Equivalent death of P-glycoprotein expressing and nonexpressing cells induced by the protein kinase C inhibitor staurosporine

P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance. In addition to its ability to efflux toxins P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We have previously demonstrated that stimuli including drugs such as hexamethylene bisacetamide (HMBA), the cytotoxic lymphocyte granule protein granzyme B, and pore-forming proteins such as perforin, kill P-gp positive cells in a caspase-independent manner. We therefore hypothesised that drugs that are not effluxed by P-gp and which induce cell death in the absence of caspase activation could induce death of P-gp expressing cells. Staurosporine has been previously shown to kill cells in the absence of caspase activation. Consistent with our hypothesis, we demonstrate here that staurosporine can equivalently kill P-gp(+ve) and P-gp(-ve) tumor cell lines in a caspase-independent manner.     

A central role for Bid in granzyme B-induced apoptosis

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Bid-deficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.     

Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation.

The protease granzyme B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.     

Filamin (280-kDa actin-binding protein) is a caspase substrate and is also cleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis

We used yeast two-hybrid screening to identify the cytoskeletal protein filamin as a ligand for the proapoptotic protease granzyme B, produced by cytotoxic T lymphocytes. Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it was also cleaved in a caspase-dependent manner following the ligation of Fas receptors. A similar pattern of filamin cleavage to polypeptides of approximately 110 and 95 kDa was observed in Jurkat cells killed by either mechanism. However, filamin cleavage in response to granzyme B was not inhibited by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations that abolished DNA fragmentation. Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following ligation of Fas receptors, coincident with the morphological changes of apoptosis. Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic survival and (51)Cr release assays, whereas death from multiple other stimuli was not affected by filamin deficiency. Thus, filamin is a functionally important substrate for granzyme B, as its cleavage may account at least partly for caspase-independent cell death mediated by the granzyme.     

Selective targeting of breast cancer cells through ROS-mediated mechanisms potentiates the lethality of paclitaxel by a novel diterpene, gelomulide K

Defects in apoptotic pathways confer resistance to tubulin-binding agents via downregulation of caspases or overexpression of antiapoptotic factors, urging the need for novel agents acting on an alternative pathway. The purpose of this study was to investigate whether induction of ROS can induce caspase-independent cell death in breast cancer cells and thereby enhance the activity of paclitaxel. Here, we report that gelomulide K acts as a caspase-independent cell death-inducing agent that synergizes with paclitaxel in breast cancer cells and has low toxicity in normal cells. Treatment with gelomulide K induced PARP-1 hyperactivation, AIF nuclear translocation, and cytoprotective autophagy. These effects were associated with increased ROS production and a decrease in cellular GSH levels in cancer cells. Furthermore, pretreatment with NAC, a precursor of intracellular GSH, effectively abrogated gelomulide K-induced caspase-independent cell death and autophagy, suggesting that ROS-mediated downstream signaling is essential to the anticancer effects of gelomulide K. Additionally, in a xenograft model, gelomulide K induced PARP-1 activation and reduced tumor growth. In terms of structure-activity relationships, analysis not only showed a correlation between ROS levels and drug activity but also highlighted the importance of the 8,14-epoxy group. Taken together, our results show that enhancement of paclitaxel activity can be achieved with gelomulide K and that the structurally relevant pharmacophore provides important insight into the development of new caspase-independent cell death-inducing agents.     

Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment.     

Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway

Granzyme B is an important mediator of cytotoxic lymphocyte granule-induced death of target cells, accomplishing this through cleavage of Bid and cleavage and activation of caspases as well as direct cleavage of downstream substrates. Significant controversy exists regarding the primary pathways used by granzyme B to induce cell death, perhaps arising from the use of different protease/substrate combinations in different studies. The primary sequence of human, rat, and mouse granzymes B is well conserved, and the substrate specificity and crystal structure of the human and rat proteases are extremely similar. Although little is known about the substrate specificity of mouse granzyme B, recent studies suggest that it may differ significantly from the human protease. In these studies we show that the specificities of human and mouse granzymes B differ significantly. Human and mouse granzyme B cleave species-specific procaspase-3 more efficiently than the unmatched substrates. The distinct specificities of human and mouse granzyme B highlight a previously unappreciated requirement for Asp(192) in the acquisition of catalytic activity upon cleavage of procaspase-3 at Asp(175). Although human granzyme B efficiently cleaves human or mouse Bid, these substrates are highly resistant to cleavage by the mouse protease, strongly indicating that the Bid pathway is not a major primary mediator of the effects of mouse granzyme B. These studies provide important insights into the substrate specificity and function of the granzyme B pathway in different species and highlight that caution is essential when designing and interpreting experiments with different forms of granzyme B.     

Pretreatment of docetaxel enhances TRAIL-mediated apoptosis in prostate cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether treatment of docetaxel (DTX) can enhance apoptotic cell death by TRAIL against androgen-independent prostate cancer (AIPC). The cell death effect of combinations of TRAIL and docetaxel on prostate cancer cell lines (androgen-dependent LNCaP and its derived androgen-independent, metastatic C4-2B) was evaluated by synergisms of apoptosis. Western blot assay and DNA fragmentation assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL induced apoptosis in the presence of docetaxel. In addition, we investigated the in vitro anti-tumor effects of combined docetaxel and TRAIL using MAP kinase inhibitors. Docetaxel itself could not induce apoptotic cell death in 24 h even in high concentration. Apoptotic cell death, however, was drastically enhanced by pretreatment of docetaxel 20 h before TRAIL treatment. Docetaxel enhanced the PARP-1 cleavage and caspases activation by TRAIL especially in androgen-independent, metastatic C4-2B cell line, mainly by phosphorylation of Bcl-2 by JNK activation. It appears that apoptotic cell death was protected by the JNK inhibitor SP600125. The results of our study show that pretreatment of docetaxel is able to enhance the apoptosis produced by TRAIL in prostate cancer cells, especially in hormone-refractory prostate cancer (HRPC).     

Does Caspase Inhibition Promote Clonogenic Tumor Growth?

Defects in the control of cell death are a major cause of resistance to tumor therapy. Until recently, components of the intrinsic apoptotic pathway that act downstream of mitochondria, such as the caspases, have been apportioned only a minor share in this business. Thus, defects in mitochondrial caspase activation were suggested to cause apoptosis inhibition but not to confer clonogenic survival. This assumption was based on the finding that chemotherapeutic agents provoke mitochondrial damage even the absence of caspases, resulting in the release various toxic mediators and a subsequent caspase-independent cell death. In contrast to these earlier observations, we recently showed that in the absence of active caspases tumor cells do not necessarily undergo caspase-independent cell death but may even survive a chemotherapeutic insult. Our findings suggest that caspase inhibition can indeed promote clonogenic tumor growth which might be not only relevant for tumor therapy but should be also considered when evaluating the safety of therapeutic caspases inhibitors.     

Caspase-dependent and -independent death pathways in cancer therapy

The majority of current anticancer therapies induce tumor cell death through the induction of apoptosis. Alterations in the apoptotic pathways may determine tumor resistance to these therapies. Activation of the proteolytic cascade involving caspase family members is a critical component of the execution of cell death in apoptotic cells. However, recent studies suggest that cell death can proceed in the absence of caspases. In this review we describe the role of caspase-dependent and -independent pathways as targets for anticancer treatment; better understanding of diverse modes of tumor cell death will help to avoid ineffective treatment and provide a molecular basis for the new strategies targeting caspase-independent death pathways in apoptosis-resistant forms of cancer.     

Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism

Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could potentially be manipulated to develop anti-cancer therapy in apoptosis resistant cells.     

