3,3'-Diindolylmethane enhances apoptosis in docetaxel-treated breast cancer cells by generation of reactive oxygen species.

CONTEXT: A major problem in the treatment of cancer is the development of toxic side effects and resistance to chemotherapy. The use of plant compounds to overcome resistance and prevent toxicity is a potential strategy for treatment. OBJECTIVE: We evaluated whether 3,3'-diindolylmethane (DIM) enhanced the sensitivity of breast cancer cells to docetaxel (DOC). MATERIALS AND METHODS: MDA-MB231 and Sk-BR-3 cells were treated with and without 25 or 50 µM of DIM and 1 nM of DOC for 48 and 72 h, respectively. MTT assay was used to measure cell survival. Apoptosis and intracellular reactive oxygen species (ROS) were determined by flow cytometry. The expression of proteins regulating ROS production and apoptosis was evaluated by immunoblotting technique. RESULTS: Combining 25 µM of DIM with 1 nM DOC decreased cell survival by 42% in MDA-MB231 cells and 59% in Sk-BR-3 cells compared to control, DIM, or DOC (p ≤ 0.05). The combination treatment increased apoptosis over 20% (p ≤ 0.01) in both cell lines, which was associated with decreased Bcl-2, increased Bax, cleaved PARP and activated JNK (p ≤ 0.01). ROS production increased by 46.5% in the MDA-MB231 and 29.3% in Sk-BR-3 cells with the combination compared to DIM or DOC alone. Pretreating cells with N-acetyl-cysteine or Tiron abrogated the anti-survival effect of the combination. The increase in ROS was associated with a 54% decrease in MnSOD and 47% increase in NOX2 protein compared to the other groups. CONCLUSIONS: Our findings indicated that DIM enhances the sensitivity of breast cancer cells to DOC treatment by increasing ROS, which led to decreased cell survival and apoptosis.

Metformin decreases growth of pancreatic cancer cells by decreasing reactive oxygen species: Role of NOX4.

BACKGROUND: Retrospective epidemiologic studies show that metformin reduced the incidence of pancreatic cancer in diabetic patients. One potential mechanism may be by altering reactive oxygen species (ROS) and apoptosis. In this in vitro study, we explore the role of ROS and apoptosis in mediating the growth-inhibitory effects of metformin in pancreatic cancer cells. METHODS: We cultured MIA PaCa and Panc1 pancreatic cancer cells in medium containing physiological concentrations of glucose (5 mM) or supra-physiological concentrations of glucose and then treated cells with metformin. Cell viability, ROS production, apoptosis, and protein levels of manganese superoxide dismutase (MnSOD), NADPH oxidase (NOX) 2, and NOX4 were measured. RESULTS: Metformin decreased viability of MIA PaCa and Panc1 cells under physiological glucose conditions in comparison to untreated cells; metformin did not have any effect on human pancreatic normal epithelial (HPNE) cells. The decrease in cell survival was associated with decreased intracellular ROS, increased protein levels of MnSOD, and decreased levels of NOX2 and NOX4 proteins in MIA PaCa and Panc1 cells but not HPNE cells. Transfecting MIA PaCa and Panc1 cells with pcDNA3NOX4 protected against the anti-survival effects of metformin. CONCLUSION: Our findings suggest that metformin decreases cell survival by reducing ROS production, in part through down regulation of NOX4 protein expression.

Nexrutine inhibits survival and induces G1 cell cycle arrest, which is associated with apoptosis or autophagy depending on the breast cancer cell line.

Breast cancers that are estrogen receptor (ER) negative or are ER negative with ErbB2/HER-2 overexpression have a poor prognosis, which emphasizes the importance of developing compounds for preventing breast cancer. Nexrutine, an herbal extract from the plant Phellodendron amurense, has been used for centuries in Asian medicine to treat inflammation, gastroenteritis, abdominal pain, and diarrhea. In this study we investigated the anticancer effects of Nexrutine on ER negative breast cancer cell lines that are positive or negative for HER-2. Nexrutine decreased the activities of 2 potential targets of breast cancer, cyclooxygenase (COX)-2, and peroxisome proliferators activated receptor gamma (PPARγ). The antiinflammatory effects of Nexrutine were evident with decreased prostaglandin (PG)E2 production, protein expression of microsomal PGE2 synthase (mPGES), and PPARγ. Nexrutine decreased cell survival and induced a G1 cell cycle arrest in SkBr3 and MDA-MB 231 cells, which were associated with reduced protein expression of Cyclin D1 and cdk2 along with increased protein expression of p21 and p27. The growth-inhibitory effect of Nexrutine was associated with apoptosis in SkBr3 cells and autophagy in MDA-MB231 cells. Based on these findings, we propose that Nexrutine may provide a novel approach for protection against breast cancer.

