Swatee Dey

Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells

The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

Cocaine exposure in vitro induces apoptosis in fetal locus coeruleus neurons by altering the Bax/Bcl-2 ratio and through caspase-3 apoptotic signaling.

Cocaine inhibits survival and growth of rat locus coeruleus (LC) neurons, which may mediate alterations in attention, following in utero exposure to cocaine. These effects are most severe in early gestation during peak neuritogenesis. Prenatal cocaine exposure may specifically decrease LC survival through an apoptotic pathway involving caspases. Dissociated fetal LC neurons or substantia nigra (SN) neurons (control) were exposed in vitro to a pharmacologically active dose of cocaine hydrochloride (500 ng/ml) and assayed for apoptosis using terminal deoxynucleotidyl transferase mediated DNA nick end labeling and Hoechst methodologies. Cocaine exposure decreased survival and induced apoptosis in LC neurons, with no changes in survival of SN neurons. Activation of apoptotic signal transduction proteins was determined using enzyme assays and immunoblotting at 30 min, 1 h, 4 h and 24 h. In LC neurons, Bax levels were induced at 30 min and 1 h, following cocaine treatment, and Bcl-2 levels remained unchanged at all time points, altering the Bax/Bcl-2 ratio. The ratio was reversed for SN neurons (elevated Bcl-2 levels and transient reduction of Bax levels). Further, cocaine exposure significantly increased caspase-9 and caspase-3 activities at all time points, without changes in caspase-8 activity in LC neurons. In addition, cleavage of caspase-3 target proteins, alpha-fodrin and poly (ADP-ribose) polymerase (PARP) were observed following cocaine treatment. In contrast, SN neurons showed either significant reductions, or no significant changes, in caspase-3, -8 or -9 activities or caspase-3 target proteins, alpha-fodrin and PARP. Thus, cocaine exposure in vitro may preferentially induce apoptosis in fetal LC neurons putatively regulated by Bax, via activation of caspases and their downstream target proteins.

Par-4, a pro-apoptotic gene, inhibits radiation-induced NF kappa B activity and Bcl-2 expression leading to induction of radiosensitivity in human prostate cancer cells PC-3.

Ionizing radiation caused induction NFκB activity and Bcl-2 protein expression in the radioresistant p53 null human prostate cancer cell line, PC-3. Exposure of PC-3 cells to Ad5-IkB super-repressor inhibited radiation-induced Bcl-2 expression indicating that radiation-induced NFκB activity is required for the induction of Bcl-2 protein. PAR-4, a novel pro-apoptotic protein is a potent down-modulator of NFκB activity and bcl-2 protein expression. This study was undertaken to investigate the impact of PAR-4 expression on radiation-induced NFκB activity and Bcl-2 expression and its resultant radiation response in PC-3 cells. Western blot analysis indicated that enforced expression of PAR-4 in PC-3 cells down regulated radiation-induced bcl-2 protein, whereas in vector transfected cells radiation caused an induction of bcl-2 protein. In both transfectant cell lines, the bax protein levels remained unaltered after radiation. When compared to PC-3/Vector cells, PC-3/PAR-4 cells showed significant sensitivity to radiation-induced clonogenic inhibition and apoptosis. Thus, the down-regulation of bcl-2 protein by ectopic PAR-4 expression altered bcl-2: bax ratio in PC-3/PAR-4 cells and this led enhanced radiosensitivity. PAR-4 was found to inhibit the radiation-induced NFκB activity and NFκB transcriptional activity is essential for bcl-2 upregulation. In PC-3/Vector cells, radiation caused an increase in NFκB activity leading to upregulation of bcl-2 protein. However, in PC-3/PAR-4 cells, the radiation-induced NFκB activity was inhibited resulting in the transrepression of bcl-2 promoter and down-modulation of bcl-2 protein. In addition, PAR-4 was found to directly inhibit the phosphorylation and degradation of IκBa, which led to the loss of NFkB activity causing repression of endogenous and radiation-induced Bcl-2 protein. Together, these mechanisms suggest that PAR-4 is functionally required to cause radiation-induced apoptosis by abrogating the survival and anti-apoptotic effects of NFκB activity and bcl-2 function respectively.

