Prognostic impact of vitamin B6 metabolism in lung cancer

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.     

Decreased vitamin B12 availability induces ER stress through impaired SIRT1-deacetylation of HSF1

Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin–oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.     

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.     

Depletion of endogenous nitric oxide enhances cisplatin-induced apoptosis in a p53-dependent manner in melanoma cell lines

The expression of inducible nitric-oxide synthase in melanoma tumor cells was recently shown to correlate strongly with poor patient survival after combination biochemotherapy (p<0.001). Furthermore, evidence suggests that nitric oxide, a reaction product of nitric oxide synthase, exhibits antiapoptotic activity in melanoma cells. We therefore hypothesized that nitric oxide antagonizes chemotherapy-induced apoptosis. Whether nitric oxide is capable of regulating cell growth and apoptotic responses to cisplatin treatment in melanoma cell lines was evaluated. We demonstrate herein that depletion of endogenously produced nitric oxide can inhibit melanoma proliferation and promote apoptosis. Moreover, our data indicate that the depletion of nitric oxide leads to changes in cell cycle regulation and enhances cisplatin-induced apoptosis in melanoma cells. Strikingly, we observed that the depletion of nitric oxide inhibits cisplatin-induced wild type p53 accumulation and p21(Waf1/Cip1/Sdi1) expression in melanoma cells. When cisplatin-induced p53 binding to the p21(Waf1/Cip1/Sdi1) promoter was examined, it was found that nitric oxide depletion significantly reduced the presence of p53-DNA complexes after cisplatin treatment. Furthermore, dominant negative inhibition of p53 activity enhanced cisplatin-induced apoptosis. Together, these data strongly suggest that endogenously produced nitric oxide is required for cisplatin-induced p53 activation and p21(Waf1/Cip1/Sdi1) expression, which can regulate melanoma sensitivity to cisplatin.     

An indirect role of vitamin B12 in regulation of thymidylate synthase in Lactobacillus leichmannii.

Vitamin B12 augments thymidylate synthase function in L. leichmannii by facilitating indirectly the availability of suitable nonmethylpolyglutamylfolate cofactors. This is effected by the demethylation of trapped methyltetrahydrofolates, catalysed by a vitamin B12 requiring methionine synthase. Deoxyuridine supplemented cells, lacking in B12, have decreased levels of methionine synthase and thymidylate synthase. Addition of active and inactive conjugase preparation as a source of mono and polyglutamylfolates indicated that the latter are the preferred cofactors for thymidylate synthase.     

Anchoring Secreted Proteins in Endoplasmic Reticulum by Plant Oleosin: The Example of Vitamin B12 Cellular Sequestration by Transcobalamin

Background

Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin.

Methodology

chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells.

Principal findings

vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration.

Conclusions/Significance

the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest.     

TCR-dependent and -independent activation underlie liver-specific regulation of NKT cells

The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.     

Uptake and physiological function of vitamin B12 in a photosynthetic unicellular coccolithophorid alga, Pleurochrysis carterae

The photosynthetic coccolithophoid alga, Pleurochrysis (Hymenomonas) carterae, could take up and accumulate exogenous vitamin B12, most of which was converted into the coenzyme forms of vitamin B12. Two vitamin B12-dependent enzyme activities (methylmalonyl-CoA mutase, 2.6+/-0.4 nmol/min/mg protein and methionine synthase, 85.1+/-38.9 pmol/min/mg protein) could be found in a cell homogenate of the vitamin B12-supplemented alga. Most of the methylmalonyl-CoA mutase activity and 19.2% of the vitamin B12 accumulated by the algal cells were recovered in the macromolecular fractions with Mr of 150 kDa, although the remaining vitamin B12 was found only in free vitamin B12 fractions.     

Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.     

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-alpha-induced apoptosis. STS activated I kappa B kinase and nuclear factor-kappa B (NF-kappa B) nuclear translocation and DNA binding but inhibited transactivation of I kappa B-alpha, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-kappa B reporter in transfected Huh-7 cells.     

Reversible effects of vitamins C and E combination on oxidative stress-induced apoptosis in melamine-treated PC12 cells

Abstract

Due to its high nitrogen content, melamine was deliberately added to raw milk for increasing the apparent protein content. Previous studies showed that melamine-induced apoptosis and oxidative damage on PC12 cells and rats’ hippocampus. Several evidences suggested that vitamin antioxidant reduced oxidative stress and improved organic function. Whether treatments with antioxidant vitamins C or E, otherwise combination of them can attenuate oxidative stress after melamine administration remains to be elucidated. In this study, the reversible effects of vitamin antioxidants was investigated on melamine-induced neurotoxicity in cultured PC12 cells, an in vitro model of neuronal cells. When comparing vitamin C and E, the combination of both statistically increased PC12 cells viability. The results further showed that vitamin complex has effectively reduced the formation of reaction oxygen species, decreased the level of malondialdehyde, and elevated the activities of antioxidative enzymes. Hoechst 33342 staining and flow cytometric analysis of apoptosis showed that vitamin combination treatment effectively prevented PC12 cells from this melamine-induced apoptosis. It revealed the apoptotic nuclear features of the melamine-induced cell death. Additionally, a combination treatment of vitamins effectively inhibited apoptosis via blocking the increased activation of caspase-3. In summary, the vitamin E and C combination treatment could rescue PC12 cells from the injury induced by melamine through the downregulation of oxidative stress and prevention of melamine-induced apoptosis.     