Calorie restriction delays the progression of lesions to pancreatic cancer in the LSL-KrasG12D; Pdx-1/Cre mouse model of pancreatic cancer.

Since pancreatic cancer is a lethal disease, developing prevention strategies is an important goal. We determined whether calorie restriction would prevent the development and delay progression of pancreatic intraepithelial neoplasms to pancreatic ductal adenocarcinoma (PDA) in LSL-KrasG12D/+; Pdx-1/Cre mice that develop all the precursor lesions that progress to PDA. Eight-week-old LSL-KrasG12D; Pdx-1/Cre mice were assigned to three groups: (1) ad libitum (AL) fed the AIN93M diet or (2) intermittently calorie restricted (ICR) a modified AIN93M at 50% of AL intake followed by one week intervals at 100% of AL intake, or (3) chronically calorie restricted (CCR) an AIN93M diet at 75% of AL intake. AL fed mice had a greater percentage of pancreatic ducts with PanIN-2 (13.6%) than did the ICR (1.0%) and CCR groups (1.6%), P < 0.0001. Calorie restriction (ICR [0%] and CCR [0.7%]) reduced the percentage of ducts with PanIN-3 lesions compared to the AL group (7.0%), P < 0.0001. The incidence of PanIN-2 or more lesions was significantly reduced in both ICR (27%; n = 16) and CCR (40%) mice (n = 15; P < 0.001) compared to AL (70%) fed mice (n = 11). The delayed progression of lesions in ICR and CCR mice was associated with reduced proliferation measured by proliferating cell nuclear antigen staining, reduced protein expression of Glut1, increased protein expression of Sirt1, increased serum adiponectin, and decreased serum leptin. CCR resulted in decreased phosphorylated mammalian target of rapamycin and decreased serum insulin-like growth factor-1. In summary, this is the first study to show in LSL-KrasG12D; Pdx-1/Cre mice that ICR and CCR delay the progression of lesions to PDA.

Curcumin enhances the anticancer effects of trichostatin a in breast cancer cells.

Breast cancer patients with HER-2 positive or estrogen receptor negative tumors have a poor prognosis because these tumors are aggressive and respond poorly to standard therapies. Histone deacetylase (HDAC) inhibitors have been shown to decreased cell survival, which suggests that HDAC inhibitors may be developed for preventing and treating breast cancer. Curcumin has anti-inflammatory and proapoptotic effects in cancer cells. We determined whether the HDAC inhibitor, Tricostatin A (TSA) in combination with curcumin would produce greater antiproliferative and apoptotic effects than either agent alone. Increasing the concentration of curcumin from 10 to 20 µM enhanced the growth inhibitory effects of the combination in SkBr3 and 435eB breast cancer cells, which was accompanied by decreased viability along with decreased phosphorylation of ERK and Akt. The decreased cell viability observed in SkBr3 cells when curcumin was combined with TSA led to a G0/G1 cell cycle arrest and increased p21 and p27, and decreased Cyclin D1 protein expression. The combination induced cleavage of caspase 3 and poly(ADP-ribose) polymerase-1, suggesting that cell death occurred by apoptosis. There were no changes in protein expression of Bcl2, Bax, or Bcl-xL and decreased expression of p53. The combination increased protein expression of phosphorylated JNK and phosphorylated p38. Pharmacological inhibition of JNK, but not p38, attenuated the decreased viability induced by the curcumin and TSA combination. We conclude that p53 independent apoptosis induced by combining curcumin and TSA involves JNK activation. These findings provide a rationale for exploring the potential benefits of the combination of curcumin with TSA for treatment of breast cancer.

Celecoxib combined with atorvastatin prevents progression of atherosclerosis.

