Selective potentiation of drug cytotoxicity by NSAID in human glioma cells: the role of COX-1 and MRP.

Here, we report that nonsteroidal anti-inflammatory drugs (NSAID) enhance the cytotoxic effects of doxorubicin and vincristine in T98G human malignant glioma cells. The cytotoxicity of BCNU, cisplatin, VM26, camptothecin, and cytarabine is unaffected by NSAID. No free radical formation is induced by doxorubicin or vincristine in the absence or presence of NSAID. Doxorubicin and vincristine cytotoxicity in the absence or presence of NSAID are unaffected by free radical scavengers. Functional inhibitors of phospholipase A2 (PLA2), such as dexamethasone and quinacrine, do not mimick the effects of NSAID. T98G cells, but not LN-18, LN-229, LN-308, or A172 glioma cells, express cyclooxygenase (COX-1) and NSAID do not modulate drug cytotoxicity in the other cell lines, except T98G. Thus, augmentation of doxorubicin and vincristine cytotoxicity by NSAID correlates with COX-1 expression. However, ectopic expression of COX-1 in LN-229 cells does not induce the phenotype of T98G cells, indicating that COX-1 inhibition does not mediate the effects of NSAID on drug cytotoxicity. In contrast, a multidrug resistance (MDR) phenotype due to expression of the multidrug resistance-associated protein (MRP) is most prominent in T98G cells and is amenable to modulation by indomethacin, suggesting that inhibition of MRP is at least in partly responsible for the potentiation of doxorubicin and vincristine cytotoxicity by NSAID.     

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-X(L). These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.     

Accumulation of Mutant p53 Modulates the Growth, Clonogenicity, and Radiochemosensitivity of Malignant Glioma Cells Independent of Endogenous p53 Status

Alterations of the p53 gene have been attributed a major role in the development and resistance to therapy of several human cancers. Accumulation of p53 in tumor cells may result from mutations associated with prolonged half-life or from stabilization of wild-type p53 by different mechanisms. To address the role of p53 accumulation in the response of malignant glioma cells to radiochemotherapy, we expressed the p53 mutant p53^V143A in five human malignant glioma cell lines with different genetic and functional p53 status. Accumulation of p53^V143A modulated proliferation in three and clonogenicity in four of five cell lines without a clear pattern with regard to their endogenous p53 status. p53^V143A inhibited the camptothecin-induced accumulation of p21^WAF1/CIP1 in cell lines with p53 functional wild-type activity, but not in cell lines lacking p53 activity, consistent with a transdominant-negative effect of p53^V143A. Irradiation induced a moderate G2/M arrest in all cell lines, irrespective of the p53 status, that was unaffected by p53^V143A. Radiosensitivity as well as sensitivity to BCNU, teniposide (VM26), topotecan, vincristine, Taxol, and cisplatin both in cytotoxic cell death and in clonogenic cell death was unchanged in p53^V143A-transfected cells with few exceptions. These data do not support the hypothesis that accumulation of mutant p53 is a major determinant of the response to adjuvant radiochemotherapy in human malignant glioma cells.     

Structurally similar diazenes exhibit significantly different biological activity

