Antiproliferative effect of a lectin- and anti-Thy-1.2 antibody-targeted HPMA copolymer-bound doxorubicin on primary and metastatic human colorectal carcinoma and on human colorectal carcinoma transfected with the mouse Thy-1.2 gene

The aim of this study was to compare the potential of two plant lectins [peanut agglutinin (PNA) and wheat germ agglutinin (WGA)], monoclonal antibody (anti-Thy-1.2), its F(ab')(2) fragments, and galactosamine as targeting moieties bound to the polymer drug carrier to deliver a xenobiotic, doxorubicin, to selected cancer cell lines. We have used primary (SW 480, HT 29) and metastatic (SW 620) human colorectal cancer cell lines and a transfectant, genetically engineered SW 620 cell line with mouse gene Thy-1.2 (SW 620/T) to test the possibility of marking human cancer with xenogeneic mouse gene and use it for effective site-specific targeting. The targeting moieties and doxorubicin were conjugated to a water-soluble copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA) acting as a carrier responsible for controlled intracellular release of the targeted drug. FACS analysis showed a strong binding of WGA-FITC to all tested cell lines. Binding of PNA-FITC was considerably weaker. The in vitro antiproliferative effect of lectin-targeted HPMA carrier-bound doxorubicin evaluated as [(3)H]TdR incorporation reflected both the intensity of the binding and the different sensitivity of the tested cancer cells lines to doxorubicin. The antiproliferative effect of conjugates targeted with WGA was comparable to that with the conjugates targeted with the anti-Thy-1.2 monoclonal antibody or their F(ab')(2) fragments. The magnitude of the cytotoxic effect of HPMA-doxorubicin targeted with PNA was lower in all tested cell lines. While the conjugates with WGA were more cytotoxic, the conjugates with PNA were more specific as their binding is limited to cancer cells and to the sites of inflammation. Noncytotoxic conjugates with a very low concentration of doxorubicin and targeted with PNA, anti-Thy-1.2, or their F(ab')(2) fragments exerted in some lines (SW 480, SW 620) low mitogenic activity. The Thy-1.2 gene-transfected SW 620 metastatic colorectal cancer cell line was sensitive to the antiproliferative effect of Thy-1.2-targeted doxorubicin as was shown for the Thy-1. 2(+) EL4 cell line and for Thy-1.2(+) concanavalin A-stimulated mouse T lymphocytes. These results represent the first indication of the suitability of transfection of human cancer cells with selected targeting genes for site-specific therapy of malignancies.     

Baicalein protects against doxorubicin-induced cardiotoxicity by attenuation of mitochondrial oxidant injury and JNK activation

The cardiotoxicity of doxorubicin limits its clinical use in the treatment of a variety of malignancies. Previous studies suggest that doxorubicin-associated cardiotoxicity is mediated by reactive oxygen species (ROS)-induced apoptosis. We therefore investigated if baicalein, a natural antioxidant component of Scutellaria baicalensis, could attenuate ROS generation and cell death induced by doxorubicin. Using an established chick cardiomyocyte model, doxorubicin (10 μM) increased cell death in a concentration- and time-dependent manner. ROS generation was increased in a dose-response fashion and associated with loss of mitochondrial membrane potential. Doxorubicin also augmented DNA fragmentation and increased the phosphorylation of ROS-sensitive pro-apoptotic kinase c-Jun N-terminal kinase (JNK). Adjunct treatment of baicalein (25 μM) and doxorubicin for 24 h significantly reduced both ROS generation (587 ± 89 a.u. vs. 932 a.u. ± 121 a.u., P < 0.01) and cell death (30.6 ± 5.1% vs. 46.8 ± 8.3%, P < 0.01). The dissipated mitochondrial potential and increased DNA fragmentation were also ameliorated. Along with the reduction of ROS and apoptosis, baicalein attenuated phosphorylation of JNK induced by doxorubicin (1.7 ± 0.3 vs. 3.0 ± 0.4-fold, P < 0.05). Co-treatment of cardiomyocytes with doxorubicin and JNK inhibitor SP600125 (10 μM; 24 h) reduced JNK phosphorylation and enhanced cell survival, suggesting that the baicalein protection against doxorubicin cardiotoxicity was mediated by JNK activation. Importantly, concurrent baicalein treatment did not interfere with the anti-proliferative effects of doxorubicin in human breast cancer MCF-7 cells. In conclusion, baicalein adjunct treatment confers anti-apoptotic protection against doxorubicin-induced cardiotoxicity without compromising its anti-cancer efficacy.     