BACKGROUND: Increased expression of cyclooxygenase (COX-2) contributes to atherosclerosis. Recent studies suggest that COX-2 inhibitors prevent early plaque development but their effects on established lesions are less clear, while the statins promote plaque stability. The purpose of this study is to investigate whether administering a combination of a COX-2 inhibitor with a statin drug alters plaque progression in apo E-/- mice. MATERIALS AND METHODS: Apo E-/- mice were fed a Western diet from 6 to 26 wk of age. At 26 wk, the Western diets supplemented with atorvastatin, celecoxib, or atorvastatin plus celecoxib were given for an additional 12 wk. RESULTS: When the mice were 38 wk of age, the total area occupied by the atherosclerotic lesion was 53% less in the mice fed the combination of atorvastatin + celecoxib P ≤ 0.05) than that of the apo E-/- mice fed the Western diet alone, atorvastatin alone, or celecoxib alone. The decreased extent of atherosclerosis observed in the apo E-/- mice fed the combination of drugs was associated with reduced levels of prostaglandin (PG) E(2,) decreased protein expression of metalloproteinase (MMP)-9, macrophage chemotactic protein (MCP-1), and COX 2, and decreased staining for MMP-9, F4-80 (a marker for macrophages), and vascular cell adhesion molecule (VCAM). CONCLUSION: This study indicates that using statins with a COX-2 inhibitor reduced the extent of atherosclerosis and inflammatory/cell adhesion molecule levels in the apo E-/- mouse model.

PPARgamma activation induces autophagy in breast cancer cells.

It has been previously shown that PPAR gamma ligands induce apoptotic cell death in a variety of cancer cells. Given the evidence that these ligands have a receptor-independent function, we further examined the specific role of PPAR gamma activation in this biological process. Surprisingly, we failed to demonstrate that MDA-MB-231 breast cancer cells undergo apoptosis when treated with sub-saturation doses of troglitazone and rosiglitazone, which are synthetic PPAR gamma ligands. Acridine orange (AO) staining showed acidic vesicular formation within ligand-treated cells, indicative of autophagic activity. This was confirmed by autophagosome formation as indicated by redistribution of LC3, an autophagy-specific protein, and the appearance of double-membrane autophagic vacuoles by electron microscopy following exposure to ligand. To determine the mechanism by which PPAR gamma induces autophagy, we transduced primary mammary epithelial cells with a constitutively active mutant of PPAR gamma and screened gene expression associated with PPAR gamma activation by genome-wide array analysis. HIF1 alpha and BNIP3 were among 42 genes up-regulated by active PPAR gamma. Activation of PPAR gamma induced HIF1 alpha and BNIP3 protein and mRNA abundance. HIF1 alpha knockdown by shRNA abolished the autophagosome formation induced by PPAR gamma activation. In summary, our data shows a specific induction of autophagy by PPAR gamma activation in breast cancer cells providing an understanding of distinct roles of PPAR gamma in tumorigenesis.

The select cyclooxygenase-2 inhibitor celecoxib reduced the extent of atherosclerosis in apo E-/- mice.

Many investigators have suggested that immune activation may trigger the atherosclerotic process. The benefits of aspirin in preventing myocardial infarction have been attributed, in part, to its anti-inflammatory effects. Several reports have documented that cyclooxygenase (COX)-2 is up-regulated in human and mouse atherosclerotic lesions. To clarify the role of COX-2 in atherosclerosis, we conducted a study to test whether the COX-2 inhibitor, celecoxib, prevents the development and progression of the atherosclerotic process. We have used the apo E-/- mouse, a relevant animal model of atherosclerosis that develops fibrofatty lesions similar to human atherosclerosis. Treatment of 4-wk old apo E-/- mice with a standard rodent no. 5020 diet supplemented with 900 ppm of celecoxib for 16 wk led to an 81% reduction in lesion size. The mean lesion area per section (mean +/- SD) of proximal aorta from the apo E-/- mice fed the diet with celecoxib (33,991 +/- 7863 microm2, P < 0.001) was significantly less than that of the untreated apo E-/- mice (183,401 +/- 36,212 microm2). There were no lesions detected in the C57B1/6 mice. Immunohistochemistry of the ileum revealed that there was 80% reduction in staining for intercellular adhesion molecule and 60% reduction in staining for vascular cell adhesion molecule in the celecoxib treated mice. The protective effect of celecoxib was not maintained when the mice were switched after feeding the celecoxib-supplemented diet to the control 5020 diet for an additional 10 wk. These findings demonstrate that selective inhibition of the COX-2 enzyme with celecoxib prevented the development of atherosclerotic lesions in the proximal aortas from apo E-/- mice. One of the possible mechanisms is reduction in expression of the endothelial cell adhesion cell molecules intercellular adhesion molecule and vascular cell adhesion molecule, which plays a key role in the recruitment of inflammatory cells during the early stages of atherogenesis.