Methyl B12 protects PC12 cells against cytotoxicity induced by Aβ25−35

Alzheimer's disease (AD) is the most common aging-associated dementia. The population of AD patients is increasing as the world age grows. Currently, there is no cure for AD. Given that methyl vitamin B12 (methylcobalamin) deficiency is related to AD and Aβ-induced oxidative damage and that methylcobalamin can scavenge reactive oxygen species (ROS) by direct or indirect ways, we studied the effect of methylcobalamin on the cytotoxicity of Aβ. PC12 cells were chronically exposed (24 hours) to Aβ_25-35 (25 μM) to establish an AD cell model. The cells were pretreated with or without methylcobalamin (1-100 μM) to investigate the role of methylcobalamin. Cell viability and apoptosis were tested, followed by testing of mitochondrial damage, oxidative stress, and mitochondrial calcium concentration. We observed that methylcobalamin improved the cell viability by decreasing the ratio of apoptosis cells in this AD cell model. Further experiments suggested that methylcobalamin functioned as an antioxidant to scavenge ROS, reducing the endoplasmic reticulum-mitochondria calcium flux through IP3R, preventing mitochondria dysfunction, ultimately protecting cells against apoptosis and cell death. Taken together, our results presented, for the first time, that methyl vitamin B12 can protect cells from Aβ-induced cytotoxicity and the mechanism was mainly relevant to the antioxidative function of methyl B12.     

Elevated L-PGDS activity contributes to PMA-induced apoptosis concomitant with downregulation of PI3-K

Recently wedemonstrated the induction of apoptosis by the addition ofrecombinant lipocalin-type prostaglandin D₂ synthase (L-PGDS) to the culture medium of LLC-PK₁ cells. Becauseprotein kinase C (PKC) has been shown to be involved in theapoptotic process of various cell types, we examined the potentialrole of L-PGDS in phorbol 12-myristate 13-acetate (PMA)-inducedapoptosis. We report here the enzymatic activation andphosphorylation of L-PGDS in response to phorbol ester in cellculture and the direct phosphorylation of recombinant L-PGDS by PKC invitro. Treatment of cells with PMA or L-PGDS decreasedphosphatidylinositol 3-kinase (PI3-K) activity and concomitantlyinhibited protein kinase B (PKB/Akt) phosphorylation, which led to thehypophosphorylation and activation of Bad. In addition,hypophosphorylation of retinoblastoma protein was also observed inresponse to L-PGDS-induced apoptosis. Cellular depletion ofL-PGDS levels by using an antisense RNA strategy prevented PI3-Kinactivation by phorbol ester and inhibited caspase-3 activation andapoptosis. We conclude that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by PKC and is accompanied by inhibition of the PI3-K/PKBanti-apoptotic signaling pathways.

     

Nitrosylcobalamin promotes cell death via S nitrosylation of Apo2L/TRAIL receptor DR4

We have previously demonstrated that nitrosylcobalamin (NO-Cbl), an analogue of vitamin B12 that delivers nitric oxide (NO), had potent antiproliferative activity against several human cancer cell lines. NO-Cbl induced apoptosis via a death receptor/caspase-8 pathway. In this study, we demonstrate that a functional Apo2L/TRAIL receptor was necessary for the induction of cell death by NO-Cbl. Furthermore, the Apo2L/TRAIL death receptor DR4 (TRAIL R1) was S nitrosylated following NO-Cbl treatment. Human melanoma (A375), renal carcinoma (ACHN), and ovarian carcinoma (NIH-OVCAR-3) cells were treated with NO-Cbl and subjected to the biotin switch assay; S-nitrosylated DR4 was detected in all three cell lines. NO-Cbl treatment did not cause S nitrosylation of DR5. The seven cysteine residues located in the cytoplasmic domain of DR4 were individually point mutated to alanines. NIH-OVCAR-3 cells expressing the DR4 C336A mutation lacked S nitrosylation following NO-Cbl treatment. Overexpression of wild-type DR4 sensitized cells to growth inhibition by NO-Cbl. Cells expressing the DR4 C336A mutant were more resistant to NO-Cbl and Apo2L/TRAIL than were the other six C-A mutations or wild-type cells. The C336A mutant also displayed blunted caspase-8 enzymatic activity following NO-Cbl treatment compared to the other mutants. Thus, DR4 residue C336 becomes S nitrosylated and promotes apoptosis following NO-Cbl treatment.     

Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells

The idea that conversion of glucose to ATP is an attractive target for cancer therapy has been supported in part by the observation that glucose deprivation induces apoptosis in rodent cells transduced with the proto-oncogene MYC, but not in the parental line. Here, we found that depletion of glucose killed normal human cells irrespective of induced MYC activity and by a mechanism different from apoptosis. However, depletion of glutamine, another major nutrient consumed by cancer cells, induced apoptosis depending on MYC activity. This apoptosis was preceded by depletion of the Krebs cycle intermediates, was prevented by two Krebs cycle substrates, but was unrelated to ATP synthesis or several other reported consequences of glutamine starvation. Our results suggest that the fate of normal human cells should be considered in evaluating nutrient deprivation as a strategy for cancer therapy, and that understanding how glutamine metabolism is linked to cell viability might provide new approaches for treatment of cancer.     

Radioresistance of human carcinoma cells is correlated to a defect in raft membrane clustering

In addition to DNA damage, exposure to irradiation involves the plasma membrane in the early phases of gamma-ray-induced cell death. The involvement of raft microdomains following gamma-radiation derives essentially from the role of ceramide as a critical component leading to apoptosis. It is demonstrated here that gamma-irradiation of a radiosensitive human head and neck squamous carcinoma cell line (SCC61) results in the triggering of raft coalescence to larger membrane platforms associated with the externalization of an acid sphingomyelinase (A-SMase), leading to ceramide release in raft, 30 min postirradiation. For the first time, we show that this structural rearrangement is defective in the radioresistant SQ20B cells and associated with the lack of A-SMase activation and translocation, a result which could explain in part their resistance to apoptosis following ionizing radiation. Moreover, we show that SQ20B are protected against radiation injury through a fivefold upper level of endogenous glutathione compared to SCC61. Overcoming the endogenous antioxidant defenses of SQ20B through either H(2)O(2) treatment or GSH depletion triggers A-SMase activation and translocation, raft coalescence, and apoptosis. On the contrary, ROS scavengers abolished these events in radiosensitive SCC61 cells. Translation of this concept to tumor biology suggests that manipulation of rafts through redox equilibrium may provide opportunities for radiosensitization of tumor cells.     

Oxidative stress and activation of proteasome protease during serum deprivation-induced apoptosis in rat hepatoma cells; inhibition of cell death by melatonin

Growth factor deprivation-induced apoptosis has been shown in various cell systems and is recognized as one of the standard models for the study of programmed cell death. The mechanism of induction of apoptosis by serum deprivation is still not clear. The objective of the present study was to investigate if serum-deprivation causes oxidative stress, which then leads to apoptotic death. We have demonstrated that indeed, there was a significant increase in reactive oxygen species following serum deprivation of 5123tc hepatoma cells. Furthermore, treatment with anti-oxidants; melatonin or vitamin E, prevented cell death caused by serum-deprivation. We also demonstrated that there was activation of proteasome proteases and decrease in glutathione levels following serum deprivation. Interestingly, melatonin treatment blocked these changes and rescued the cells from apoptosis induced by serum-deprivation. These results indicated that oxidative stress may play a causal role in the induction of apoptosis induced by serum deprivation.     

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ)

Abstract

Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2–5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.     

Characterization of the Kallikrein-Kinin System Post Chemical Neuronal Injury: An In Vitro Biochemical and Neuroproteomics Assessment

Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain provoking mild, moderate or severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neural injury is not fully elucidated. Some studies have highlighted the pivotal role of the Kallikrein-Kinin System (KKS) in brain trauma but the results are still controversial and inconclusive. In this study, we investigated both the expression and the role of Bradykinin 1 and 2 receptors (B1R and B2R), in mediating neuronal injury under chemical neurotoxicity paradigm in PC12 cell lines. The neuronal cell line PC12 was treated with the apoptotic drug Staurosporine (STS) to induce cell death. Intracellular calcium release was evaluated by Fluo 4-AM staining and showed that inhibition of the B2R prevented calcium release following STS treatment. Differential analyses utilizing immunofluorescence, Western blot and Real-time Polymerase Chain Reaction revealed an upregulation of both bradykinin receptors occurring at 3h and 12h post-STS treatment, but with a higher induction of B2R compared to B1R. This implies that STS-mediated apoptosis in PC12 cells is mainly conducted through B2R and partly via B1R. Finally, a neuroproteomics approach was conducted to find relevant proteins associated to STS and KKS in PC12 cells. Neuroproteomics results confirmed the presence of an inflammatory response leading to cell death during apoptosis-mediated STS treatment; however, a “survival” capacity was shown following inhibition of B2R coupled with STS treatment. Our data suggest that B2R is a key player in the inflammatory pathway following STS-mediated apoptosis in PC12 cells and its inhibition may represent a potential therapeutic tool in TBI.