2)O(2)-induced apoptosis in the H9c2 cells was significantly inhibited. These observations demonstrate that factors released from ES cells contain antiapoptotic factors and that the effects are mediated by TIMP-1. Moreover, these findings suggest that released factors might be useful for therapeutic applications in ischemic heart disease as well as for many other diseases.