Quercetin selectively inhibits bioreduction and enhances apoptosis in melanoma cells that overexpress tyrosinase.

Tyrosinase is expressed in melanoma cells and catalyzes the formation of 3,3',4',5,7-pentahydroxyflavone (quercetin) into reactive quinone species and subsequent glutathionyl adducts. Therefore, we examined the effect of quercetin metabolism on the glutathione (GSH) bioreduction pathway and cell viability in DB-1 melanoma cells that express varying levels of tyrosinase (Tyr+). In a cell-free system, GSH was significantly decreased by quercetin, which coincided with the formation of glutathionyl adducts. In Tyr+ clones, quercetin decreased bioreduction capacity and increased reactive oxygen species (ROS) to a greater degree compared to control cells. The antioxidant/electrophile response element-induced enzymes, glutathione-S-transferase (GST), and nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 were expressed at high levels in Tyr+ cells and contributed to pro-oxidant quercetin metabolism. The basal level of ROS and apoptosis was higher in Tyr+ cells and were selectively increased after exposure to quercetin. The increase in apoptosis following quercetin exposure was p53/Bax mediated and correlated with a decrease in GST-driven bioreduction capacity and an increase in ROS. In conclusion, quercetin can selectively sensitize Tyr+ expressing melanoma cells to apoptosis and may serve as an adjuvant to chemotherapy by enhancing cell death and interfering with GST-mediated drug resistance.

Effect of simultaneous inhibition of epidermal growth factor receptor and cyclooxygenase-2 in HER-2/neu-positive breast cancer.

PURPOSE: HER-2/erbB2/neu is overexpressed in 25% to 30% of all invasive breast cancers and is associated with an aggressive course and reduced survival. HER-2/erbB2/neu breast tumors are frequently associated with up-regulation of cyclooxygenase (COX)-2 and activation of the epidermal growth factor receptor (EGFR) pathway, which promote enhanced cell growth and resistance to apoptosis. This study investigated whether simultaneously blocking both EGFR and COX-2 pathways with ZD1839 and celecoxib, respectively, would be more effective in inhibiting cell growth and inducing apoptosis than either agent alone. EXPERIMENTAL DESIGN: The effects of ZD1839, celecoxib, and their combination on cell survival, cell cycle progression, and apoptosis were determined in NMF11.2 mouse mammary tumor cells. We also investigated the potential downstream EGFR-activated pathways that are altered by simultaneous inhibition of COX-2 and EGFR. RESULTS: Celecoxib alone and ZD1839 alone produced a concentration- and time-dependent inhibition of cell survival. Both agents combined produced supra-additive inhibitory effects on cell survival and apoptosis compared with either agent alone. This effect was associated with increased cleaved poly(ADP-ribose)polymerase and reduced protein expression of bcl-2. Phosphorylation of extracellular signal-regulated kinase 1/2 was partially blocked by ZD1839 and celecoxib alone and was completely blocked by the combination of both agents. The enhanced proapoptotic effects of the combined agents were also associated with decreased phosphorylation of Akt and increased phosphorylation of p38. CONCLUSIONS: These findings show that both COX-2 and EGFR are important targets for inhibiting survival and inducing apoptosis in breast cancer. The data suggest a potential cross-talk between COX-2 and EGFR signaling in breast cancer cells overexpressing HER-2/erbB2/neu.

Cyclooxygenase (COX)-2-dependent effects of the inhibitor SC236 when combined with ionizing radiation in mammary tumor cells derived from HER-2/neu mice.

