Antonio Macho

Selective induction of apoptosis by capsaicin in transformed cells: the role of reactive oxygen species and calcium

Capsaicin is a vanilloid quinone analog that inhibits the plasma membrane electron transport (PMOR) system and induces apoptosis in transformed cells. Using a cytofluorimetric approach we have determined that capsaicin induces a rapid increase of reactive oxygen species (ROS) followed by a subsequent disruption of the transmembrane mitochondrial potential (ΔΨm) and DNA nuclear loss in transformed cell lines and in mitogen activated human T cells. This apoptotic pathway is biochemically different from the typical one induced by either ceramide or edelfosine where, in our system, the ΔΨm dissipation precedes the generation of reactive oxygen species. Neither production of ROS nor apoptosis was found in capsaicin-treated resting T cells where the activity of the PMOR system is minimal when compared with mitogen activated or transformed T cells. Capsaicin also induces Ca2+ mobilization in activated but not in resting T cells. However, preincubation of cells with BAPTA-AM, which chelate cytosolic free calcium, did not prevent ROS generation or apoptosis induced by capsaicin, suggesting that ROS generation in capsaicin treated cells is not a consequence of calcium signaling and that the apoptotic pathway may be separated from the one that mobilizes calcium. Moreover, we present data for the implication of a possible vanilloid receptor in calcium mobilization, but not in ROS generation. These results provide evidence that the PMOR system may be an interesting target to design antitumoral and anti-inflammatory drugs.

Immunosuppressive activity of capsaicinoids: capsiate derived from sweet peppers inhibits NF-κB activation and is a potent antiinflammatory compound in vivo

Capsiate and its dihydroderivatives are the major capsaicinoids of sweet pepper. These new capsaicinoids do not activate the vanilloid receptor type 1 (VR1) but they share with capsaicin (CPS)some biological activities mediated in a VR1‐independent fashion. In this study we show that CPS and nordihydrocapsiate (CPT) inhibit early and late events in T cell activation, including CD69, CD25 and ICAM‐1 cell surface expression, progression to the S phase of the cell cycle and proliferation in response to TCR and CD28 co‐engagement. Moreover, both CPS and CPT inhibit NF‐κB activation in response to different agents including TNF‐α. CPS itself does not affect the DNA‐binding ability of NF‐κB but it prevents IκB kinase activation and IκBα degradation in a dose‐dependent manner, without inhibiting the activation of the mitogen‐activated protein kinases, p38, extracellular regulated kinase and c‐Jun N‐terminal protein kinase. Moreover, intraperitoneal pretreatment with CPT prevented mice from lethal septic shock induced by lipopolysaccharide. In a second model of inflammation CPT pretreatment greatly reduced the extensive damage in the glandular epithelium observed in the bowel of DSS‐treated mice. Taken together, these results suggest that CPT and related synthetic analogues target specific pathways involved in inflammation, and hold considerable potential for dietary health benefits as well as for pharmaceutical development.

Involvement of mitochondria and caspase-3 in ET-18-OCH3-induced apoptosis of human leukemic cells

The induction of cell death in leukemic HL‐60 cells by the ether lipid 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3; edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET‐18‐OCH3 induced disruption of the mitochondrial transmembrane potential (ΔΨm) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET‐18‐OCH3 also induced caspase‐3 activation in human leukemic cells, as assessed by cleavage of caspase‐3 into the p17 active form and cleavage of the caspase‐3 substrate poly(ADP‐ribose) polymerase (PARP). ET‐18‐OCH3 analogues unable to induce apoptosis failed to disrupt ΔΨm and to activate caspase‐3. ET‐18‐OCH3‐resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase‐3 activation and did not undergo ΔΨm disruption upon ET‐18‐OCH3 incubation. Cyclosporin A partially inhibited ΔΨm dissipation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic cells. Overexpression of bcl‐2 by gene transfer prevented ΔΨm collapse, ROS generation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic T cells. Pretreatment with the caspase inhibitor Z‐Asp‐2,6‐dichlorobenzoyloxymethylketone prevented ET‐18‐OCH3‐induced PARP proteolysis and DNA fragmentation, but not ΔΨm dissipation. ET‐18‐OCH3 did not affect the expression of caspases and bcl‐2‐related genes. ET‐18‐OCH3‐induced apoptosis did not require protein synthesis. Our data indicate that ΔΨm dissipation and caspase‐3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET‐18‐OCH3, and that the sequence of events in the apoptotic action of ET‐18‐OCH3 on human leukemic cells is: ΔΨm disruption, caspase‐3 activation and internucleosomal DNA degradation. Int. J. Cancer 86:208–218, 2000.