We have previously synthesized various diazenecarboxamides (subsequently referred to as diazenes) that were cytotoxic to several tumor cell lines. To increase their biological activity, the structure has been modified appropriately. In the present study we examined the effects of N¹-phenyl-N²-(2-pyridinylmethyl)diazenedicarboxamide (RL-337) obtained from the previously examined cytotoxic compound N¹-phenyl-N²-(2-pyridinyl)diazenecarboxamide (JK-279), and compared them with those of diazene JK-279. Using a modified colorimetric MTT assay, the cytotoxicity of RL-337 was determined on human cervical carcinoma HeLa cells, glioblastoma A1235 cells, and prostate adenocarcinoma PC-3 cells. The possible synergistic effect of diazene RL-337 with cisplatin, doxorubicin, and vincristine, and its influence on intracellular GSH content was examined on HeLa cells. Diazene RL-337 was cytotoxic against all three human tumor cell lines, being more cytotoxic to HeLa cells than diazene JK-279. The higher efficacy of RL-337 than of JK-279 can be connected with higher basicity of the 2-picoline moiety present in the former diazene comparing with the pyridine fragment that is a part of the latter. The diazene RL-337 acted synergistically with cisplatin, doxorubicin, and vincristine (diazene JK-279 exhibited synergistic effect only with cisplatin). Glutathione (determined by Tietze's method) was not a target molecule of diazene RL-337 (but was for JK-279, as shown earlier). After just 1 h treatment with diazene RL-337, the cells started to lose membrane integrity. There was no cleavage of caspase-3 in RL-337-treated samples, and the majority of cells died 6 h after the treatment through necrosis (previously, apoptosis-like cell death was detected for diazene JK-279). Thus, although diazenes JK-279 and RL-337 are very similar in their structure, they exhibit widely different biological activity.     

p53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells

Induction of CD95 (Fas/APO-1) and CD95 ligand during chemotherapeutic treatment may contribute to the death by apoptosis of some tumor cells. In this study, we have analyzed the role of the CD95 system in genotoxic drug-induced death of human breast tumor cells. Incubation of the breast tumor cell lines MCF-7 and EVSA-T with doxorubicin or methotrexate caused apoptosis after 48 h of treatment. These drugs induced a marked increase in the level of CD95 mRNA and protein in wild-type p53-expressing MCF-7 cells. On the contrary, the breast cancer cell line EVSA-T that expresses high levels of an inactive form of p53, did not up-regulate CD95 upon drug treatment. Elevation of CD95 expression by DNA-damaging drugs was notably blocked in MCF-7 cells expressing the human papillomavirus type 16 E6 protein (E6 cells) which prevented p53 accumulation upon DNA damage. However, E6 cells were still killed by the drugs. Furthermore, the genotoxic drugs did not induce the expression of CD95 ligand in MCF-7 cells at doses that caused apoptosis in these breast tumor cells. Moreover, drug-induced apoptosis of breast tumor cells was not prevented in the presence of either a CD95 antagonistic antibody or a CD95 ligand blocking antibody. We also observed a strong synergism between lower doses of DNA-damaging drugs and CD95 agonistic antibody in the induction of apoptosis in MCF-7 cells. In summary, our data indicate that drug-induced apoptosis of breast tumor cells occurs by a CD95/CD95L-independent mechanism although by elevating the tumor suppressor proteins p53 and CD95, genotoxic drugs may sensitize breast tumor cells to CD95-mediated apoptosis.     

The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.     

Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing

Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis.     

Accumulation of mutant p53(V143A) modulates the growth, clonogenicity, and radiochemosensitivity of malignant glioma cells independent of endogenous p53 status

Alterations of the p53 gene have been attributed a major role in the development and resistance to therapy of several human cancers. Accumulation of p53 in tumor cells may result from mutations associated with prolonged half-life or from stabilization of wild-type p53 by different mechanisms. To address the role of p53 accumulation in the response of malignant glioma cells to radiochemotherapy, we expressed the p53 mutant p53(V143A) in five human malignant glioma cell lines with different genetic and functional p53 status. Accumulation of p53(V143A) modulated proliferation in three and clonogenicity in four of five cell lines without a clear pattern with regard to their endogenous p53 status. p53(V143A) inhibited the camptothecin-induced accumulation of p21(WAF1/CIP1) in cell lines with p53 functional wild-type activity, but not in cell lines lacking p53 activity, consistent with a transdominant-negative effect of p53(V143A). Irradiation induced a moderate G2/M arrest in all cell lines, irrespective of the p53 status, that was unaffected by p53(V143A). Radiosensitivity as well as sensitivity to BCNU, teniposide (VM26), topotecan, vincristine, Taxol, and cisplatin both in cytotoxic cell death and in clonogenic cell death was unchanged in p53(V143A)-transfected cells with few exceptions. These data do not support the hypothesis that accumulation of mutant p53 is a major determinant of the response to adjuvant radiochemotherapy in human malignant glioma cells.     

