The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer.

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.

IRF-3 partners Bax in a viral-induced dance macabre.

In this issue of The EMBO Journal, Chattopadhyay et al (2010) describe a surprising new mechanism for how viral dsRNA detection by the RIG-I/MAVS signalling complex can initiate apoptosis. Independent of its transcriptional function, a pool of interferon regulatory factor (IRF)-3 activated downstream of MAVS can bind to and activate cytosolic Bax, resulting in Bax translocation to the mitochondria and initiation of the intrinsic apoptotic pathway.

Clic4, a novel protein that sensitizes ß-cells to apoptosis.

Introduction: La préservation et/ou l'expansion de la masse des cellules ß pourraient constituer des approches prometteuses dans le traitement du diabète. L'une des stratégies clés serait de réduire l'apoptose des cellules ß. Le chloride intracellular channel protein 4 (Clic4) est une protéine exprimée de manière ubiquitaire et supposée agir dans de nombreux processus cellulaires tels que le contrôle du cycle cellulaire, la différenciation cellulaire et l'apoptose. Ici, nous avons étudié le rôle de Clic4 dans l'apoptose des cellules ß pancréatiques en utilisant des cellules ßTC-tet et des îlots de Langerhans issus de souris knockout pour Clic4 (ßClic4KO). Résultats: L'expression de l'ARNm et de la protéine Clic4 était augmentée par un traitement aux cytokines dans les cellules ßTC-tet et encore plus fortement dans des îlots isolés de souris. De plus, la sous-expression de Clic4 dans les cellules ßTC-tet diminuait leur sensibilité à l'apoptose induite par les cytokines. La sous-expression de Clic4 dans les cellules ßTC-tet n'affectait pas l'expression des ARNm de Bcl-2 et Bad, mais augmentait leur expression protéique ainsi que la forme phosphorylée de Bad. Les mêmes résultats ont été obtenus sur des îlots isolés de souris contrôles et ßClic4KO. De plus, les îlots issus de souris ßClic4KO présentaient une augmentation de l'expression de la protéine Bcl-xL. Dans le but de déterminer si Clic4 augmentait l'expression de Bcl-2 et Bad via une interaction protéique directe, nous avons immunoprécipité Clic4 à partir de cellules ßTC-tet à l'aide d'anticorps dirigés contre la partie C- ou N-terminale de la protéine, puis nous avons soumis les immunoprécipités à une analyse de spectrométrie de masse. Aucune co- immunoprécipitation avec Bcl-2 ou d'autres protéines de la famille Bcl-2 n'a été détectée. Cependant, de manière intéressante, Clic4 était co-purifié avec plusieurs protéines du protéasome suggérant un rôle de Clic4 dans la dégradation des protéines. Par conséquent, nous avons étudié la demi-vie de Bcl-2 et Bad, et avons observé que la sous-expression de Clic4 dans les cellules ßTC-tet augmentait la demi-vie de ces protéines. De plus, l'expression de l'ARNm et de la protéine Clic4 était également augmentée lors d'un stress du réticulum endoplasmique induit par la thapsigargine dans les cellules ßTC-tet. La sous-expression de Clic4 dans les cellules ßTC-tet ou chez les KO diminuait la sensibilité des cellules ß à l'apoptose induite par la thapsigargine ou l'acide palmitique, respectivement. Conclusion: Ces résultats suggèrent que Clic4 sensibilise les cellules ß à l'apoptose induite par les cytokines ou l'acide palmitique/thapsigargine (stress du réticulum endoplasmique). De plus, la sous-expression de Clic4 améliore la survie des cellules ß en diminuant la dégradation de Bcl-2 et Bcl-xL, et en augmentant le niveau total de Bad phosphorylé, peut-être suite à une interaction de Clic4 avec le protéasome.

Multifunctional mitoxantrone-conjugated magnetic nanosystem for targeted therapy of folate receptor-overexpressing malignant cells.

