Good guy or bad guy: the opposing roles of microRNA 125b in cancer.

MicroRNAs (miRNAs) are a class of non-coding RNAs that post-transcriptionally silence target mRNAs. Dysregulation of miRNAs is a frequent event in several diseases, including cancer. One miRNA that has gained special interest in the field of cancer research is miRNA-125b (miR-125b). MiR-125b is a ubiquitously expressed miRNA that is aberrantly expressed in a great variety of tumors. In some tumor types, e.g. colon cancer and hematopoietic tumors, miR-125b is upregulated and displays oncogenic potential, as it induces cell growth and proliferation, while blocking the apoptotic machinery. In contrast, in other tumor entities, e.g. mammary tumors and hepatocellular carcinoma, miR-125b is heavily downregulated. This downregulation is accompanied by de-repression of cellular proliferation and anti-apoptotic programs, contributing to malignant transformation. The reasons for these opposing roles are poorly understood. We summarize the current knowledge of miR-125b and its relevant targets in different tumor types and offer several hypotheses for the opposing roles of miR-125b: miR-125b targets multiple mRNAs, which have diverse functions in individual tissues. These target mRNAs are tissue and tumor specifically expressed, suggesting that misregulation by miR-125b depends on the levels of target gene expression. Moreover, we provide several examples that miR-125b upregulation dictates oncogenic characteristics, while downregulation of miR-125b corresponds to the loss of tumor suppressive functions. Thus, in different tumor entities increased or decreased miR-125b expression may contribute to carcinogenesis.

Xanthohumol-induced transient superoxide anion radical formation triggers cancer cells into apoptosis via a mitochondria-mediated mechanism.

Oxidative stress and increased release of reactive oxygen species (ROS) are associated with apoptosis induction. Here we report ROS-mediated induction of apoptosis by xanthohumol (XN) from hops. XN at concentrations of 1.6-25 microM induced an immediate and transient increase in superoxide anion radical (O(2)(-*)) formation in 3 human cancer cell lines (average+/-SD EC(50) of maximum O(2)(-*) induction=3.1+/-0.8 microM), murine macrophages (EC(50)=4.0+/-0.3 microM), and BPH-1 benign prostate hyperplasia cells (EC(50)=4.3+/-0.1 microM), as evidenced by the O(2)(-*)-specific indicator dihydroethidium. MitoSOX Red costaining and experiments using isolated mouse liver mitochondria (EC(50)=11.4+/-1.8 microM) confirmed mitochondria as the site of intracellular O(2)(-*) formation. Antimycin A served as positive control (EC(50)=12.4+/-0.9 microM). XN-mediated O(2)(-*) release was significantly reduced in BPH-1 rho(0) cells harboring nonfunctional mitochondria (EC(50)>25 microM) and by treatment of BPH-1 cells with vitamin C, N-acetylcysteine (NAC), or the superoxide dismutase mimetic MnTMPyP. In addition, we demonstrated a rapid 15% increase in oxidized glutathione and a dose-dependent overall thiol depletion within 6 h (IC(50)=24.3+/-11 microM). Respiratory chain complexes I-III were weakly inhibited by XN in bovine heart submitochondrial particles, but electron flux from complex I and II to complex III was significantly inhibited in BPH-1 cells, with IC(50) values of 28.1 +/- 2.4 and 24.4 +/- 5.2 microM, respectively. Within 15 min, intracellular ATP levels were significantly reduced by XN at 12.5 to 50 microM concentrations (IC(50)=26.7+/-3.7 microM). Concomitantly, XN treatment caused a rapid breakdown of the mitochondrial membrane potential and the release of cytochrome c, leading to apoptosis induction. Pre- or coincubation with 2 mM NAC and 50 microM MnTMPyP at various steps increased XN-mediated IC(50) values for cytotoxicity in BPH-1 cells from 6.7 +/- 0.2 to 12.2 +/- 0.1 and 41.4 +/- 7.6 microM, and it confirmed XN-induced O(2)(-*) as an essential trigger for apoptosis induction. In summary, we have identified mitochondria as a novel cellular target of XN action, resulting in increased O(2)(-*) production, disruption of cellular redox balance and mitochondrial integrity, and subsequent apoptosis.

Differential selection for B-raf and Ha-ras mutated liver tumors in mice with high and low susceptibility to hepatocarcinogenesis.

Activation of the Ras/Raf/MEK/ERK pathway is frequently observed in animal and human tumors. In our study, we analyzed B-raf codon 637 (formerly 624) and Ha-ras codon 61 mutations in liver tumors from C3H, B6C3F1 and C56BL mice which differ considerably with regard to their susceptibility to hepatocarcinogenesis. In total, 73% (102/140) of tumors induced by a single application of N-nitrosodiethylamine or 7,12-dimethylbenz[a]anthracene contained either B-raf or Ha-ras mutations and only <3% (4/140) were mutated in both genes. In addition, B-raf mutations were present in 76% (19/25) of early precancerous liver lesions. The prevalence of Ha-ras mutated tumors was significantly higher in the susceptible C3H and B6C3F1 mouse strains (39-50%) than in the comparatively resistant C57BL mouse (7%). B-raf mutated tumors, by contrast, were more frequent in C57BL mice (68%) than in the other two strains (17-45%). Taken together, our findings indicate that alterations affecting the Ras/Raf/MEK/ERK signalling pathway are a hallmark of carcinogen-induced liver tumors in mice. Moreover, our results show that mutational activation of B-raf in liver tumors of different mouse strains is, by contrast to Ha-ras, inversely related to their susceptibility to hepatocarcinogenesis. Although activated Ras and Raf proteins are assumed to have similar biological effects because they feed into the same signalling pathway, there seem to be subtle strain-specific differences in selection processes favouring the preferential outgrowth of either B-raf or Ha-ras mutated tumor populations in mouse liver.