C2-ceramide signaling in glioma cells: synergistic enhancement of CD95-mediated, caspase-dependent apoptosis

Most human malignant glioma cell lines are susceptible to CD95 ligand (CD95L)-induced apoptosis. Here, we report that glioma cells are also susceptible to the cytotoxic effects of exogenous C2-ceramide. This form of cell death exhibits some morphological features of apoptosis as assessed by electron microscopy, but is unaffected by the broad spectrum caspase inhibitor, zVAD-fmk. Further, CD95L-induced apoptosis is synergistically enhanced by coexposure of the glioma cells to CD95L and C2-ceramide. CD95L-induced caspase 3-like activity, cytochrome c release and cleavage of caspases 3, 8, 9 and poly(ADP-ribose)polymerase (PARP) increase substantially after cotreatment with CD95L and C2-ceramide compared with CD95L treatment alone. None of these events occur in response to cytotoxic concentrations of C2-ceramide alone. C2-ceramide does not alter CD95 expression. Gene transfer-mediated enhancement of CD95 expression results not only in increased susceptibility to CD95L, but also in increased sensitivity to C2-ceramide. We conclude that (i) synergistic induction of apoptosis by C2-ceramide and CD95L depend on a cross-talk between the two signal transduction pathways and that (ii) C2-ceramide, independently of its sensitizing effects on CD95-dependent caspase activation, is also capable of triggering an apoptotic signaling cascade that is unaffected by zVAD-fmk-mediated caspase inhibition, but promoted by high levels of CD95 expression.     

A novel p53 rescue compound induces p53-dependent growth arrest and sensitises glioma cells to Apo2L/TRAIL-induced apoptosis

Reactivation of mutant p53 in tumours is a promising strategy for cancer therapy. Here we characterise the novel p53 rescue compound P53R3 that restores sequence-specific DNA binding of the endogenously expressed p53(R175H) and p53(R273H) mutants in gel-shift assays. Overexpression of the paradigmatic p53 mutants p53(R175H), p53(R248W) and p53(R273H) in the p53 null glioma cell line LN-308 reveals that P53R3 induces p53-dependent antiproliferative effects with much higher specificity and over a wider range of concentrations than the previously described p53 rescue drug p53 reactivation and induction of massive apoptosis (PRIMA-1). Furthermore, P53R3 enhances recruitment of endogenous p53 to several target promoters in glioma cells bearing mutant (T98G) and wild-type (LNT-229) p53 and induces mRNA expression of numerous p53 target genes in a p53-dependent manner. Interestingly, P53R3 strongly enhances the mRNA, total protein and cell surface expression of the death receptor death receptor 5 (DR5) whereas CD95 and TNF receptor 1 levels are unaffected. Accordingly, P53R3 does not sensitise for CD95 ligand- or tumour necrosis factor alpha-induced cell death, but displays synergy with Apo2L.0 in 9 of 12 glioma cell lines. Both DR5 surface induction and synergy with Apo2L.0 are sensitive to siRNA-mediated downregulation of p53. Thus this new p53 rescue compound may open up novel perspectives for the treatment of cancers currently considered resistant to the therapeutic induction of apoptosis.     

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.     

NOXA as critical mediator for drug combinations in polychemotherapy

During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug–drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.     

Induction of CD95 ligand and apoptosis by doxorubicin is modulated by the redox state in chemosensitive- and drug-resistant tumor cells.