BACKGROUND: Targeted delivery of anticancer chemotherapeutics such as mitoxantrone (MTX) can significantly intensify their cytotoxic effects selectively in solid tumors such as breast cancer. In the current study, folic acid (FA)-armed and MTX-conjugated magnetic nanoparticles (MNPs) were engineered for targeted eradication of folate receptor (FR)-positive cancerous cells. Polyethylene glycol (PEG), FA and MTX were covalently conjugated onto the MNPs to engineer the PEGylated FA-MTX-MNPs. The internalization studies were performed using fluorescein isothiocyanate (FITC)-labeled FA-decorated MNPs (FA-FITC-MNPs) in both FR-positive MCF-7 cells and FR-negative A549 cells by means of fluorescence microscopy and flow cytometry. The cellular and molecular impacts of FA-MTX-MNPs were examined using trypan blue cell viability and FITC-labeled annexin V apoptosis assays and 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder and quantitative polymerase chain reaction (qPCR) assays. RESULTS: The FR-positive MCF-7 cells showed significant internalization of the FA-FITC-MNPs, but not the FR-negative A549 cells. The FR-positive cells treated with the PEGylated FA-MTX-MNPs exhibited the IC50 values of 3 μg/mL and 1.7 μg/mL, 24 h and 48 h post-treatment, respectively. DAPI staining and DNA ladder assays revealed significant condensation of nucleus and fragmentation of genomic DNA in the FR-positive MCF-7 cells treated with the PEGylated FA-MTX-MNPs as compared to the FR-negative A549 cells. The FITC-labeled annexin V assay confirmed emergence of late apoptosis (>80%) in the FR-positive MCF-7 cells treated with the PEGylated FA-MTX-MNPs, but not in the FR-negative A549 cells. The qPCR analysis confirmed profound cytotoxic impacts via alterations of apoptosis-related genes induced by MTX-FA-MNPs in MCF-7 cells, but not in the A549 cells. CONCLUSION: Our findings evince that the engineered PEGylated FA-MTX-MNPs can be specifically taken up by the FR-positive malignant cells and effectively demolish them through up-regulation of Bcl-2-associated X protein (Bax) and Caspase 9 and down-regulation of AKt. Hence, the engineered nanosystem is proposed for simultaneous targeted imaging and therapy of various cancers overexpressing FRs.

Expressão da proteína Bax no tecido mamário normal de mulheres no menacme tratadas com raloxifeno

OBJETIVO: avaliar a expressão do antígeno Bax no epitélio mamário normal de mulheres na pré-menopausa tratadas com raloxifeno. MÉTODOS: estudo randomizado duplo-cego, envolvendo 33 mulheres pré-menopáusicas com fibroadenoma. As pacientes foram divididas em dois grupos: Placebo, (n=18) e Raloxifeno 60 mg, (n=15). A medicação foi usada durante 22 dias, começando no primeiro dia do ciclo menstrual. Uma biópsia foi realizada no 23º dia do ciclo menstrual, durante a qual uma amostra do tecido mamário normal adjacente ao fibroadenoma foi coletada e submetida a estudo imuno-histoquímico utilizando o anticorpo policlonal anti-Bax para avaliar a expressão da proteína Bax. A imunorreação para a proteína Bax foi avaliada, levando-se em consideração a intensidade e a fração de células coradas, cuja combinação resultou em um escore final de 0 a 6. Os casos com escore final >3 foram classificados como positivos para proteína Bax. O teste do c2 foi usado para análise estatística dos dados (p<0,05). RESULTADOS: a porcentagem de positividade da proteína Bax foi 66,7 e 73,3% nos Grupos A e B, respectivamente. Não houve diferença significante na expressão do Bax entre os dois grupos (p=0,678). CONCLUSÕES: o raloxifeno administrado por 22 dias na dose de 60 mg/dia não alterou a expressão da proteína Bax no tecido mamário normal de mulheres no menacme.

