Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKCδ in cell culture and animal models of Parkinson's disease

The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ^D327A and kinase dead PKCδ^K376R or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ^D327A protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.     

Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis

Gallic acid (GA) is widely distributed in various plants and foods, and its various biological effects have been reported. Here, we evaluated the effects of GA on HeLa cells in relation to cell growth inhibition and death. HeLa cell growth was diminished with an IC₅₀ of approximately 80 μM GA at 24 h whereas an IC₅₀ of GA in human umbilical vein endothelial cells (HUVEC) was approximately 400 μM. GA-induced apoptosis and/or necrosis in HeLa cells and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). The percents of MMP (ΔΨ_m) loss cells and death cells were lower in HUVEC than HeLa cells. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from GA-induced cell death. GA increased reactive oxygen species (ROS) level and GSH (glutathione) depleted cell number in HeLa cells. Caspase inhibitors reduced GSH depleted cell number but not ROS level in GA-treated HeLa cells. In conclusion, GA inhibited the growth of HeLa cells and HUVEC via apoptosis and/or necrosis. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA-induced HeLa cell death was accompanied by ROS increase and GSH depletion.     

Heterocyclic organobismuth(III) induces apoptosis of human promyelocytic leukemic cells through activation of caspases and mitochondrial perturbation

We have synthesized novel heterocyclic organobismuth compounds that have potent antibacterial properties. In this study, we examined their anticancer activity and addressed the cellular mechanisms involved. Heterocyclic organobismuth compounds showed anticancer activities in various human cancer cell lines. These compounds have particularly potent anticancer activities against leukemia cell lines. One of them, bi-chlorodibenzo [c,f][1,5] thiabismocine (compound 3), inhibited the growth of the human promyelocytic leukemia cell line HL-60 at a concentration of 0.22 μM. Low concentrations of compound 3 (0.22-0.44 μM) induced apoptosis, whereas at a higher concentration (>1.1 μM) it causes acute necrosis. During the apoptosis, caspase-3, -8, and -9 were activated but caspase-12 was not. A broad caspase inhibitor (z-VAD-fmk), and caspase-3 (z-DEVD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors suppressed the compound 3-induced apoptosis, but a caspase-8 inhibitor (z-IETD-fmk) was less effective, suggesting that the caspase-8 activity only partially participates in the apoptosis. In the apoptotic cells, cytochrome c was released from mitochondria to cytosol and a loss of mitochondrial transmembrane potential (ΔΨ_m) was detected. Compound 3-induced apoptosis was associated with enhanced generation of intracellular reactive oxygen species (ROS). Pretreatment of the cells with N-acetyl-l-cysteine or catalase suppressed the apoptosis. On the other hand, buthionine sulfoximine enhanced the compound 3-induced collapse of ΔΨ_m and apoptosis. Taken together, these results indicate that compound 3 is a potent inducer of apoptosis, triggering a caspase-3-mediated mechanism via the generation of ROS and release of cytochrome c from mitochondria, suggesting a potential mechanism for the anticancer activity of compound 3.     

Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKCδ) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKCδ negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKCδ-PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3-10 μM) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKCδ specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1-1 μM Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKCδ kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKCδ inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKCδ and PP2A activity.     

Differential responses to docosahexaenoic acid in primary and immortalized cardiac cells

The importance of dietary polyunsaturated fatty acids (PUFAs) in the reduction of cardiovascular disease has been recognized for many years. Docosahexaenoic acid (22:6n3, DHA) is an n-3 PUFA known to affect numerous biological functions and provide cardioprotection; however, the exact molecular and cellular protective mechanism(s) remain unknown. In contrast, DHA also possesses many anti-tumorgenic properties including suppressing cell growth and inducing apoptosis. In the present study, we investigated the effect of DHA toward H9c2 cells (an immortalized cardiac cell line) and neonatal primary cardiomyocytes (NCM). Cells were treated with 0 μM, 10 μM or 100 μM DHA for upto 48 h. Cell viability and mitochondrial activity were assayed at different time points. DHA caused a significant time- and dose-dependent decrease in cell viability and mitochondrial activity in H9c2 cells but not NCM. In addition, DHA decreased levels of TGF-β1 but increased IL-6 release in H9c2 cells. Significant induction of apoptosis was observed only in H9c2 cells, which involved activation of caspase-8 and -3 activities with a marked release of cytochrome c from mitochondria. DHA-induced severe mitochondrial damage resulting in a fragmented and punctated morphology with corresponding loss of mitochondrial membrane potential within 3 h, prior to activation of caspases and cytochrome c release at 6 h in H9c2 cells. Our data indicate that DHA treatment targets mitochondria, triggering collapse of mitochondrial membrane potential, increasing cellular stress and mitochondrial fragmentation resulting in apoptosis in immortalized cardiac cells, H9c2, but not neonatal primary cardiomyocyte.     