Involvement of Yes‐associated protein 1 (YAP1) in doxorubicin‐induced cytotoxicity in H9c2 cardiac cells

Cardiac cell death is one of the major events implicated in doxorubicin‐induced cardiotoxicity, which leads to heart failure. We recently reported that Yes‐associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin‐induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin‐induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin‐treated mice with increased caspase‐3 activation. Doxorubicin also caused cell death by increasing caspase‐3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase‐3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1–5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin‐induced cell death by decreasing caspase‐3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin‐ and YAP1 short interfering RNA (siRNA)‐treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin‐induced cardiac cell death is mediated in part by down‐regulation of YAP1 and YAP1‐targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin‐induced cardiotoxicity.     

Star structure of antibody-targeted HPMA copolymer-bound doxorubicin: a novel type of polymeric conjugate for targeted drug delivery with potent antitumor effect

The aim of this study was to compare the properties and antitumor potential of a novel type of antibody-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin conjugates with star structure with those of previously described classic antibody-targeted or lectin-targeted HPMA copolymer-bound doxorubicin conjugates. Classic antibody-targeted conjugates were prepared by aminolytic reaction of the multivalent HPMA copolymer containing side-chains ending in 4-nitrophenyl ester (ONp) reactive groups with primary NH(2) groups of the antibodies. The star structure of antibody-targeted conjugates was prepared using semitelechelic HPMA copolymer chains containing only one reactive N-hydroxysuccinimide group at the end of the backbone chain. In both types of conjugates, B1 monoclonal antibody (mAb) was used as a targeting moiety. B1 mAb recognizes the idiotype of surface IgM on BCL1 cells. The star structure of the targeted conjugate had a narrower molecular mass distribution than the classic structure. The peak in the star structure was around 300-350 kDa, while the classic structure conjugate had a peak around 1300 kDa. Doxorubicin was bound to the HPMA copolymer via Gly-Phe(D,L)-Leu-Gly spacer to ensure the controlled intracellular delivery. The release of doxorubicin from polymer conjugates incubated in the presence of cathepsin B was almost twice faster from the star structure of targeted conjugate than from the classic one. The star structure of the targeted conjugate showed a lower binding activity to BCL1 cells in vitro, but the cytostatic activity measured by [(3)H]thymidine incorporation was three times higher than that seen with the classic conjugate. Cytostatic activity of nontargeted and anti-Thy 1.2 mAb (irrelevant mAb) modified HPMA copolymer-bound doxorubicin was more than hundred times lower as compared to the star structure of B1 mAb targeted conjugate. In vivo, both types of conjugates targeted with B1 mAb bound to BCL1 cells in the spleen with approximately the same intensity. The classic structure of the targeted conjugate bound to BCL1 cells in the blood with a slightly higher intensity than the star structure. Both types of targeted conjugates had a much stronger antitumor effect than nontargeted HPMA copolymer-bound doxorubicin and free doxorubicin. The star structure of targeted conjugate had a remarkably higher antitumor effect than the classic structure: a single intravenous dose of 100 microg of doxorubicin given on day 11 completely cured five out of nine experimental animals whereas the classic structure of targeted conjugate given in the same schedule only prolonged the survival of experimental mice to 138% of control mice. These results show that the star structure of antibody-targeted HPMA copolymer-bound doxorubicin is a suitable conjugate for targeted drug delivery with better characterization, higher cytostatic activity in vitro, and stronger antitumor potential in vivo than classic conjugates.     