Cyclooxygenase (COX)-2-derived prostaglandins (PGs) are thought to contribute to tumor growth and resistance to radiation therapy. COX-2 protein expression is increased in many tumors including those of the breast. COX-2-derived PGs have been shown to protect cells from radiation damage. This study evaluated the role of COX-2-derived PG in radiation treatment by using the NMF11.2 mammary tumor cell line originally obtained from HER-2/neu mice that overexpress HER-2/neu. We determined whether the effects of the COX-2 inhibitor SC236 on cell growth, radiation-induced PGE2 production and COX expression, cell cycle redistribution, and vascular endothelial growth factor (VEGF) were acting through COX-2-dependent mechanisms. The NMF11.2 cells expressed both COX-1 and COX-2 protein and mRNA. The radiation treatment alone led to a dose-dependent increase in the levels of COX-2 mRNA and COX-2 protein, which was associated with an increase in the production of PGE2 and prostacyclin (PGI2). Treating NMF11.2 cells with high concentrations (20 microM) of SC236 for 48 h reduced the radiation-induced increase in COX-2 activity and also decreased cell growth. SC236 (20 microM) increased the accumulation of the cells in the radiosensitive G2-M phase of the cell cycle. However, a low concentration (5 microM) of SC236 was adequate to reduce COX-2 activity. The lower concentration of SC236 (5 microM) also decreased cell growth after a longer incubation period (96 h) and, in combination with a 2 or 5 Gy dose, led to an accumulation of cells in G2-M phase. Restoring PG to control values in cells treated with 5 microM SC236 prevented the growth inhibition and G2-M cell cycle arrest. Radiation treatment of NMF11.2 cells also increased VEGF protein expression and VEGF secretion in a dose-dependent manner, which was blocked in those cells pretreated with 20 microM SC236 but not in those pretreated with 5 microM SC236. These findings indicate that the COX-2 inhibitor SC236 reduced cell growth and arrested cells in the G2-M phase of the cell cycle by mechanisms that are both dependent and independent of PG production while its effects on VEGF appear to be independent of COX-2.

The cyclooxygenase-2 inhibitor, celecoxib, prevents the development of mammary tumors in Her-2/neu mice.

Evidence is now available showing that cyclooxygenase (COX)-2, which is involved in prostaglandin production, is overexpressed in many types of tumors including breast. Several reports have indicated that HER-2/neu-positive breast tumors are associated with an increased amount of COX-2 protein. In this study, we evaluated the effectiveness of the select COX-1 and COX-2 inhibitors in preventing mammary tumor development in HER-2/neu transgenic mice. At 4 weeks of age, female HER-2/neu mice were fed a #5020 rodent diet supplemented with 900 ppm celecoxib, a COX-2 inhibitor, 64 ppm of SC560, a COX-1 inhibitor, or the unsupplemented #5001 diet (control). The incidence of mammary tumors was significantly lower in the celecoxib-fed mice (71%; P = 0.001 versus control) than in the control mice (95%) or in the SC560-fed mice (91%). Celecoxib-treated mice also developed fewer tumors (1.3 +/- 1.1 SD; P = 0.039 versus control) than the control mice (2.2 +/- 1.2) or the SC560 treated mice (2.3 +/- 1.3). The median time to tumor development was 266 days in the control group versus 291 days in the celecoxib-treated group (P = 0.003 versus control). Lung metastasis was also reduced by treatment with celecoxib. The COX-1 inhibitor SC560 had no protective effect. The protection offered by celecoxib was associated with significantly lower concentrations of prostacyclin and prostaglandin E(2) in mammary tumors and their adjacent mammary glands. Our findings provide additional preclinical evidence to support the clinical studies to investigate the potential effectiveness of COX-2 inhibitors in protecting woman who are at high risk for breast cancer.

Hyperlipoproteinemic low-density lipoprotein receptor-deficient mice are more susceptible to sepsis than corresponding wild-type mice.

High circulating concentrations of lipoproteins have been shown to modify the cytokine response and reduce mortality after endotoxin or live bacterial challenge. Sepsis, however, is more complex than endotoxemia, and it is not clear whether elevated plasma lipoproteins will be protective. Previous studies have shown that the low-density-lipoprotein receptor deficient (LDLR-/-) mice with increased circulating LDL are protected against the lethal effects of endotoxemia and Gram-negative infection. We evaluated whether the LDLR-/- mice would be protected against the effects of sepsis induced by cecal ligation and puncture (CLP). Mortality was greater in LDLR-/-mice than in control C57Bl/6J mice. At 120 h after inducing sepsis, 20% of the control mice survived whereas none of theLDLR-/-mice were alive. Prior to inducing sepsis, serum concentrations of amyloid A protein and lipopolysaccharide binding protein (LBP) were significantly elevated in the LDLR-/-mice in comparison to the C57Bl/6J mice. Protein expression of sCD14 was also greater in the serum from the LDLR-/-mice than the C57Bl/6J mice. The elevated serum concentrations of LBP and CD14 were not associated with increases in the levels of liver CD14 mRNA and LBP mRNA. After inducing sepsis, serum concentration of interleukin (IL)-1beta was also significantly higher in LDLR-/-mice than in the control C57Bl/6J mice. These findings indicate that the LDLR-/-mice were more susceptible to the lethal effects of sepsis induced by CLP. The LDLR-/-mice also had higher serum concentrations of baseline, acute phase response proteins, SAA and LBP, and increased production of IL-1beta in response to CLP.