A clinically relevant in vitro model for evaluating the effects of aerosolized vesicants

The chemical warfare vesicant sulfur mustard (HD) is a known toxic agent to the human respiratory tract and the major airways are considered to be a primary target of HD-induced injury. However, there is no consensus regarding which model systems are most appropriate for studying the effects of aerosolized vesicants on human airway epithelium. In this study, we evaluated the consequences of exposure of differentiated human respiratory epithelial cells in air–liquid interface to mechlorethamine (HN2), an HD functional analog. HN2 challenge was administered via the apical (air) interface over a wide dose range (20–400 μM) to differentiated HBE1 cells. Cultures were observed over 1–48 h for evidence of HN2-induced morphologic abnormalities as well as for possible cellular cytotoxicity, apoptotic changes, and induction of cytokine secretion. HN2 at concentrations of ≥200 μM caused disruption and denudation of the airway epithelial architecture within 24 h of exposure. Moreover, HN2-induced cytotoxic and apoptotic changes in HBE1 cells in a dose- and time-dependent fashion. HN2 challenge also induced secretion of chemokines and proinflammatory cytokines including TNF-α, IL-1α, IL-1β, IL-6, IL-8, RANTES, MCP-1, IP-10, G-CSF, GM-CSF and IL-15. These observations parallel those described in the lungs of HD-exposed victims and underscore the utility and potential applicability of this model to future mechanistic studies of vesicant-induced pulmonary injury.     

Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC₅₀ of approximately 6.64 ± 1.84 μM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 μM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.     

Coactivation of the PI3K/Akt and ERK signaling pathways in PCB153-induced NF-κB activation and caspase inhibition

Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxicity, endocrine disruption and reproductive abnormalities. In order to verify the hypothesis that the PI3K/Akt and MAPK pathways play important roles in hepatotoxicity induced by PCBs, Sprague–Dawley (SD) rats were dosed with PCB153 intraperitoneally at 0, 4, 16 and 32 mg/kg for five consecutive days; BRL cells (rat liver cell line) were treated with PCB153 (0, 1, 5, and 10 μM) for 24 h. Results indicated that the PI3K/Akt and ERK pathways were activated in vivo and in vitro after exposure to PCB153, and protein levels of phospho-Akt and phospho-ERK were significantly increased. Nuclear factor-κB (NF-κB) activation and caspase-3, -8 and -9 inhibition caused by PCB153 were also observed. Inhibiting the ERK pathway significantly attenuated PCB153-induced NF-κB activation, whereas inhibiting the PI3K/Akt pathway hardly influenced phospho-NF-κB level. However, inhibiting the PI3K/Akt pathway significantly elevated caspase-3, -8 and -9 activities, while the ERK pathway only synergistically regulated caspase-9. Proliferating cell nuclear antigen (PCNA), a reliable indicator of cell proliferation, was also induced. Moreover, PCB153 led to hepatocellular hypertrophy and elevated liver weight. Taken together, PCB153 leads to aberrant proliferation and apoptosis of hepatocytes through NF-κB activation and caspase inhibition, and coactivated PI3K/Akt and ERK pathways play critical roles in PCB153-induced hepatotoxicity.     

Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥ 3 μM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 μM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 μM PM. Numbers of oocytes with ≥ 5 γH2AX foci were significantly increased both 1 and 8 days after exposure to ≥ 1 μM PM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥ 30 μM) and increased γH2AX foci (≥ 3 μM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.     