Effect of alpha-tocopherol on peroxidative membrane damage caused by doxorubicin: an in vitro study in human erythrocytes

Level of lipid peroxidation in doxorubicin treated human erythrocytes was studied and compared with that of cells pretreated with alpha-tocopherol. Erythrocytes treated with alpha-tocopherol had reduced level of lipid peroxidation with concomitantly lowered membrane damage. The membrane damage was monitored by the levels of conjugated diene absorption, lipid hydroperoxides and lipid peroxides. alpha-tocopherol was not effective in inhibiting the conjugated diene formation, but the lipid hydroperoxides and the lipid peroxide levels were significantly decreased. Methemoglobin level was found to be increased in alpha-tocopherol pretreated cells, which protects the membrane from damage. Erythrocyte membrane lipids were found to be decreased during doxorubicin treatment and alpha-tocopherol significantly reduced the membrane lipid breakdown. Level of reduced glutathione was maintained in alpha-tocopherol pretreated cells. These results are discussed with reference to the antioxidant property of alpha-tocopherol.     

Proliferative activity of Echinacea angustifolia root extracts on cancer cells: Interference with doxorubicin cytotoxicity

Doxorubicin is an anticancer drug that causes apoptosis in cells, but cardiotoxicity limits the cumulative dose that can remain in the blood. Echinacea extracts have been prescribed to supplement cancer chemotherapy. In a recent study, it was reported that Echinacea purpurea extracts protected noncancerous cells from apoptosis. Our study aimed to determine interference with doxorubicin chemotherapy, and if fractions and compounds from Echinacea angustifolia roots protected the cells. Cervical and breast cancer cells were treated with the Echinacea samples and doxorubicin. At 0.05 and 0.5 microM doxorubicin concentration, cynarine increased HeLa cell growth by 48-125% and 29-101%, respectively (p<0.01). At 0.05 microM doxorubicin concentration, chicoric acid increased cell growth by 23-100% (p<0.01). When MCF-7 cells were treated with Echinacea and doxorubicin, the ethyl acetate fraction increased cell growth by 20-25%, and chicoric acid increased cell growth by 10-15%. Cynarine showed proliferative activity on HeLa cells, but showed antiproliferative activity on MCF-7 cells. Results indicate that phenolic compounds are responsible for proliferative activity. Studies with individual compounds show that chicoric acid and cynarine interfered with cells treated with 0.5 microM doxorubicin. The results of this study show that Echinacea herbal medicines affect cell proliferation despite cancer treatment, and that herbal medicines require further study with respect to anticancer drugs.     

IL-1β induces ER stress in a JNK dependent manner that determines cell death in human pancreatic epithelial MIA PaCa-2 cells

The proinflammatory cytokine, IL-1β (Interleukin-1β) is a significant determinant of pancreatic apoptosis and cell death that are common characteristics during diabetes. Using human derived pancreatic MIA PaCa-2 cells, we describe one of the underlying molecular mechanisms behind this observation. Incubation of these cells with IL-1β at doses from 0.5 to 3.0 ng/ml caused significant cell death at 36 h. This was accompanied with marked increases in JNK and p38 phosphorylation together with increased levels of the endoplasmic reticulum (ER) stress markers, namely BiP, CHOP, GADD34, ATF4 and sXBP1. IL-1β also led to increased phosphorylation of eIF2α and all these events could be prevented by pretreatment with the JNK inhibitor, SP600125. A time course study indicated that while IL-1β mediated JNK phosphorylation was induced as early as 2 h of IL-1β treatment, induction of the ER stress markers was evident at later time points. IL-1β stimulated JNK phosphorylation was observed even in the presence of the ER stress inhibitor, 4-phenyl butyrate and the decrease in cell viability was significantly prevented in the presence of the JNK inhibitor. All these suggest that JNK activation is a pre-requisite for ER stress induction and cell death. Reports till date have consistently demonstrated JNK activation as a consequence of ER stress induction by IL-1β in the pancreas. We show here for the first time that the activation of JNK by IL-1β is a prelude to the subsequent induction of ER stress and cell death. These therefore suggest that the JNK-ER stress axis is critical in deciding the decreased survival status by IL-1β in MIA PaCa-2 cells.     

HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.     