Effects of hyperprolactinemia and ovariectomy on the tibial epiphyseal growth plate and bone formation in mice

PURPOSE: To evaluate the effects of ovariectomy and the hyperprolactinemia procedure in the tibial epiphyseal growth plate of female mice.METHODS: In this study, the epiphyseal growth plate of ovariectomized (OVX) and/or rendered hyperprolactinemic female mice by 50 days of treatment with 200 μg metoclopramide (M) was evaluated morphologically, morphometrically and immuno-histochemically. Forty female and adult mice were divided into four groups according to treatment: V group - animals treated with saline solution; H group - hyperprolactinemic animals; Ovx/V group - ovariectomized animals and treated with saline solution; Ovx/H group - hyperprolactinemic and ovariectomized animals. After the treatment period, the animals were sacrificed, tibia was removed and fixed in 10% buffered formalin and decalcified in 10% formic acid. The material was immersed in paraffin and subjected to histological processing in paraffin. The sections were stained with Masson's trichrome and immunohistochemistry was carried out for the pro-apoptotic protein BCL-2. The images for the morphological and morphometric study were analyzed with the imaging program AxioVision 4.8 (Carl-Zeiss(r), Germany).RESULTS: The combination of hyperprolactinemia and the ovariectomy procedure decreased the number of resting chondrocytes 1.5-fold, the number of proliferative chondrocytes 1.8-fold; the percentage of resting cartilage 2.4-fold and the percentage of trabecular bone 2.1-fold, compared with respective control animals.CONCLUSION: The procedure of ovariectomy combined with the metoclopramide-induced hyperprolactinemia in female mice has showed marked bone degeneration due to significant decrease of cell proliferation in the epiphyseal growth plate and bone formation.

Apoptose na maturação placentária de vacas em diferentes estágios de gestação: evidenciação imuno-histoquímica e bioquímica

Apoptose tem um papel importante na manutenção da homeostase placentária, e o desequilíbrio desse processo pode comprometer a gestação. O objetivo do presente estudo foi avaliar a ocorrencia de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomos de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imuno-histoquímica e isolamento de DNA. As lâminas obtidas foram coradas em HE, ou submetidas à análise imuno-histoquímica das proteínas pró-apoptóticas caspase-3 e Bax, e da proteína anti-apoptótica Bcl-2. O DNA isolado foi submetido à eletroforese em gel de agarose para detecção da fragmentação internucleossômica do genoma. Os resultados de histomorfometria revelaram que as células apoptóticas aumentaram progressivamente com o avanço da gestação. Confirmou-se a apoptose pela fragmentação característica do DNA genômico, visualizada pelo clássico "padrão em escada" na eletroforese em gel de agarose. Adcionalmente, a imunoexpressão de caspase-3, Bax e Bcl-2 foram observadas em todas as amostras. Entretanto, a proteína caspase-3 apresentou marcação mais intensa em todos os tempos gestacionais, quando comparada com a marcação das proteínas Bcl-2 e Bax. Esses resultados confirmam e reforçam a importância da apoptose na maturação placentária. Além disto, indica que caspase-3, Bax e Bcl-2 estão envolvidas nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial ao longo da gestação, contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.

Prognostic Factors In Breast Cancer. With Special Reference to Cyclins A, B1, D1 and E, MMP-1 and Decorin