Induction of apoptosis in human mitogen‐activated peripheral blood T‐lymphocytes by the ether phospholipid ET‐18‐OCH3: Involvement of the Fas receptor/ligand system

Activated T‐cells constitute a target for treatment of autoimmune diseases. We have found that the antitumour ether phospholipid 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3; edelfosine) induced dose‐ and time‐dependent apoptosis in human mitogen‐activated peripheral blood T‐lymphocytes, but not in resting T‐cells. T‐lymphocytes were stimulated with phytohemagglutinin and interleukin‐2 or with concanavalin A. Apoptosis was assessed by DNA fragmentation through cell cycle and TUNEL analyses, as well as through visualization of internucleosomal DNA fragmentation in agarose gels. The ET‐18‐OCH3‐mediated apoptotic response in activated T‐lymphocytes was less intense than in human leukaemic T cell lines, such as Jurkat cells and Peer cells; namely about 25% apoptosis in activated T‐cells versus about 46–61% apoptosis in T leukaemic cells after 24 h treatment with 10 μM ET‐18‐OCH3. The ET‐18‐OCH3 thioether analogue BM 41.440 (ilmofosine) showed a similar apoptotic capacity to that found with ET‐18‐OCH3 in activated T‐cells, whereas the phospholipid analogue hexadecylphosphocholine (miltefosine) failed to promote this response. The uptake of [3H]‐ET‐18‐OCH3 was much larger in activated T‐cells than in resting lymphocytes. Using a cytofluorimetric approach we have found that ET‐18‐OCH3 induced disruption of the mitochondrial transmembrane potential and production of reactive oxygen species in activated T‐cells, but not in resting lymphocytes. ET‐18‐OCH3 induced an increase in Fas (APO‐1/CD95) ligand mRNA expression in activated T‐cells, and incubation with a blocking anti‐Fas (APO‐1/CD95) antibody partially inhibited the ET‐18‐OCH3‐induced apoptosis of activated T‐lymphocytes. These results demonstrate that mitogen‐activated T‐cells, unlike resting lymphocytes, are able to take up significant amounts of ET‐18‐OCH3, and are susceptible to undergo apoptosis by the ether lipid via, in part, the Fas (APO‐1/CD95) receptor/ligand system. This ET‐18‐OCH3 apoptotic action can be of importance in the therapeutic action of this ether lipid in certain autoimmune diseases.

Immunosuppressive Activity of Endovanilloids: N-Arachidonoyl-Dopamine Inhibits Activation of the NF-κB, NFAT, and Activator Protein 1 Signaling Pathways

Endogenous N-acyl dopamines such as N-arachidonoyldopamine (NADA) and N-oleoyldopamine have been recently identified as a new class of brain neurotransmitters sharing endocannabinoid and endovanilloid biological activities. As endocannabinoids show immunomodulatory activity, and T cells play a key role in the onset of several diseases that affect the CNS, we have evaluated the immunosuppressive activity of NADA and N-oleoyldopamine in human T cells, discovering that both compounds are potent inhibitors of early and late events in TCR-mediated T cell activation. Moreover, we found that NADA specifically inhibited both IL-2 and TNF-α gene transcription in stimulated Jurkat T cells. To further characterize the inhibitory mechanisms of NADA at the transcriptional level, we examined the DNA binding and transcriptional activities of NF-κB, NF-AT, and AP-1 transcription factors in Jurkat cells. We found that NADA inhibited NF-κB-dependent transcriptional activity without affecting either degradation of the cytoplasmic NF-κB inhibitory protein, IκBα, or DNA binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by NADA in stimulated cells. In addition, NADA inhibited both binding to DNA and the transcriptional activity of NF-AT and AP-1, as expected from the inhibition of NF-AT1 dephosphorylation and c-Jun N-terminal kinase activation in stimulated T cells. Finally, overexpression of a constitutively active form of calcineurin demonstrated that this phosphatase may represent one of the main targets of NADA. These findings provide new mechanistic insights into the anti-inflammatory activities of NADA and highlight their potential to design novel therapeutic strategies to manage inflammatory diseases.