Differential regulation of HSP70 expression by the JNK kinases SEK1 and MKK7 in mouse embryonic stem cells treated with cadmium

JNK, a member of the mitogen-activated protein kinases (MAPKs), is activated by the MAPK kinases SEK1 and MKK7 in response to environmental stresses. In the present study, the effects of CdCl2 treatment on MAPK phosphorylation and HSP70 expression were examined in mouse embryonic stem (ES) cells lacking the sek1 gene, the mkk7 gene, or both. Following CdCl2 exposure, the phosphorylation of JNK, p38, and ERK was suppressed in sek1-/- mkk7-/- cells. When sek1-/- or mkk7-/- cells were treated with CdCl2, JNK phosphorylation, but not the phosphorylation of either p38 or ERK, was markedly reduced, while a weak reduction in p38 phosphorylation was observed in sek1-/- cells. Thus, both SEK1 and MKK7 are required for JNK phosphorylation, whereas their role in p38 and ERK phosphorylation could overlap with that of another kinase. We also observed that CdCl2-induced HSP70 expression was abolished in sek1-/- mkk7-/- cells, was reduced in sek1-/- cells, and was enhanced in mkk7-/- cells. Similarly, the phosphorylation of heat shock factor 1 (HSF1) was decreased in sek1-/- mkk7-/- and sek1-/- cells, but was increased in mkk7-/- cells. Transfection with siRNA specific for JNK1, JNK2, p38, ERK1, or ERK2 suppressed CdCl2-induced HSP70 expression. In contrast, silencing of p38 or p38 resulted in further accumulation of HSP70 protein. These results suggest that HSP70 expression is up-regulated by SEK1 and down-regulated by MKK7 through distinct MAPK isoforms in mouse ES cells treated with CdCl2.     

Ultrasmall gold-doxorubicin conjugates rapidly kill apoptosis-resistant cancer cells

Ultrasmall (mean diameter, 2.7 nm) gold nanoparticles conjugated to doxorubicin (Au-Dox) are up to 20-fold more cytotoxic to B16 melanoma cells than the equivalent concentration of doxorubicin alone, and act up to six times more quickly. Ultrasmall Au-Dox enters the cell endocytic vesicles and is also seen free in the cytoplasm and nuclei. This is in distinct contrast to larger particles reported in previous studies, which are excluded from the nucleus and which show no increased toxicity over Dox alone. Cell death with Au-Dox is confirmed to be apoptotic by TUNEL staining and ultrastructural examination using transmission electron microscopy. To further explore the mechanism of action, two other cell lines were examined: HeLa cells which are highly sensitive to Dox, and HeLa cells overexpressing Bcl-2 which show impaired apoptosis and Dox resistance. Interestingly, the Dox-sensitive cells show a slightly decreased sensitivity to Au-Dox relative to Dox alone, whereas the Dox-resistant cells are not resistant to Au-Dox. These results have implications for the design of chemotherapeutic nanoparticles, suggesting that it is possible to selectively target apoptosis-resistant cancer cells while at the same time reducing cytotoxicity to normal cells.     

Parkin deficiency increases the resistance of midbrain neurons and glia to mild proteasome inhibition: the role of autophagy and glutathione homeostasis

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals and abnormal neurotransmitter release. In this study, we have investigated whether partial proteasomal inhibition by epoxomicin, an ubiquitin proteasomal system (UPS) irreversible inhibitor, further aggravates the cellular effects of parkin suppression in midbrain neurons and glia. We observed that parkin null (PK‐KO) midbrain neuronal cultures are resistant to epoxomicin‐induced cell death. This resistance is due to increased GSH and DJ‐1 protein levels in PK‐KO mice. The treatment with epoxomicin increases, in wild type (WT) cultures, the pro‐apoptotic Bax/Bcl‐2 ratio, the phosphorylation of tau, and the levels of chaperones heat‐shock protein 70 and C‐terminal Hsc‐interacting protein, but none of these effects took place in epoxomicin‐treated PK‐KO cultures. Poly‐ubiquitinated proteins increased more in WT than in PK‐KO‐treated neuronal cultures. Parkin accumulated in WT neuronal cultures treated with epoxomicin. Markers of autophagy, such as LC3II/I, were increased in naïve PK‐KO cultures, and further increased after treatment with epoxomicin, implying that the blockade of the proteasome in PK‐KO neurons triggers the enhancement of autophagy. The treatment with l ‐buthionine‐S,R‐sulfoximine and the inhibition of autophagy, however, reverted the increase resistance to epoxomicin of the PK‐KO cultures. We also found that PK‐KO glial cells, stressed by growth in defined medium and depleted of GSH, were more susceptible to epoxomicin induced cell death than WT glia treated similarly. This susceptibility was linked to reduced GSH levels and less heat‐shock protein 70 response, and to activation of p‐serine/threonine kinase protein signaling pathway as well as to increased poly‐ubiquitinated proteins. These data suggest that mild UPS inhibition is compensated by other mechanisms in PK‐KO midbrain neurons. However the depletion of GSH, as happens in stressed glia, suppresses the protection against UPS inhibition‐induced cell death. Furthermore, GSH inhibition regulated differentially UPS activity and in old PK‐KO mice, which have depletion of GSH, UPS activity is decreased in comparison with that of old‐WT.     

