Although the impact of tumor immunology on the clinical management of most cancers is still negligible, there is increasing evidence that anticancer immune responses may contribute to the control of cancer after conventional chemotherapy. Thus, radiotherapy and some chemotherapeutic agents, in particular anthracyclines, can induce specific immune responses that result either in immunogenic cancer cell death or in immunostimulatory side effects. This anticancer immune response then helps to eliminate residual cancer cells (those that fail to be killed by chemotherapy) or maintains micrometastases in a stage of dormancy. Based on these premises, in this Review we address the question, How may it be possible to ameliorate conventional therapies by stimulating the anticancer immune response? Moreover, we discuss the rationale of clinical trials to evaluate and eventually increase the contribution of antitumor immune responses to the therapeutic management of neoplasia.
Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIFLps2 mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.
doxorubicin; TLRs; apoptosis; neutrophils; DAMPs; immunogenic cell death
Paclitaxel is one of the chemotheraputic drugs widely used for the treatment of nonsmall cell lung cancer (NSCLC) patients. Here, we tested the ability of α-tocopheryl succinate (TOS), another promising anticancer agent, to enhance the paclitaxel response in NSCLC cells. We found that sub-apoptotic doses of TOS greatly enhanced paclitaxel-induced growth suppression and apoptosis in the human H460 NSCLC cell lines. Our data revealed that this was accounted for primarily by an augmented cleavage of poly(ADP-ribose) polymerase (PARP) and enhanced activation of caspase-8. Pretreatment with z-VAD-FMK (a pan-caspase inhibitor) or z-IETD-FMK (a caspase-8 inhibitor) blocked TOS/paclitaxel cotreatment-induced PARP cleavage and apoptosis, suggesting that TOS potentiates the paclitaxel-induced apoptosis through enforced caspase 8 activation in H460 cells. Furthermore, the growth suppression effect of TOS/paclitaxel combination on human H460, A549 and H358 NSCLC cell lines were synergistic. Our observations indicate that combination of paclitaxel and TOS may offer a novel therapeutic strategy for improving paclitaxel drug efficacy in NSCLC patient therapy as well as for potentially lowering the toxic side effects of paclitaxel through reduced drug dosage.
apoptosis; carcinoma, non-small-cell lung; caspase 8; paclitaxel; α-tocopherol
Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.
Methodology and Principal Findings
The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.
Conclusions and Significance
Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.
Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1– and caspase-3–like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk–dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.
In order to know how caspases contribute to the intracellular fate of Mycobacterium tuberculosis and host cell death in the infected macrophages, we examined the effect of benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethane (z-VAD-fmk), a broad-spectrum caspase inhibitor, on the growth of M. tuberculosis H37Rv in RAW 264 cells. In the cells treated with z-VAD-fmk, activation of caspase-8, caspase-3/7, and caspase-9 was clearly suppressed, and DNA fragmentation of the infected cells was also reduced. Under this experimental condition, it was found that the treatment markedly inhibited bacterial growth inside macrophages. The infected cells appeared to undergo cell death of the necrosis type in the presence of z-VAD-fmk. We further found that z-VAD-fmk treatment resulted in the generation of intracellular reactive oxygen species (ROS) in the infected cells. By addition of a scavenger of ROS, the host cell necrosis was inhibited and the intracellular growth of H37Rv was significantly restored. Among inhibitors specific for each caspase, only the caspase-9-specific inhibitor enhanced the generation of ROS and induced necrosis of the infected cells. Furthermore, we found that severe necrosis was induced by infection with H37Rv but not H37Ra in the presence of z-VAD-fmk. Caspase-9 activation was also detected in H37Rv-infected cells, but H37Ra never induced such caspase-9 activation. These results indicated that caspase-9, which was activated by infection with virulent M. tuberculosis, contributed to the inhibition of necrosis of the infected host cells, presumably through suppression of intracellular ROS generation.
Certain caspase-8 null cell lines demonstrate resistance to
Fas-induced apoptosis, indicating that the Fas/FasL apoptotic
pathway may be caspase-8-dependent. Some reports, however, have
shown that Fas induces cell death independent of caspase-8. Here
we provide evidence for an alternative, caspase-8-independent,
Fas death domain-mediated apoptotic pathway. Murine 12B1-D1 cells
express procaspase-3, -8, and -9, which were activated upon the
dimerization of Fas death domain. Bid was cleaved and
mitochondrial transmembrane potential was disrupted in this
apoptotic process. All apoptotic events were completely blocked
by the broad-spectrum caspase inhibitor Z-VAD-FMK, but not by
other peptide caspase inhibitors. Cyclosporin A (CsA), which
inhibits mitochondrial transition pore permeability, blocked
neither pore permeability disruption nor caspase activation.