Breast cancer is a highly heterogenous malignancy, which despite of the similar histological type shows different clinical behaviour and response to therapy. Prognostic factors are used to estimate the risk for recurrence and the likelihood of treatment effectiveness. Because breast cancer is one of the most common causes of cancer death in women worldwide, identification of new prognostic markers are needed to develop more specific and targeted therapies. Cancer is caused by uncontrolled cell proliferation. The cell cycle is controlled by specific proteins, which are known as cyclins. They function at important checkpoints by activating cyclin-dependent kinase enzymes. Overexpression of different cyclins has been linked to several cancer types and altered expression of cyclins A, B1, D1 and E has been associated with poor survival. Little is known about the combined expression of cyclins in relation to the tumour grade, breast cancer subtype and other known prognostic factors. In this study cyclins A, B1 and E were shown to correlate with histological grade, Ki-67 and HER2 expression. Overexpression of cyclin D1 correlated with receptor status and non-basal breast cancer suggesting that cyclin D1 might be a marker of good prognosis. Proteolysis in the surrounding tumour stroma is increased during cancer development. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading extracellular matrix proteins. Increased expression and activation of several MMPs have been found in many cancers and MMPs appear to be important regulators of invasion and metastasis. In this study MMP-1 expression was analysed in breast cancer epithelial cells and in cancer associated stromal cells. MMP-1 expression by breast cancer epithelial cells was found to carry an independent prognostic value as did Ki-67 and bcl-2. The results suggest that in addition to stromal cells MMP-1 expression in tumour cells control breast cancer progression. Decorin is a small proteoglycan and an important component of the extracellular matrix. Decorin has been shown to inhibit growth of tumour cells and reduced decorin expression is associated with a poor prognosis in several cancer types. There has been some suspicion wheather different cancer cells express decorin. In this study decorin expression was shown to localize only in the cells of the original stroma, while breast cancer epithelial cells were negative for decorin expression. However, transduction of decorin in decorin-negative human breast cancer cells markedly modulated the growth pattern of these cells. This study provides evidence that targeted decorin transduction to breast cancer cells could be used as a novel adjuvant therapy in breast malignancies.

Immunological effects of donor lymphocyte infusion in patients with chronic myelogenous leukemia relapsing after bone marrow transplantation

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.

Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression

Curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. However, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. In the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma HT-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and DNA ladder formation. At the same time, p53, phospho-p53 (Ser15), and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, pro-caspase-3, and pro-caspase-9 were determined by Western blot analysis. The colon adenocarcinoma cells were treated with curcumin (0-75 µM) for 0-24 h. We observed that p53 was highly expressed in HT-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. An increase in expression of the pro-apoptotic factor Bax and a decrease in expression of the anti-apoptotic factor Bcl-2 were also observed in a time-dependent manner after exposure of 50 µM curcumin, while the expression of the anti-apoptotic factor Bcl-xL was unchanged. Curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. These data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human HT-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. This property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients.

A mechanistic view of mitochondrial death decision pores

Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

Beneficial effect of recombinant human growth hormone on the intestinal mucosa barrier of septic rats

The objective of the present study was to investigate the effects of recombinant human growth hormone (rhGH) on the intestinal mucosa barrier of septic rats and explore its possible mechanism. Female Sprague-Dawley rats were randomized into three groups: control, Escherichia coli-induced sepsis (S) and treatment (T) groups. Groups S and T were subdivided into subgroups 1d and 3d, respectively. Expression of liver insulin-like growth factor-1 (IGF-1) mRNA, Bcl-2 and Bax protein levels and the intestinal Bax/Bcl-2 ratio, and plasma GH and IGF-1 levels were determined. Histological examination of the intestine was performed and bacterial translocation was determined. rhGH significantly attenuated intestinal mucosal injuries and bacterial translocation in septic rats, markedly decreased Bax protein levels, inhibited the decrease of Bcl-2 protein expression and maintained the Bax/Bcl-2 ratio in the intestine. rhGH given after sepsis significantly improved levels of plasma GH (T1d: 1.28 ± 0.24; T3d: 2.14 ± 0.48 µg/L vs S1d: 0.74 ± 0.12; S3d: 0.60 ± 0.18 µg/L; P < 0.05) and IGF-1 (T1d: 168.94 ± 65.67; T3d: 201.56 ± 64.98 µg/L vs S1d: 116.72 ± 13.96; S3d: 107.50 ± 23.53 µg/L; P < 0.05) and expression of liver IGF-1 mRNA (T1d: 0.98 ± 0.20; T3d: 1.76 ± 0.17 vs S1d: 0.38 ± 0.09; S3d: 0.46 ± 0.10; P < 0.05). These findings indicate that treatment with rhGH had beneficial effects on the maintenance of the integrity of the intestinal mucosa barrier in septic rats.