Phorboid 20-homovanillates induce apoptosis through a VR1-independent mechanism

BACKGROUND Vanilloids, such as capsaicin and resiniferatoxin (RTX), are recognized at the cell surface by vanilloid receptor type 1 (VR1), which has recently been cloned. VR1 mediates the effects of capsaicin and RTX in VR1-expressing cells, but vanilloids can induce apoptosis through a pathway not mediated by VR1. Phorboid 20-homovanillates can be used to investigate cell death induced by vanilloids. RESULTS 12,13-Diacylphorbol-20 homovanillates were prepared by the sequential esterification of the natural polyol. Phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) induced apoptosis in Jurkat cells to the same extent as RTX. Apoptosis was preceded by an increase in intracellular reactive oxygen species and by the loss of mitochondrial transmembrane potential. PPAHV-induced apoptosis was mediated by a pathway involving caspase-3 activation and was initiated at the S phase of the cell cycle. The cell-death pathway triggered by VR1 activation was studied in 293T cells transfected with the cloned rat vanilloid receptor. In this system, capsaicin and PPAHV induced cell death by an apparent necrotic mechanism, which was selectively inhibited by the competitive vanilloid receptor antagonist capsazepine. Interestingly, phorbol-12, 13-bisnonanoate-20-homovanillate, an analogue of PPAHV, induced cell death in VR1-transfected cells but could not trigger apoptosis in the Jurkat cell line. CONCLUSIONS Vanilloids can induce cell death through different signalling pathways. The cell death induced in a VR1-independent manner has the hallmark of apoptosis, whereas the cell death mediated by vanilloids binding to VR1 is seemingly necrotic. Phorboid homovanillates that have antitumour and anti-inflammatory activities but lack the undesirable side effects of the natural vanilloids could be developed as potential drugs.

Involvement of reactive oxygen species in capsaicinoid-induced apoptosis in transformed cells

Some varieties of sweet pepper accumulate non-pungent isosters of capsaicin, a type of compounds exemplified by capsiate. The only structural difference between capsaicin and capsiate is the link between the vanillyl and the acyl moieties, via an amide bond in the former and via an ester bond in the latter. By flow cytometry analyses we have determined that nor-dihydrocapsiate, a simplified analogue of capsiate, is a pro-oxidant compound that induces apoptosis in the Jurkat tumor cell line. The nuclear DNA fragmentation induced by nor-dihydrocapsiate is preceded by an increase in the production of reactive oxygen species and by a subsequent disruption of mitochondria transmembrane potential. Capsiate-induced apoptosis is initiated at the S phase of the cell cycle and is mediated by a caspase-3-dependent pathway. The accumulation of intracellular reactive oxygen species in capsiate-treated cells is greatly prevented by the presence of ferricyanide, suggesting that capsiates target a cellular redox system distinct from the one involved in the mitochondrial electron-chain transport. Methylation of the phenolic hydroxyl of nor-dihydrocapsiate completely abrogated the ability to induce reactive oxygen species and apoptosis, highlighting the relevance of the presence of a free phenolic hydroxyl for the pro-oxidant properties of capsaicinoids.

Ingenol esters induce apoptosis in Jurkat cells through an AP-1 and NF-κB independent pathway

BACKGROUND Ingenol derivatives have received constant and multidisciplinary attention on account of their pleiotropic pattern of biological activity. This includes activation of protein kinase C (PKC), tumour-promotion, anticancer, and anti-HIV properties, and the possibility of dissecting co-cancerogenic and clinically useful activities has been demonstrated. Certain ingenol esters show powerful anticancer activity, and a structure-activity relationship model to discriminate between their apoptotic and non-apoptotic properties has been developed. RESULTS The polyhydroxylated southern region of ingenol was selectively modified, using the anticancer and PKC activator ingenol 3,20-dibenzoate (IDB) as a lead compound. The evaluation of IDB analogues in apoptosis assays showed strict structure-activity relationships, benzoylation of the 20-hydroxyl being required to trigger apoptosis through a pathway involving caspase-3 and occurring at the specific cell cycle checkpoint that controls the S-M phase transition. Conversely, a study on the activation of the PKC-dependent transcription factors AP-1 and NF-kappaB by IDB analogues showed significant molecular flexibility, including tolerance to changes at the 3- and 20-hydroxyls. IDB-induced apoptosis was independent of activation of PKC, since it was not affected by treatment with the non-isoform-selective PKC inhibitor GF 109230X0. CONCLUSIONS Remarkable deviations from the tumour-promotion pharmacophore were observed for both the apoptotic and the PKC-activating properties of IDB analogues, showing that ingenol is a viable template to selectively target crucial pathways involved in tumour promotion and development. Since the apoptotic and the PKC-activating properties of ingenoids are mediated by different pathways and governed by distinct structure-activity relationships, it is possible to dissect them by suitable chemical modification. In this context, the esterification pattern of the 5- and 20-hydroxyls is critical.