A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-induced paw edema in mice

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase is a key enzyme in the biosynthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) in inflammatory cells. Substances which inhibit this enzyme are of therapeutic interest. In this study, we screened for new inhibitors of lyso-PAF acetyltransferase with anti-inflammatory effects. In a metabolite from Penicillium sp. F33, we isolated an acetyltransferase inhibitor identified as dihydrofumigatin (2-methoxy-1,3,4-trihydroxy-5-methylbenzene) from high resolution mass spectrometer and NMR data. Dihydrofumigatin had strong acetyltransferase inhibitory activity, but was not stable in aqueous solution. Thus, we chemically synthesized its oxidized form fumigatin (3-hydroxy-2-methoxy-5-methyl-1,4-benzoquinone) and derivatives thereof, and evaluated their inhibitory effects. Strong inhibitory activity was observed for saturated fatty acid esters of fumigatin; the order of inhibition was 3-decanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (termed FUD-7, IC₅₀ = 3 μM) > 2-methoxy-5-methyl-3-tetradecanoyloxy-1,4-benzoquinone (termed FUD-8, IC₅₀ = 20 μM) > 3-hexanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (IC₅₀ = 139 μM). Interestingly, these compounds also significantly suppressed the gene expression of lyso-PAF acetyltransferase/LPCAT2 in mouse bone marrow-derived macrophages stimulated by lipopolysaccharide (LPS). We further evaluated the effect of these substances on anti-inflammatory activity in vivo using the carrageenan-induced mouse paw edema test. FUD-7 and FUD-8 at 2.5 mg/kg showed significant, 47.9–51.7%, inhibition stronger than that of prednisolone at 10 mg/kg (41.9%). These results suggest that FUD-7 and FUD-8 are potent inhibitors with anti-inflammatory activity.     

Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.     

Neurotoxic injury pathways in differentiated mouse motor neuron-neuroblastoma hybrid (NSC-34D) cells in vitro--limited effect of riluzole on thapsigargin, but not staurosporine, hydrogen peroxide and homocysteine neurotoxicity

The neuroblastoma-spinal motor neuron fusion cell line, NSC-34, in its differentiated form, NSC-34D, permits examining the effects of riluzole, a proven treatment for amyotrophic lateral sclerosis (ALS) on cell death induction by staurosporine (STS), thapsigargin (Thaps), hydrogen peroxide (H₂O₂) and homocysteine (HCy). These neurotoxins, applied exogenously, have mechanisms of action related to the various proposed molecular pathogenetic pathways in ALS and are differentiated from endogenous cell death that is associated with cytoplasmic aggregate formation in motor neurons. Nuclear morphology, caspase-3/7 activation and high content imaging were used to assess toxicity of these neurotoxins with and without co-treatment with riluzole, a benzothiazole compound with multiple pharmacological actions. STS was the most potent neurotoxin at killing NSC-34D cells with a toxic concentration at which 50% of maximal cell death is achieved (TC₅₀ = 0.01 μM), followed by Thaps (TC₅₀ = 0.9 μM) and H₂O₂ (TC₅₀ = 15 μM) with HCy requiring higher concentrations to kill at the same level (TC₅₀ = 2200 μM). Riluzole provided neurorescue with a 20% absolute reduction (47.6% relative reduction) in apoptotic cell death against Thaps-induced NSC-34D cell (p ≤ 0.05), but had no effect on STS-, H₂O₂- and HCy-induced NSC-34D cell death. This effect of riluzole on Thaps induction of cell death was independent of caspase-3/7 activation. Riluzole mitigated a toxin that can cause intracellular calcium dysregulation associated with endoplasmic reticulum (ER) stress but not toxins associated with other cell death mechanisms.     

Genistein enhances the effect of trichostatin A on inhibition of A549 cell growth by increasing expression of TNF receptor-1

Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell number and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12 h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and ‐10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells.     