Proteome analysis of chemically induced mouse liver tumors with different genotype.

Mouse liver tumors frequently harbor mutations in Ha-ras, B-raf, or Ctnnb1 (encoding beta-catenin). We conducted a proteome analysis with protein extracts from normal mouse liver and from liver tumors which were induced by a single injection of N-nitrosodiethylamine (DEN) as initiator followed by multiple injections of two different polychlorinated biphenyls (PCBs) as tumor promoters, or corn oil as a control. Liver tumors were stratified into two classes: they were either mutated in Ctnnb1 and positive for the marker glutamine synthetase (GS(+)), or they lacked Ctnnb1 mutations and were therefore GS-negative (GS(-)). Proteome analysis by 2-DE and MS revealed 98 significantly deregulated proteins, 44 in GS(+) and 54 in GS(-) tumors. Twelve of these proteins showed expression changes in both tumor types, but only seven of them were deregulated in the same direction. Several of the identified enzymes could be assigned to fundamental metabolic or other cellular pathways with characteristically different alterations in GS(+) and GS(-) tumors such as ammonia and amino acid turnover, cellular energy supply, and calcium homeostasis. Our data suggest that GS(+) and GS(-) tumor cells show a completely different biology and use divergent evolutionary strategies to gain a selective advantage over normal hepatocytes.

Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane on proliferation of human colon cancer cells in vitro.

Isothiocyanates (ITCs) and indoles derived from cruciferous vegetables possess growth-inhibiting and apoptosis-inducing activities in cancer cell lines in vitro. ITCs like sulforaphane (SFN) are cytotoxic, whereas indoles including indole-3-carbinol or its condensation product 3,3'-diindolylmethane (DIM) are acting by cytostatic mechanisms in human colon cancer cell lines. In the present study, we have investigated the impact of defined combinations of SFN and DIM (ratio 1:4, 1:2, 1:1, 2:1 and 4:1) on cell proliferation, cell-cycle progression and apoptosis induction in cultured 40-16 colon carcinoma cells. Calculations of combination effects were based on the method of Chou et al. (1984) Adv. Enzyme Regul., 22, 27-55, and were expressed as a combination index (CI) with CI < 1, CI = 1 or CI > 1 representing synergism, additivity or antagonism, respectively. Interestingly, at a total drug concentration of 2.5 microM, all combinations of SFN and DIM were antagonistic. With increasing concentrations, the antagonistic effect gradually turned into a synergistic interaction at the highest combined cytotoxic concentration of 40 microM. Cell-cycle analyses with SFN:DIM ratios of 1:1, 1:2 and 1:4 and total concentrations between 10 and 25 microM confirmed antagonism at low and additive effects at higher doses. SFN (10 microM) in combination with DIM (10 microM) resulted in strong G(2)/M cell-cycle arrest, which was not observed with either compound alone. Our results indicate that cytotoxic concentrations of SFN:DIM combinations affect cell proliferation synergistically. At low total concentrations (below 20 microM), which are physiologically more relevant, the combined broccoli compounds showed antagonistic interactions in terms of cell growth inhibition. These data stress the need for elucidating mechanistic interactions for better predicting beneficial health effects of bioactive food components.

PCB 153, a non-dioxin-like tumor promoter, selects for beta-catenin (Catnb)-mutated mouse liver tumors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental toxicants which act as liver tumor promoters in rodents and can be classified as either dioxin-like or non-dioxin (phenobarbital [PB])-like inducers of cytochrome P-450. Since we have previously shown that tumor promotion by PB leads to clonal outgrowth of beta-catenin (Catnb)-mutated but not Ha-ras-mutated mouse liver tumors, we were interested to know whether the non-dioxin-like tumor promoter 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) shows the same selective pressure during tumor promotion. Male B6129SF2/J mice were given a single injection of N-nitrosodiethylamine (90 mg/kg body weight) at 9 weeks of age, followed by 39 weeks of treatment with PCB 153 (20 biweekly ip injections of 300 mumol/kg body weight) or corn oil as a control. Animals were killed 15 weeks after the last PCB 153 injection and liver tumors were identified by immunohistochemical staining of glutamine synthetase (GS) and analyzed for Catnb, Ha-ras, and B-raf mutations. Quantitative analyses revealed that GS-positive tumors were much larger and more frequent in livers from PCB 153-treated mice than in control animals, whereas GS-negative tumors were similar in both groups. Almost 90% (34/38) of all tumors from PCB 153-treated animals contained Catnb mutations, which compares to approximately 45% (17/37) of tumors in the control group. Ha-ras- and B-raf-mutated liver tumors were rare and not significantly different between treatment groups. These results clearly indicate that PCB 153 strongly selects for Catnb-mutated, GS-positive liver tumors, which is similar to the known action of PB, a prototypical tumor promoter in rodent liver.