Contribution of endoplasmic reticulum stress,MAPK and PI3K/Akt pathways to the apoptotic death induced by a penicillin derivative in melanoma cells

We have previously examined the in vitro and in vivo antitumor action of TAP7f, a synthetic triazolylpeptidyl penicillin, on murine melanoma cells. In this work, we explored the signal transduction pathways modulated by TAP7f in murine B16-F0 and human A375 melanoma cells, and the contribution of some intracellular signals to the apoptotic cell death. TAP7f decreased ERK1/2 phosphorylation and increased phospho-p38, phospho-JNK and phospho-Akt levels. ERK1/2 blockage suppressed cell growth, while inhibition of p38, JNK and PI3K-I pathways reduced the antitumor effect of TAP7f. Pharmacological inhibition of p38 and JNK, or blockage of PI3K-I/Akt cascade with a dominant negative PI3K-I mutant diminished Bax expression levels and PARP-1 cleavage, indicating the involvement of these pathways in apoptosis. PI3K-I/Akt inhibition also favored an autophagic response, as evidenced by the higher expression levels of Beclin-1 and LC3-II detected in transfected cells exposed to TAP7f. However, although PI3K-I/Akt blockage promoted an autophagic survival response, this mechanism appears not to be critical for TAP7f antitumor action. It was also shown that TAP7f induced ER stress by enhancing the expression of ER stress-related genes and proteins. Downregulation of CHOP protein with specific siRNA increased cell growth and decreased cleavage of PARP-1, supporting its role in apoptosis. Furthermore, it was found that activation of p38, JNK and Akt occurred downstream ER perturbation. In summary, our results showed that TAP7f triggers an apoptotic cell death in melanoma cells through induction of ER stress and activation of p38, JNK and PI3K-I/Akt pathways.

     

Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft

Melanoma is very aggressive and major cause of mortality due to skin cancer. Herein, we studied the anticancer effects of berberine, a plant alkaloid, in combination with doxorubicin on murine melanoma B16F10 cells in vitro and in vivo. This drug combination strongly inhibited cell growth and induced cell death, and caused G2/M arrest in cell cycle together with a decrease in Kip1/p27. Berberine showed stronger inhibitory effect on ERK1/2 phosphorylation as compared to Akt phosphorylation, whereas the combination of the drugs showed greater inhibitory effect on Akt phosphorylation. In murine B16F10 xenograft, cells were implanted into mice and treated with vehicle (methyl cellulose) or berberine (100 mg/kg of body weight/day by oral gavage) or doxorubicin (4 mg/kg of body weight/week by intraperitoneal injection) or combination of berberine and doxorubicin. Berberine alone did not show any considerable effect on tumor growth as observed with doxorubicin, however, the combination of the two drugs resulted in a significant and strong decrease in tumor volume (85%, p < 0.005) and tumor weight (78%, p < 0.05) as compared to control. Immunohistochemical analysis of tumor samples showed that drug combination decreased PCNA-positive cells (82%, p < 0.001) and increased cleaved caspase-3 positive cells (3-fold, p < 0.05) indicating inhibition of proliferation and an increase in apoptosis, respectively. Overall, our findings suggest that berberine and doxorubicin could be a novel combination to inhibit melanoma tumor growth.     