However, CsA plus caspase-8 inhibitor blocked all apoptotic
events of 12B1-D1 induced by Fas death domain dimerization. Our
data therefore suggest that there is a novel,
caspase-8-independent, Z-VAD-FMK-inhibitable, apoptotic pathway in
12B1-D1 cells that targets mitochondria directly.
The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2Apro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2Apro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2Apro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2Apro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2Apro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2Apro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2Apro, suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.
Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.
Apoptosis; brain tumor; JAK2; neuroblastoma; NFκB; proliferation; Sorafenib; STAT3
There are limited chemotherapy options for hepatocellular carcinoma (HCC). The heparin-degrading endosulfatase SULF1 functions as a liver tumor suppressor. We investigated the effects of the histone deacetylase inhibitor apicidin in combination with doxorubicin in SULF1-expressing HCC cells in vitro and in SULF1-expressing xenografts in nude mice.
We evaluated the effects of apicidin alone or combined with doxorubicin on apoptosis, caspase activity, and phosphorylation of Erk and Akt in SULF1-transfected Huh7 and Hep3B cells in vitro and in vivo.
Apicidin induced HCC cell apoptosis and caspase activation in a dose- and time-dependent manner. Apicidin-induced caspase activation was significantly inhibited by the caspase inhibitor Z-Vad-fmk. Apicidin also decreased phosphorylation of both Erk and Akt. Expression of constitutively-active Mek1 and Akt significantly decreased apicidin-induced apoptosis. The combination of doxorubicin with apicidin significantly increased the anti-tumor effect in the SULF1-expressing Huh7 and Hep3B cells as compared to either apicidin or doxorubicin alone, both in vitro and in vivo.
The combination of a histone deacetylase inhibitor with doxorubicin may be a novel and promising therapeutic modality for HCCs, particularly for SULF1-expressing HCCs.
Histone deacetylase inhibitor; Doxorubicin; Hepatocellular carcinoma; Sulfatase 1 (SULF1); Apoptosis; Akt kinase; MAP kinase
Cytolethal distending toxin (CDT) induces apoptosis using the caspase-dependent classical pathway in the majority of human leukemic T cells (MOLT-4). However, we found the process to cell death is only partially inhibited by pretreatment of the cells with a general caspase inhibitor, z-VAD-fmk. Flow cytometric analysis using annexin V and propidium iodide showed that a 48-h CDT treatment decreased the living cell population by 35% even in the presence of z-VAD-fmk. z-VAD-fmk completely inhibited caspase activity in 24 h CDT-intoxicated cells. Further, CDT with z-VAD-fmk treatment clearly increased the cell population that had a low level of intracellular reactive oxygen. This is a characteristic opposite to that of caspase-dependent apoptosis. Overexpression of bcl2 almost completely inhibited cell death using CDT treatment in the presence of z-VAD-fmk. The data suggest there are at least two different pathways used in CDT-induced cell death: conventional caspase-dependent (early) apoptotic cell death and caspase-independent (late) death. Both occur via the mitochondrial membrane disruption pathway.
IL-17 production by γδ T cells is required for tumor cell infiltration by IFN-γ–producing CD8+ T cells and inhibition of tumor growth in response to anthracyclines.
By triggering immunogenic cell death, some anticancer compounds, including anthracyclines and oxaliplatin, elicit tumor-specific, interferon-γ–producing CD8+ αβ T lymphocytes (Tc1 CTLs) that are pivotal for an optimal therapeutic outcome. Here, we demonstrate that chemotherapy induces a rapid and prominent invasion of interleukin (IL)-17–producing γδ (Vγ4+ and Vγ6+) T lymphocytes (γδ T17 cells) that precedes the accumulation of Tc1 CTLs within the tumor bed. In T cell receptor δ−/− or Vγ4/6−/− mice, the therapeutic efficacy of chemotherapy was compromised, no IL-17 was produced by tumor-infiltrating T cells, and Tc1 CTLs failed to invade the tumor after treatment. Although γδ T17 cells could produce both IL-17A and IL-22, the absence of a functional IL-17A–IL-17R pathway significantly reduced tumor-specific T cell responses elicited by tumor cell death, and the efficacy of chemotherapy in four independent transplantable tumor models. Adoptive transfer of γδ T cells restored the efficacy of chemotherapy in IL-17A−/− hosts. The anticancer effect of infused γδ T cells was lost when they lacked either IL-1R1 or IL-17A. Conventional helper CD4+ αβ T cells failed to produce IL-17 after chemotherapy. We conclude that γδ T17 cells play a decisive role in chemotherapy-induced anticancer immune responses.