High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60Co-γ-rays. NIH 3T3 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 60Co-γ-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 ± 6.51 vs 66.0 ± 3.51 and 67.7 ± 7.37; transfection: 121.7 ± 6.50 vs 65.3 ± 3.51 and 69.0 ± 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, c-myc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes.

Efficacy of geraniol but not of β-ionone or their combination for the chemoprevention of rat colon carcinogenesis

β-ionone (βI), a cyclic isoprenoid, and geraniol (GO), an acyclic monoterpene, represent a promising class of dietary chemopreventive agents against cancer, whose combination could result in synergistic anticarcinogenic effects. The chemopreventive activities of βI and GO were evaluated individually or in combination during colon carcinogenesis induced by dimethylhydrazine in 48 3-week-old male Wistar rats (12 per group) weighing 40-50 g. Animals were treated for 9 consecutive weeks with βI (16 mg/100 g body weight), GO (25 mg/100 g body weight), βI combined with GO or corn oil (control). Number of total aberrant crypt foci (ACF) and of ACF ≥4 crypts in the distal colon was significantly lower in the GO group (66 ± 13 and 9 ± 2, respectively) compared to control (102 ± 9 and 17 ± 3) and without differences in the βI (91 ± 11 and 14 ± 3) and βI+GO groups (96 ± 5 and 19 ± 2). Apoptosis level, identified by classical apoptosis morphological criteria, in the distal colon was significantly higher in the GO group (1.64 ± 0.06 apoptotic cells/mm²) compared to control (0.91 ± 0.07 apoptotic cells/mm²). The GO group presented a 0.7-fold reduction in Bcl-2 protein expression (Western blot) compared to control. Colonic mucosa concentrations of βI and GO (gas chromatography/mass spectrometry) were higher in the βI and GO groups, respectively, compared to the control and βI+GO groups. Therefore, GO, but not βI, represents a potential chemopreventive agent in colon carcinogenesis. Surprisingly, the combination of isoprenoids does not represent an efficient chemopreventive strategy.

The cytotoxicity of methacryloxylethyl cetyl ammonium chloride, a cationic antibacterial monomer, is related to oxidative stress and the intrinsic mitochondrial apoptotic pathway

Antibacterial monomers incorporated in dentin bonding systems may have toxic effects on the pulp. Thus, the cytotoxicity of antibacterial monomers and its underlying mechanisms must be elucidated to improve the safety of antibacterial monomer application. The influence of an antibacterial monomer, methacryloxylethyl cetyl ammonium chloride (DMAE-CB), on the vitality of L929 mouse fibroblasts was tested using MTT assay. Cell cycle progression was studied using flow cytometry. Production of intracellular reactive oxygen species (ROS) after DMAE-CB treatment was measured using 2,7-dichlorodihydrofluorescein diacetate staining and flow cytometry analysis. Loss of mitochondrial membrane potential, disturbance of Bcl-2 and Bax expression, as well as release of cytochrome C were also measured using flow cytometry analysis or Western blot to explore the possible involvement of the mitochondrial-related apoptotic pathway. DMAE-CB elicited cell death in a dose-dependent manner and more than 50% of cells were killed after treatment with 30 µM of the monomer. Both necrosis and apoptosis were observed. DMAE-CB also induced G1- and G2-phase arrest. Increased levels of intracellular ROS were observed after 1 h and this overproduction was further enhanced by 6-h treatment with the monomer. DMAE-CB may cause apoptosis by disturbing the expression of Bcl-2 and Bax, reducing the mitochondrial potential and inducing release of cytochrome C. Taken together, these findings suggest that the toxicity of the antibacterial monomer DMAE-CB is associated with ROS production, mitochondrial dysfunction, cell cycle disturbance, and cell apoptosis/necrosis.