Identification of small-molecule inhibitors of ricin and shiga toxin using a cell-based high-throughput screen

The Category B agents, ricin and shiga toxin (Stx), are RNA N-glycosidases that target a highly conserved adenine residue within the sarcin-ricin loop of eukaryotic 28S ribosomal RNA. In an effort to identify small-molecule inhibitors of these toxins that could serve as lead compounds for potential therapeutics, we have developed a simple Vero cell-based high-throughput cytotoxicity assay and have used it to screen ∼81,300 compounds in 17 commercially available chemical libraries. This initial screen identified ∼300 compounds with weak (≥30 to <50%), moderate (≥50 to <80%), or strong (≥80%) ricin inhibitory activity. Secondary analysis of 244 of these original “hits” was performed, and 20 compounds that were capable of reducing ricin cytotoxicity by >50% were chosen for further study. Four compounds demonstrated significant dose-dependent ricin inhibitory activity in the Vero cell-based assay, with 50% effective inhibitory concentration (EC₅₀) values ranging from 25 to 60 μM. The same 20 compounds were tested in parallel for the ability to inhibit ricin’s and Stx1’s enzymatic activities in an in vitro translation reaction. Three of the 20 compounds, including the most effective compound in the cell-based assay, had discernible anti-toxin activity. One compound in particular, 4-fluorophenyl methyl 2-(furan-2-yl)quinoline-4-carboxylate (“compound 8”), had 50% inhibitory concentration (IC₅₀) of 30 μM, a value indicating >10-fold higher potency than is the case for previously described ricin-Stx1 inhibitors. Computer modeling predicted that compound 8 is capable of docking within the ricin active site. In conclusion, we have used a simple high-throughput cell-based method to identify several new small-molecule inhibitors of ricin and Stx.     

Protective effects of memantine and epicatechin on catechol-induced toxicity on Müller cells in vitro

This study evaluates the toxic effects of catechol (a component from cigarette smoke) on Müller cells (MIO-M1) in vitro, and investigates the inhibitors memantine and epicatechin to determine if they can reverse the catechol toxic effects. MIO-M1 cells were exposed to varying concentrations of catechol with or without memantine or epicatechin. Cell viability (CV) was measured by a trypan blue dye-exclusion assay. Caspase-3/7 activity was measured by fluorochrome assay. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with 2′,7′-dichlorodihydrofluorescein diacetate dye assay. Mitochondrial membrane potential (ΔΨm) was measured using JC-1 assay. Intracellular ATP content was determined by the ATPLite kit. MIO-M1 cells showed significant decrease in cell viability, increased caspase-3/7 activity, elevated ROS/RNS levels, decreased ΔΨm value, and decreased intracellular ATP content after exposure to catechol 150, 300, and 600 μM compared with control. Pre-treatment with memantine 10 μM or epicatechin 15 μM reversed loss of cell viability in catechol 150 μM-treated cultures (22.3%, p < 0.01 and 17.8%, p < 0.05), respectively. Similarly, pre-treatment with memantine 10 μM and epicatechin 15 μM prior to catechol resulted in decreased caspase-3/7 activities (77% and 64.2%, p < 0.001), decreased ROS/RNS levels (82.3% and 79%, p < 0.001), increased ΔΨm value (76.4% and 72.2%, p < 0.001), and increased ATP levels (46.6% and 40.4%, p < 0.001) compared to 150 μM catechol-treated cultures. Catechol, a component of smoking, can diminish cell viability and mitochondrial function in MIO-M1 cells in vitro. However, memantine and epicatechin can partially reverse the cytotoxic effect of catechol. Their administration may reduce or prevent Müller cells degeneration in AMD or other retinal degenerative disorders.     

Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.     

The pyranoxanthone inophyllin A induces oxidative stress mediated-apoptosis in Jurkat T lymphoblastic leukemia cells