Induction of CD95 ligand (CD95-L) may contribute to drug-induced apoptosis in chemosensitive leukemias and solid tumors. Here we report that induction of CD95-L and apoptosis by doxorubicin in leukemic and neuroblastoma cells is regulated by the redox state and reactive oxygen species (ROS). Preincubation of chemosensitive cells with antioxidants such as N-acetyl-cysteine (NAC) or glutathione (GSH), significantly reduced doxorubicin-induced apoptosis, hyperexpression of ROS, loss of mitochondrial membrane potential (DeltaPsim) and upregulation of CD95-L expression. Doxorubicin-resistant cells exhibited higher levels of GSH in comparison to chemosensitive cells and were deficient in hyperproduction of ROS, loss of DeltaPsim and upregulation of CD95-L in response to cytotoxic drugs. Downregulation of intracellular GSH concentrations reversed deficient drug-induced hyperproduction of ROS and CD95-L upregulation. In addition, overexpression of Bcl-XL in CEM cells blocked doxorubicin-triggered ROS and CD95-L expression. These findings suggest that induction of CD95-L by cytotoxic drugs is modulated by the cellular redox state and mitochondria derived ROS.     

Preparation and functional evaluation of new doxorubicin immunoconjugates containing an acid-sensitive linker on small-cell lung cancer cells

 The anthracycline doxorubicin (DOX) is one of the most effective drugs for the treatment of small-cell lung cancer (SCLC), but its clinical application is limited by unspecific side-effects like cardiotoxicity. In the present study doxorubicin was conjugated to the monoclonal antibodies (mAb) SEN7, MOC31, and SWA11 via a novel acid-sensitive hydrazone linker. These mAb recognize SCLC-associated antigens of cluster 1 (NCAM), cluster 2 (EGP-2/GA733-2), and cluster 4 (CD24) respectively. To assess their potential therapeutic use against SCLC, the antigen-binding activities, the rates of internalization and the cytotoxic effects of the immunoconjugates were examined on tumour cell lines. The preparation procedure preserved the antigen-binding activities of the mAb and yielded immunoconjugates with average drug : mAb ratios of 7 : 1. The hydrazone linker was found to be stable at neutral pH but to release doxorubicin under acidic conditions. In contrast to SEN7-DOX, MOC31-DOX and SWA11-DOX were rapidly internalized into SCLC target cells upon binding to their specific cell-surface antigens. Accordingly, both immunoconjugates proved to be highly cytotoxic agents, inhibiting thymidine incorporation by 50% at concentrations between 0.5 μM and 1 μM and were 100-fold more selective than free doxorubicin. The results suggest that binding to selective cell-surface antigens, rapid internalization and efficient release of doxorubicin from the mAb by acid hydrolysis are required for the selective and potent function of the immunoconjugates. In particular, the use of MOC31-DOX for targeted cytotoxic therapy might be promising because of the limited cross-reactivity of the mAb with normal human tissues and its recently demonstrated tumour localization potential in SCLC patients. Received: 22 September 1995 / Accepted: 23 November 1995     

Resveratrol enhances the cytotoxic profile of docetaxel and doxorubicin in solid tumour cell lines in vitro

Objectives: Resveratrol, with its robust antioxidant activity, has frequently been suggested as potentially having activity in cancer prevention and some recent reports have indicated that it has cancer treatment potential for several types of neoplasia. It has been found to block p‐glycoprotein and to protect against several chemotherapeutic agents’ side effects. In this study, we assessed interactive characteristics of resveratrol with docetaxel and doxorubicin and further investigated molecular bases of this interaction in cells of three different solid tumour lines (MCF‐7, HeLa and HepG2). Materials and methods and results: Resveratrol per se was found to have anti‐cancer properties, but with relatively low potency in all tested cell lines (IC₅₀ ranged from 35.1 to 83.8 μM). Doxorubicin and docetaxel showed IC₅₀ ranging from 0.48 to 0.72 μM and from 25.9 to 77.8 nM, respectively. Resveratrol in combination with doxorubicin and docetaxel significantly increased potencies of both chemotherapeutic agents showing IC₅₀ ranging from 0.12 to 0.34 μM and from 7.2 to 53.02 nM, respectively. The combination index showed synergistic interaction between resveratrol and doxorubicin or docetaxel on MCF‐7 cells, and additive interactions on HeLa and HepG2 cells. Real time PCR revealed that expression of Bax and Bcl‐2 was simultaneously elevated on combination of resveratrol with doxorubicin or docetaxel in all tested cell lines, whereas p53 exhibited marginal elevation in MCF‐7 and HepG2 cells. In addition, p‐glycoprotein efflux activity was significantly inhibited, with subsequent accumulation of p‐glycoprotein substrate in intracellular compartments. Expression level of mdr1 gene was downregulated after resveratrol combined with doxorubicin or docetaxel in all tested cell lines. Conclusion: Resveratrol potentiates cytotoxic properties of both cancer drugs used in the study through increasing their intracellular level due to p‐glycoprotein inhibition and downregulation of mdr1 gene.     