The CB1/VR1 agonist arvanil induces apoptosis through an FADD/caspase-8-dependent pathway.

Arvanil (N‐arachidonoylvanillamine), a nonpungent capsaicin–anandamide hybrid molecule, has been shown to exert biological activities through VR1/CB1‐dependent and ‐independent pathways. We have found that arvanil induces dose‐dependent apoptosis in the lymphoid Jurkat T‐cell line, but not in peripheral blood T lymphocytes. Apoptosis was assessed by DNA fragmentation through cell cycle and TUNEL analyses. Arvanil‐induced apoptosis was initiated independently of any specific phase of the cell cycle, and it was inhibited by specific caspase‐8 and ‐3 inhibitors and by the activation of protein kinase C. In addition, kinetic analysis by Western blots and fluorimetry showed that arvanil rapidly activates caspase‐8, ‐7 and ‐3, and induces PARP cleavage. The arvanil‐mediated apoptotic response was greatly inhibited in the Jurkat‐FADDDN cell line, which constitutively expresses a negative dominant form of the adapter molecule Fas‐associated death domain (FADD). This cell line does not undergo apoptosis in response to Fas (CD95) stimulation. Using a cytofluorimetric approach, we have found that arvanil induced the production of reactive oxygen species (ROS) in both Jurkat‐FADD+ and Jurkat‐FADDDN cell lines. However, ROS accumulation only plays a residual role in arvanil‐induced apoptosis. These results demonstrate that arvanil‐induced apoptosis is essentially mediated through a mechanism that is typical of type II cells, and implicates the death‐inducing signalling complex and the activation of caspase‐8. This arvanil‐apoptotic activity is TRPV1 and CB‐independent, and can be of importance for the development of potential anti‐inflammatory and antitumoral drugs.

Mechanisms of HIV-1 Inhibition by the Lipid Mediator N -Arachidonoyldopamine

Several linear fatty acid dopamides (N-acyldopamines) have been identified recently in the brain. Among them, N-arachidonoyldopamine (NADA) is an endogenous lipid mediator sharing endocannabinoid and endovanilloid biological activities. We have reported previously that NADA exerts some of its biological activities through inhibition of the NF-κB pathway and, because this transcription factor plays a key role in HIV-1-long terminal repeat (LTR) trans activation, we have evaluated the anti-HIV-1 activity of NADA. In this study, we show that NADA inhibits vesicular stomatitis virus-pseudotyped HIV-1 infection in the human leukemia T cell line Jurkat, in primary T cells, and in the human astrocytic cell line U373-MG. Other endocannabinoids such as anandamide, 2-arachidonoylglycerol, and noladin ether did not show inhibitory activity in the HIV-1 replication assays. The anti-HIV-1 activity of NADA was independent of known cannabinoid and vanilloid receptor activation. In addition, NADA did not affect reverse transcription and integration steps of the viral cycle, and its inhibitory effect was additive with that of the reverse transcriptase inhibitor azidothymidine. NADA inhibited both TNF-α and HIV-1 trans activator protein-induced HIV-1-LTR activation. We also show that NADA counteracts the TNF-α-mediated trans activation capacity of the p65 NF-κB subunit without affecting its physical association to the HIV-1-LTR promoter. Moreover, NADA inhibited the p65 transcriptional activity by specifically targeting the phosphorylation of this NF-κB subunit at Ser536. These findings provide new mechanistic insights into the biological activities of NADA, and highlight the potential of lipid mediators for the management of AIDS.