Low dose fractionated radiation potentiates the effects of taxotere in nude mice xenografts of squamous cell carcinoma of head and neck

This study evaluated the combined effect of Low Dose Fractionated Radiation (LDFRT) and Taxotere (TXT) therapy on the growth of SCCHN (squamous cell carcinoma of head and neck; SQ-20B, a p53 mutant SCCHN cell line) tumors in a nude mouse model to exploit the increased hyper radiation sensitivity (HRS) phenomenon present in G2M cell cycle phase when induced by low doses of radiation that was demonstrated in in-vitro settings. Seventy-eight animals were randomized into one control group and 5 treatment groups (treatments were administered weekly for six weeks). Tumor regression was observed in all the groups, however, tumor regression was not significant in 2 Gy or TXT or 2 Gy plus TXT treated groups when compared to control group. The tumor regression was significant in both the LDFRT group (p

Farnesyltransferase inhibitor (L-744,832) restores TGF-β type II receptor expression and enhances radiation sensitivity in K-ras mutant pancreatic cancer cell line MIA PaCa-2

Activated ras is known to dysregulate TGF-β signaling by altering the expression of TGF-β type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-β signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 μM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 μM concentrations (P>0.004). The TGF-β effector gene p21waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-β responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-β signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT–PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-β signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

The Impact of TNF-α Induction on Therapeutic Efficacy following High Dose Spatially Fractionated (GRID) Radiation

A variety of cytokines especially TNF-α and TGF-β are known to be released in response to high dose radiation of tumors. However, these are not normally measurable in systemic circulation unless high levels of these cytokines are produced by tumor cells. This study was undertaken to see if circulating levels of these cytokines could be measured in the serum of patients treated with high dose spatially fractionated (GRID) radiation and to correlate the finding of these cytokines with clinical response to treatment. Thirty-four patients (31 patients had single treatment site and 3 patients had 2 treatment sites) treated with spatially fractionated (GRID) radiation were entered in this study. Serum samples were collected before treatment and at 24, 48 and 72 hours after GRID radiation. Sandwich enzyme linked immunosorbent assay (ELISA) was performed to estimate the levels of TNF-α and activated TGF-β1 proteins. Seven of 37 patients studied had no TNF-α protein before treatment but showed induction of TNF-α after GRID radiation. Three patients showed faint basal level of TNF-α protein before treatment and these levels were induced after treatment. Three patients showed a basal level of TNF-α protein before treatment and these levels decreased after treatment. In 21 cases no TNF-α protein was detected before or after treatment at the time points measured. In the case of TGF-β1 protein, 2 patients showed no TGF-β1 protein before GRID radiation and an induction of TGF-β1 protein was observed after treatment. Seven patients showed basal level of TGF-β1 protein prior to treatment and these levels were induced after treatment. Seventeen patients showed a basal level of TGF-β1 protein before treatment and these levels decreased after treatment. In 8 cases no TGF-β1 protein was detected before or after treatment. Complete clinical response (CR) to GRID therapy was seen in 12/37 (32%) treatment sites and partial response (PR) in 18/37 (49%) treatment sites. A strong correlation was observed between clinical CR rate and TNF-α induction. The rate of CR was 6/10 (60%) in patients where TNF-α was induced as compared to 6/27 (23%) treatment sites in patients where TNF-α induction was not seen (p = 0.029). No significant correlation with CR rate and TGF-β1 induction (44% vs. 28%, p = 0.36) was observed. High dose spatially fractionated (GRID) radiation results in significant induction of TNF-α that can be measured in serum of some patients 24 – 72 hours after radiation. Complete tumor response strongly correlated with the induction of TNF-α levels in the serum.

Cocaine exposure in vitro induces apoptosis in fetal locus coeruleus neurons through TNF-α-mediated induction of Bax and phosphorylated c-jun NH2-terminal kinase

Cocaine exposure results in aberrant outgrowth and decreased survival for locus coeruleus (LC), a noradrenergic population of neurons that putatively regulates attentional function; however, the underlying mechanisms for these events are not known. We previously showed that cocaine exposure in vitro activates pro‐apoptotic Bax, caspase‐9, and caspase‐3 in LC neurons dissected from embryonic day 14 rats, implicating that apoptosis may be orchestrated via signal transduction events. In the current study in vitro, we examined upstream events to determine the role of the pro‐inflammatory cytokine, tumor necrosis factor alpha (TNF‐α), on LC signal transduction, because cocaine exposure to LC neurons triggered TNF‐α expression at 30 min as measured by ELISA. Exposure of LC neurons to recombinant‐TNF‐α resulted in decreased metabolic activity, an indicator of reduced neuron viability [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay], and increased apoptosis (terminal deoxynucleotidyl transferase‐mediated DNA nick end labeling assay). Pro‐apoptotic caspase‐3 was induced by cocaine starting at 30 min. Recombinant‐TNF‐α induced caspase‐3 activity earlier than cocaine (15 and 20 min). The caspase‐3 levels were significantly reduced when cocaine and TNF‐α were combined with neutralizing‐TNF‐α (nTNF‐α), respectively. Further, cocaine alone elevated phospho‐p38‐mitogen‐activated protein kinases that persisted when combined with nTNF‐α. However, both cocaine and TNF‐α independently increased phospho‐c‐Jun NH2‐terminal kinase and Bax levels at concurrent time periods (30 min and 1 h), and this elevation was attenuated in the presence of nTNF‐α. These simultaneous molecular events triggered by cocaine and TNF‐α implicate a potential apoptotic signal transduction pathway via induction of phospho‐c‐Jun NH2‐terminal kinase and Bax that may lead to caspase‐3 activation and apoptosis in cocaine‐exposed fetal LC neurons.