Involvement of Microtubules in the Regulation of Bcl2 Phosphorylation and Apoptosis through Cyclic AMP-Dependent Protein Kinase

The Bcl2 family of proteins plays a significant role in regulation of apoptosis. In this study, the microtubule-damaging drugs paclitaxel, vincristine, and vinblastine induced Bcl2 hyperphosphorylation and apoptosis in MCF-7 and MDA-MB-231 cells and reduced Bcl2-Bax dimerization. Paclitaxel or vincristine induced increased expression of Bax, while overexpression of Bcl2 in these cell lines counteracted the effects of low doses of these drugs. In addition, paclitaxel- and vincristine-induced activation of cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) induced Bcl2 hyperphosphorylation and apoptosis, which were blocked by the PKA inhibitor Rp diastereomers of cAMP (Rp-cAMP). This finding suggests that activation of PKA due to microtubule damage is an important event in Bcl2 hyperphosphorylation and induction of apoptosis. These microtubule-damaging drugs caused growth arrest in G₂-M phase of the cell cycle and had no effect on p53 induction, suggesting that hyperphosphorylation mediated inactivation of Bcl2 and apoptosis without the involvement of p53. By comparison, the DNA-damaging drugs methotrexate and doxorubicin had no effect on Bcl2 hyperphosphorylation but induced p53 expression. Interestingly, paclitaxel or vincristine induced activation of caspase 3 and cleavage of poly(ADP-ribose) polymerase downstream of Bcl2 hyperphosphorylation. These data suggest that there may be a signaling cascade induced by agents that disrupt or damage the cytoskeleton that is distinct from (i.e., p53 independent), but perhaps related to (i.e., involves kinase activation and leads to apoptosis), the cellular response to DNA damage.     

Cell-mediated cytotoxicity can be regulated by p53 tumor suppressor gene activity in vitro

Summary— The wild-type human p53 tumor suppressor gene was tested for its ability to modulate cytotoxic activity of in vitro activated peripheral blood lymphocytes. Peripheral blood mononuclear cells (PBMCs) were stimulated by phytohemagglutinin (PHA), interferon α2b (IFNα2b), interleukin 2 (IL-2) or their combinations to induce cytotoxicity. This stimulation significantly increased the percentage of cells expressing p53, which was at its maximum when induced by IL-2 combined with IFNα2b. The role of p53 in the modulation of different aspects of cytotoxic activity of these cells was analyzed by studying the effects of p53 abrogation by antisense oligonucleotide (p53 AS) treatment in comparison with p53 sense or scrambled (missense) oligonucleotide (p53 S or p53 MS) treatment. We show that p53 plays a key role through induction of apoptosis in target cells (tumor necrosis factor pathway) rather than through osmolytic degeneration (perforin pathway) which is only slightly increased by p53 abrogation. Meanwhile, in vitro abrogation of p53 expression in PBL was found to be accompanied by an increase of CD8+ lymphocytes and an important increase of the CD56 ‘bright’ NK cell sub-population.     