Inophyllin A (INO-A), a pyranoxanthone isolated from the roots of Calophyllum inophyllum represents a new xanthone with potential chemotherapeutic activity. In this study, the molecular mechanism of INO-A-induced cell death was investigated in Jurkat T lymphoblastic leukemia cells. Assessment of phosphatidylserine exposure confirmed apoptosis as the primary mode of cell death in INO-A-treated Jurkat cells. INO-A treatment for only 30 min resulted in a significant increase of tail moment which suggests that DNA damage is an early apoptotic signal. Further flow cytometric assessment of the superoxide anion level confirmed that INO-A induced DNA damage was mediated with a concomitant generation of reactive oxygen species (ROS). Investigation on the thiols revealed an early decrease of free thiols in 30 min after 50 μM INO-A treatment. Using tetramethylrhodamine ethyl ester, a potentiometric dye, the loss of mitochondrial membrane potential (MPP) was observed in INO-A-treated cells as early as 30 min. The INO-A-induced apoptosis progressed with the simultaneous activation of caspases-2 and -9 which then led to the processing of caspase-3. Taken together, these data demonstrate that INO-A induced early oxidative stress, DNA damage and loss of MMP which subsequently led to the activation of an intrinsic pathway of apoptosis in Jurkat cells.     

Suicidal erythrocyte death triggered by cisplatin

Cisplatin, a cytotoxic drug for the treatment of cancer, induces suicidal death or apoptosis of nucleated cells. Side effects of cisplatin include anemia, which, at least in theory, could similarly result from suicidal cell death. Erythrocyte suicidal death or eryptosis is characterized by cell shrinkage and cell membrane scrambling, the latter leading to exposure of phosphatidylserine (PS) at the cell surface. PS-exposing cells are rapidly cleared from circulating blood. The present experiments explored whether cisplatin could trigger eryptosis. According to forward scatter in FACS analysis, a 48 h exposure to cisplatin (≥1 μM) indeed decreased cell volume and, according to annexin V-binding, cisplatin (≥1 μM, 48 h) indeed increased PS exposure at the cell surface. Cisplatin did not induce hemolysis. According to Fluo3 fluorescence, cisplatin increased cytosolic Ca²⁺ activity, a known stimulator of eryptosis. In the absence of extracellular Ca²⁺, the effect of cisplatin on annexin V-binding was blunted. Cisplatin did not significantly modify the formation of ceramide, another stimulator of eryptosis. Cisplatin moderately decreased the cellular concentration of ATP, which is known to favour eryptosis. In conclusion, cisplatin triggers suicidal erythrocyte death at least partially by increasing cytosolic Ca²⁺ activity. The effect contributes to or even accounts for the development of anemia during cisplatin treatment.     

Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways

Drug induced thyroid effects were evaluated in organotypic models utilizing either a rat thyroid lobe or human thyroid slices to compare rodent and human response. An inhibition of thyroid peroxidase (TPO) function led to a perturbation in the expression of key genes in thyroid hormone synthesis and release pathways. The clinically used thiourea drugs, methimazole (MMI) and 6-n-propyl-2-thioruacil (PTU), were used to evaluate thyroid drug response in these models. Inhibition of TPO occurred early as shown in rat thyroid lobes (2 h) and was sustained in both rat (24-48 h) and human (24 h) with ≥ 10 μM MMI. Thyroid from rats treated with single doses of MMI (30-1000 mg/kg) exhibited sustained TPO inhibition at 48 h. The MMI in vivo thyroid concentrations were comparable to the culture concentrations (~ 15-84 μM), thus demonstrating a close correlation between in vivo and ex vivo thyroid effects. A compensatory response to TPO inhibition was demonstrated in the rat thyroid lobe with significant up-regulation of genes involved in the pathway of thyroid hormone synthesis (Tpo, Dio1, Slc5a5, Tg, Tshr) and the megalin release pathway (Lrp2) by 24 h with MMI (≥ 10 μM) and PTU (100 μM). Similarly, thyroid from the rat in vivo study exhibited an up-regulation of Dio1, Slc5a5, Lrp2, and Tshr. In human thyroid slices, there were few gene expression changes (Slc5a5, ~ 2-fold) and only at higher MMI concentrations (≥ 1500 μM, 24 h). Extended exposure (48 h) resulted in up-regulation of Tpo, Dio1 and Lrp2, along with Slc5a5 and Tshr. In summary, TPO was inhibited by similar MMI concentrations in rat and human tissue, however an increased sensitivity to drug treatment in rat is indicated by the up-regulation of thyroid hormone synthesis and release gene pathways at concentrations found not to affect human tissue.