The action of antifeins with different memory effects on the cAMP-independent protein kinases of brain chromatin]

The two cAMP-independent protein kinases (PK I and PK II) were obtained from neuronal chromatin of rat brain. Nonhistone HMG-proteins were used as phosphorylation substrates. The possibility of antifeines direct action on PK II was revealed. Ethylnorantifeine and its demethylated analogues increased enzyme activity whereas allylnorantifeine decreased it. The action of ethylnorantifeine and its structural analogues were similar to its influence on neuronal RNA-synthesizing activity and long-term memory. The conclusion was made that cAMP-independent PK II may be the target of antifeines action during realization of its mnestic effects.     

Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth.     

Induction of premature senescence in cardiomyocytes by doxorubicin as a novel mechanism of myocardial damage

Cellular senescence is an important phenomenon in decreased cellular function. Recently, it was shown that cellular senescence is induced in proliferating cells within a short period of time by oxidative stresses. This phenomenon is known as premature senescence. However, it is still unknown whether premature senescence can be also induced in cardiomyocytes. The aim of the present study was to investigate whether a senescence-like phenotype can be induced in cardiomyocytes by oxidative stress. In cardiomyocytes obtained from aged rats (24 months of age), the staining for senescence-associated beta-galactosidase increased significantly and the protein or RNA levels of cyclin-dependent kinase inhibitors increased compared to those of young rats. Decreased cardiac troponin I phosphorylation and telomerase activity were also observed in aged cardiomyocytes. Treatment of cultured neonatal rat cardiomyocytes with a low concentration of doxorubicin (DOX) (10(-7) mol L(-1)) did not induce apoptosis but did induce oxidative stress, which was confirmed by 2',7'-dichlorofluorescin diacetate staining. In DOX-treated neonatal cardiomyocytes, increased positive staining for senescence-associated beta-galactosidase, cdk-I expression, decreased cardiac troponin I phosphorylation, and decreased telomerase activity were observed, as aged cardiomyocytes. Alterations in mRNA expression typically seen in aged cells were observed in DOX-treated neonatal cardiomyocytes. We also found that promyelocytic leukemia protein and acetylated p53, key proteins involved in stress-induced premature senescence in proliferating cells, were associated with cellular alterations of senescence in DOX-treated cardiomyocytes. In conclusion, cardiomyocytes treated with DOX showed characteristic changes similar to cardiomyocytes of aged rats. promyelocytic leukemia-related p53 acetylation may be an underlying mechanism of senescence-like alterations in cardiomyocytes. These findings indicate a novel mechanism of myocardial dysfunction induced by oxidative stress.     

The RhoGDI-α/JNK signaling pathway plays a significant role in mycophenolic acid-induced apoptosis in an insulin-secreting cell line

Mycophenolic acid (MPA)-induced β-cell toxicity is an important factor for islet graft function. The signal transduction mechanisms underlying this process have not been fully explored. Using a proteomics approach, we examined protein expression patterns in MPA-treated RIN-5 cells and found that RhoGDI-α expression is altered by MPA-treatment. We examined the relationship between RhoGDI-α expression and activated JNK during MPA-induced apoptosis. Cells were treated with N-acetyl-cysteine (NAC), caspase inhibitor, JNK inhibitor, guanosine or GTP for 1 h before being treated with MPA. To investigate the regulatory effects of RhoGDI-α on JNK activity, we examined cells showing either elevated or reduced expression of RhoGDI-α as a result of transfection with cDNA or siRNA constructs, respectively. MPA significantly increased cell death, caspase-3 expression and JNK activation, but it decreased the expression of a protein spot 25 observed by two-dimensional electrophoresis. This protein 25 was identified as RhoGDI-α by mass spectrometry. MPA-induced cell death and down-regulation of RhoGDI-α were prevented by guanosine, GTP or a JNK inhibitor. However, MPA-induced cell death was partially restored by treatment with a caspase inhibitor, but not by NAC treatment. RhoGDI-α expression was not affected by treatment with NAC or caspase inhibitor. Over-expression of RhoGDI-α increased cell viability and decreased activated JNK expression following exposure to MPA, whereas knockdown of RhoGDI-α enhanced MPA-induced cell death and increased the activation of JNK. In conclusion, MPA induces significant apoptosis in insulin-secreting cells via down-regulation of RhoGDI-α linked with increased JNK expression. This RhoGDI-α/JNK pathway might be the focus of therapeutic target for the prevention of MPA-induced islet apoptosis.