NF-kappaB-independent actions of sulfasalazine dissociate the CD95L- and Apo2L/TRAIL-dependent death signaling pathways in human malignant glioma cells

Death receptor-mediated apoptosis of human malignant glioma cells triggered by CD95 ligand (CD95L) or Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) share several features, including processing of multiple caspases and mitochondrial cytochrome c release. We here report that CD95L-induced cell death is inhibited by sulfasalazine (SS) in all of four human glioma cell lines, both in the absence and presence of cycloheximide (CHX). Coexposure to CD95L and SS prevents the CD95L-evoked processing of caspases 2, 3, 8 and 9, the release of cytochrome c from mitochondria, and the loss of BCL-x(L) protein. This places the protective effect of SS proximal to most known events triggered by the CD95-dependent signaling pathway in glioma cells. CD95L promotes the accumulation of nuclear factor kappa B (NF-kappaB) in the nucleus and induces the DNA-binding activity of NF-kappaB assessed by electrophoretic mobility shift assay. The total levels of p50, p65 and IkappaBalpha remain unchanged, but the levels of phosphorylated IkappaBalpha and of nuclear p65 increase, in response to CD95L. IkappaBalpha phosphorylation as well as nuclear NF-kappaB translocation and DNA binding are blocked by SS. However, unlike SS, dominant-negative IkappaBalpha (IkappaBdn) does not block apoptosis, suggesting that SS inhibits CD95L-mediated apoptosis in an NF-kappaB-independent manner. In contrast to CD95L, the cytotoxic effects of Apo2L/TRAIL are enhanced by SS, and SS facilitates Apo2L/TRAIL-evoked caspase processing, cytochrome c release, and nuclear translocation of p65. These effects of SS are nullified in the presence of CHX, suggesting that the effects of SS and CHX are redundant or that enhanced apoptosis mediated by SS requires protein synthesis. IkappaBdn fails to modulate Apo2L/TRAIL-induced apoptosis. Similar effects of SS on CD95L- and Apo2L/TRAIL-induced apoptosis are observed in MCF-7 breast and HCT116 colon carcinoma cells. Interestingly, HCT cells lacking p21 (80S14(p21-/-)) are only slightly protected by SS from CD95L-induced apoptosis, but sensitized to Apo2L/TRAIL-induced apoptosis, indicating a link between the actions of SS and p21. Thus, SS modulates the death cascades triggered by CD95L and Apo2L/TRAIL in opposite directions in an NF-kappaB-independent manner, and SS may be a promising agent for the augmentation of Apo2L/TRAIL-based cancer therapies.     

Topotecan enhances immune clearance of gliomas

Despite aggressive surgery, radiation therapy, and chemotherapy, glioblastoma multiforme (GBM) is refractory to therapy, recurs quickly, and results in a median survival time of only 14 months. The modulation of the apoptotic receptor Fas with cytotoxic agents could potentiate the response to therapy. However, Fas ligand (FasL) is not expressed in the brain and therefore this Fas-inducing cell death mechanism cannot be utilized. Vaccination of patients with gliomas has shown promising responses. In animal studies, brain tumors of vaccinated mice were infiltrated with activated T cells. Since activated immune cells express FasL, we hypothesized that combination of immunotherapy with chemotherapy can activate Fas signaling, which could be responsible for a synergistic or additive effect of the combination. When we treated the human glioma cell line U-87 and GBM tumor cells isolated from patients with TPT, Fas was up regulated. Subsequent administration of soluble Fas ligand (sFasL) to treated cells significantly increased their cell death indicating that these Fas receptors were functional. Similar effect was observed when CD3⁺ T cells were used as a source of the FasL, indicating that the up regulated Fas expression on glioma cells increases their susceptibility to cytotoxic T cell killing. This additive effect was not observed when glioma cells were pre-treated with temozolomide, which was unable to increase Fas expression in tumor. Inhibition of FasL activity with the antagonistic antibody Nok-1 mitigated these effects confirming that these responses were specifically mediated by the Fas-FasL interaction. Furthermore, the CD3⁺ T cells co-cultured with topotecan treated U-87 and autologous GBM tumor cells showed a significant increase in expression in IFN-γ, a key cytokine produced by activated T cells, and accordingly enhanced tumor cytotoxicity. Based on our data we conclude that drugs, such as topotecan, which cause up regulation of Fas on glioma cells can be potentially exploited with immunotherapy to enhance immune clearance of tumors via Fas signaling. Jun Wei and Guillermo DeAngulo are Co-lead authors.