A caspase 8–deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (ΔΨm), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of ΔΨm and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti–mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD–fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in ΔΨm, as well as necrotic morphological changes. The presence of z-VAD–fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.
apoptosis; caspase; Fas; mitochondrial membrane potential; necrosis
Caspases are central to the execution of programmed cell death and their activation constitutes the biochemical hallmark of apoptosis. In this article, we report the successful adaptation of a high content assay method utilizing the DEVD-NucView488™ fluorogenic substrate, and for the first time, we show caspase activation in live cells induced either by drugs or siRNA. The fluorogenic substrate was found to be non-toxic over an exposure period of several days; during which we demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the anti-apoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to Doxorubicin, Etoposide or cell death siRNA. Our method was further validated against two well characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to Erlotinib; where we show a differential time dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, our results demonstrate the feasibility of using this newly adapted and validated high content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells.
High content assay; RNAi HT screening; Chemical HT screening; caspase; apoptosis; cancer; live cells
The alphavirus Semliki Forest virus (SFV) and its derived vectors induce apoptosis in mammalian cells. Here, we show that apoptosis is associated with the loss of mitochondrial membrane potential followed by the activation of caspase-3, caspase-8, and caspase-9. Cell death can be partially suppressed by treatment with the pan-caspase inhibitor zVAD-fmk. To determine the role of SFV structural proteins in cell death, the temporal course of cell death was compared in cells infected with SFV and cells infected with SFV virus replicon particles (VRPs) lacking some or all of the virus structural genes. In the absence of virus structural proteins, cell death was delayed. The endoplasmic reticulum (ER) stress response, as determined by the splicing of X-box binding protein 1 (XBP1) transcripts and the activation of caspase-12, was activated in virus-infected cells but not in VRP (SFV lacking structural genes)-infected cells. The C/EBP-homologous protein (CHOP) was upregulated by both virus and VRP infections. The virus envelope proteins but not the virus capsid protein triggered ER stress. These results demonstrate that in NIH 3T3 cells, SFV envelope glycoproteins trigger the unfolded protein response of the ER and accelerate apoptotic cell death initiated by virus replicase activity.
Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.
To evaluate the role of ceramide (Cer) in apoptosis signaling, we examined Cer formation induced by CD95, etoposide, or γ-radiation (IR) in relation to caspase activation and mitochondrial changes in Jurkat T cells. The Cer response to all three stimuli was mapped in between caspases sensitive to benzoyloxycarbonyl-VAD-fluoromethylketone (zVAD-fmk) and acetyl-DEVD-aldehyde (DEVD-CHO). Cer production was independent of nuclear fragmentation but associated with the occurrence of other aspects of the apoptotic morphology. Caspase-8 inhibition abrogated Cer formation and apoptosis induced by CD95 but did not affect the response to etoposide or IR, placing CD95-induced Cer formation downstream from caspase-8 and excluding a role for caspase-8 in the DNA damage pathways. CD95 signaling to the mitochondria required caspase-8, whereas cytochrome c release in response to DNA damage was caspase-independent. These results indicate that the caspases required for the Cer response to etoposide and IR reside at or downstream from the mitochondria. Bcl-2 overexpression abrogated the Cer response to etoposide and IR and reduced CD95-induced Cer accumulation. We conclude that the Cer response to DNA damage fully depends on mitochondrion-dependent caspases, whereas the response to CD95 partially relies on these caspases. Our data imply that Cer is not instrumental in the activation of inducer caspases or signaling to the mitochondria. Rather, Cer formation is associated with the execution phase of apoptosis.
NF-κB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-κB activation is essential for macrophage survival. Blocking the constitutive activation of NF-κB with pyrrolidine dithiocarbamate or expression of IκBα induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-κB activation induced a time-dependent loss of mitochondrial transmembrane potential (ΔΨm) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD.fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on ΔΨm collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to ΔΨm loss and that ectopic expression of A1 protected against cell death following inactivation of NF-κB. These data suggest that inhibition of NF-κB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-κB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.
Understanding the immune response to the death of malignant cells is critical for the development of therapeutic strategies designed to stimulate the immune system against cancer. We have developed an inducible caspase-3-mediated death switch model to explore the effects of apoptosis on the host immune system, demonstrating that the synchronous apoptotic demise of established tumors can be immunogenic and elicit anticancer T-cell responses.
apoptosis; caspase 3; cancer; death switch; HMGB1; immunogenic cell death
In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H2O2), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.