Specificity of prenatal cocaine on inhibition of locus coeruleus neurite outgrowth

Prenatal cocaine exposure induces alterations in attentional function that presumably involve locus coeruleus noradrenergic neurons and their projections. Previous reports indicate that embryonic rat locus coeruleus neurons exposed to cocaine, both in vitro and in vivo, showed in decreased cell survival and inhibition of neurite outgrowth, and that the effects were most deleterious during early gestation. The present study performed in vitro addressed the specificity of the inhibitory effects of cocaine by comparing locus coeruleus neurite formation and extension to that of dopaminergic substantia nigra neurons following exposure to a physiologically-relevant dose of cocaine (500 ng/ml, two times a day, for four days) during peak neuritogenesis. Following cocaine treatment, immunocytochemistry (anti-norepinephrine antibody to locus coeruleus; anti-tyrosine hydroxylase antibody to substantia nigra) and image analysis were performed to measure a variety of neurite outgrowth parameters. For locus coeruleus neurons, cocaine treatment decreased the 1) number of cells initiating neurites [P<0.001], 2) mean number [P<0.05] and length of neurites [P<0.0001], 3) mean number [P<0.0016] and length of branched neurites [P<0.0006], and 4) mean length of the longest neurites [P<0.0001]. In comparison, substantia nigra neurons were not significantly affected by cocaine for any of the parameters examined. More importantly, a significant interaction between cocaine treatment and brain region was observed [P<0.0002] indicating greater vulnerability of locus coeruleus, relative to substantia nigra neurons, to cocaine exposure. These data support our hypothesis that cocaine targets the noradrenergic system by negatively regulating locus coeruleus neuronal outgrowth, which likely affects pathfinding, synaptic connectivity, and ultimately attentional behavior in cocaine-exposed offspring.

The impact of TNF-α induction on therapeutic efficacy following high dose spatially fractionated (GRID) radiation

A variety of cytokines especially TNF-alpha and TGF-beta are known to be released in response to high dose radiation of tumors. However, these are not normally measurable in systemic circulation unless high levels of these cytokines are produced by tumor cells. This study was undertaken to see if circulating levels of these cytokines could be measured in the serum of patients treated with high dose spatially fractionated (GRID) radiation and to correlate the finding of these cytokines with clinical response to treatment. Thirty-four patients (31 patients had single treatment site and 3 patients had 2 treatment sites) treated with spatially fractionated (GRID) radiation were entered in this study. Serum samples were collected before treatment and at 24, 48 and 72 hours after GRID radiation. Sandwich enzyme linked immunosorbent assay (ELISA) was performed to estimate the levels of TNF-alpha and activated TGF-beta1 proteins. Seven of 37 patients studied had no TNF-alpha protein before treatment but showed induction of TNF-alpha after GRID radiation. Three patients showed faint basal level of TNF-alpha protein before treatment and these levels were induced after treatment. Three patients showed a basal level of TNF-alpha protein before treatment and these levels decreased after treatment. In 21 cases no TNF-alpha protein was detected before or after treatment at the time points measured. In the case of TGF-beta1 protein, 2 patients showed no TGF-beta1 protein before GRID radiation and an induction of TGF-beta1 protein was observed after treatment. Seven patients showed basal level of TGF-beta1 protein prior to treatment and these levels were induced after treatment. Seventeen patients showed a basal level of TGF-beta1 protein before treatment and these levels decreased after treatment. In 8 cases no TGF-beta1 protein was detected before or after treatment. Complete clinical response (CR) to GRID therapy was seen in 12/37 (32%) treatment sites and partial response (PR) in 18/37 (49%) treatment sites. A strong correlation was observed between clinical CR rate and TNF-alpha induction. The rate of CR was 6/10 (60%) in patients where TNF-alpha was induced as compared to 6/27 (23%) treatment sites in patients where TNF-alpha induction was not seen (p = 0.029). No significant correlation with CR rate and TGF-beta1 induction (44% vs. 28%, p = 0.36) was observed. High dose spatially fractionated (GRID) radiation results in significant induction of TNF-alpha that can be measured in serum of some patients 24 72 hours after radiation. Complete tumor response strongly correlated with the induction of TNF-alpha levels in the serum.