cysteine peptidase; cathepsin B; biotin-VAD-FMK; Leishmania; trypanosomes; unicellular organisms
Photodynamic therapy (PDT) has been developed as an anticancer treatment, which is based on the tumor-specific accumulation of a photosensitizer that induces cell death after irradiation of light with a specific wavelength. Depending on the subcellular localization of the photosensitizer, PDT could trigger various signal transduction cascades and induce cell death such as apoptosis, autophagy, and necrosis. In this study, we report that both AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) signaling cascades are activated following 5-aminolevulinic acid (ALA)-mediated PDT in both PC12 and CL1-0 cells. Although the activities of caspase-9 and -3 are elevated, the caspase inhibitor zVAD-fmk did not protect cells against ALA-PDT-induced cell death. Instead, autophagic cell death was found in PC12 and CL1-0 cells treated with ALA-PDT. Most importantly, we report here for the first time that it is the activation of AMPK, but not MAPKs that plays a crucial role in mediating autophagic cell death induced by ALA-PDT. This novel observation indicates that the AMPK pathway play an important role in ALA-PDT-induced autophagy.
The advancement of cancer immunotherapy faces barriers which limit its efficacy. These include weak immunogenicity of the tumor, as well as immunosuppressive mechanisms which prevent effective antitumor immune responses. Recent studies suggest that aberrant expression of cancer testis antigens (CTAs) can generate robust antitumor immune responses, which implicates CTAs as potential targets for immunotherapy. However, the heterogeneity of tumor cells in the presence and quantity of CTA expression results in tumor escape from CTA-specific immune responses. Thus, the ability to modulate the tumor cell epigenome to homogenously induce expression of such antigens will likely render the tumor more immunogenic. Additionally, emerging studies suggest that suppression of antitumor immune responses may be overcome by reprogramming innate and adaptive immune cells. Therefore, this paper discusses recent studies which address barriers to successful cancer immunotherapy and proposes a strategy of modulation of tumor-immune cell crosstalk to improve responses in carcinoma patients.
Anthracyclines remain the cornerstone of treatment in many malignancies but these agents have a cumulative dose relationship with cardiotoxicity. Development of cardiomyopathy and congestive heart failure induced by anthracyclines are typically dose-dependent, irreversible, and cumulative. Although past studies of cardiotoxicity have focused on anthracyclines, more recently interest has turned to anticancer drugs that target many proteins kinases, such as tyrosine kinases. An attractive model to explain the mechanism of this cardiotoxicity could be myocyte loss through cell death pathways. Inhibition of mitochondrial transition permeability is a valuable tool to prevent doxorubicin-induced cardiotoxicity. In response to anthracycline treatment, activation of several protein kinases, neuregulin/ErbB2 signaling, and transcriptional factors modify mitochondrial functions that determine cell death or survival through the modulation of mitochondrial membrane permeability. Cellular response to anthracyclines is also modulated by a myriad of transcriptional factors that influence cell fate. Several novel targeted chemotherapeutic agents have been associated with a small but worrying risk of left ventricular dysfunction. Agents such as trastuzumab and tyrosine kinase inhibitors can lead to cardiotoxicity that is fundamentally different from that caused by anthracyclines, whereas biological effects converge to the mitochondria as a critical target.
D609 is known to modulate death receptor-induced ceramide generation and cell death. We show that in Jurkat cells, non-toxic D609 concentrations inhibit sphingomyelin synthase and, to a lesser extent, glucosylceramide synthase, and transiently increase the intracellular ceramide level. D609 significantly enhanced FasL-induced caspase activation and apoptosis. D609 stimulated FasL-induced cell death in caspase-8-deficient Jurkat cells, indicating that D609 acts downstream of caspase-8. At high FasL concentration (500 ng/mL), cell death was significantly, but not completely, inhibited by zVAD-fmk, a broad-spectrum caspase inhibitor, indicating that FasL can activate both caspase-dependent and -independent cell death signaling pathways. FasL-induced caspase activation was abolished by zVAD-fmk, whereas ceramide production was only partially impaired. D609 enhanced caspase-independent ceramide increase and cell death in response to FasL. Also, D609 overcame zVAD-fmk-conferred resistance to a FasL concentration as low as 50 ng/mL and bypassed RIP deficiency. It is likely that mitochondrial events were involved, since Bcl-xL over-expression impaired D609 effects. In PHA-activated human T lymphocytes, D609 enhanced FasL-induced cell death in the presence or absence of zVAD-fmk. Altogether, our data strongly indicate that the inhibition of ceramide conversion to complex sphingolipids by D609 is accompanied by an enhancement of FasL-induced caspase-dependent and -independent cell death in T lymphocytes.
CD95; apoptosis; necrosis; sphingomyelin synthase; glucosylceramide synthase; ALPS
Nonsteroidal anti-inflammatory drugs (NSAID) reduce the risk for cancer, due to their antiproliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.
aspirin; apoptosis; cytochrome c; mitochondria; NSAID