Triggering caspase-independent cell death to combat cancer

Caspase-mediated apoptosis is a major hindrance to tumour growth and metastasis. Accordingly, defects in signalling pathways leading to the activation of caspases are common in tumours. Moreover, many tumour cells can unexpectedly survive the activation of caspases. As a result, caspase-independent cell death programmes are gaining increasing interest among cancer researchers. The heterogeneity of cancer cells with respect to their sensitivity to various death stimuli further emphasizes the need for additional death pathways in the therapeutic control of cell death. An understanding of the molecular control of alternative death pathways is beginning to emerge, being comparable with that of the molecular anatomy of apoptosis at the time of the discovery of caspases less than a decade ago. Here, newly discovered triggers and molecular regulators of alternative cell death programmes are reviewed and their potential in future cancer therapy is discussed.     

Caspase-independent killing of Burkitt lymphoma cell lines by rituximab

Caspase-independent cell death may have a critical role to play in the therapeutic destruction of tumours. Recently it has been suggested that one of the mechanisms by which rituximab, a therapeutic anti-CD20 antibody, kills B cells is caspase-independent. In this study we show that rituximab can induce death in a variety of Burkitt lymphoma derived cell lines. Rituximab-treated cells show leakage of adenylate kinase, surface expression of phosphatidylserine, upregulation of the cellular stress protein HSP70, phosphorylation of the survival protein Akt, and depolarisation of the mitochondrial membrane but no loss of cytochrome c or apoptosis inducing factor. Caspase inhibitors do not block these events. In support of these data there is no cleavage of caspases 3, 8 and 9, poly(ADP-ribose) polymerase, BH3 interacting domain death agonist or genomic DNA. Morphologically, cells show nuclear enlargement and cytoplasmic vacuolisation. Triggering of receptor mediated death in CD95 responsive lines results in “classical” apoptosis indicating that the internal machinery necessary for apoptosis is intact in these lines. The results suggest that rituximab can kill human B cells via a caspase-independent form of programmed cell death that shares features of apoptosis and necrosis. This pathway may be relevant to the clinical efficacy of rituximab.     

Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.     

Human and murine granzyme B exhibit divergent substrate preferences

The cytotoxic lymphocyte protease granzyme B (GzmB) can promote apoptosis through direct processing and activation of members of the caspase family. GzmB can also cleave the BH3-only protein, BID, to promote caspase-independent mitochondrial permeabilization. Although human and mouse forms of GzmB exhibit extensive homology, these proteases diverge at residues predicted to influence substrate binding. We show that human and mouse GzmB exhibit radical differences in their ability to cleave BID, as well as several other key substrates, such as ICAD and caspase-8. Moreover, pharmacological inhibition of caspases clonogenically rescued human and mouse target cells from apoptosis initiated by mouse GzmB, but failed to do so in response to human GzmB. These data demonstrate that human and murine GzmB are distinct enzymes with different substrate preferences. Our observations also illustrate how subtle differences in enzyme structure can radically affect substrate selection.     

Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells

Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8⁺ T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.     

Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins

Caspase activation and apoptosis can be initiated by the introduction of serine proteinases into the cytoplasm of a cell. Cytotoxic lymphocytes have evolved at least one serine proteinase with specific pro-apoptotic activity (granzyme B), as well as the mechanisms to deliver it into a target cell, and recent evidence suggests that other leucocyte granule proteinases may also have the capacity to kill if released into the interior of cells. For example, the monocyte/granulocyte proteinase cathepsin G can activate caspases in vitro, and will induce apoptosis if its entry into cells is mediated by a bacterial pore-forming protein. The potent pro-apoptotic activity of granzyme B and cathepsin G suggests that cells producing these (or other) proteinases would be at risk from self-induced death if the systems involved in packaging, degranulation or targeting fail and allow proteinases to enter the host cell cytoplasm. The purpose of the present review is to describe recent work on a group of intracellular serine proteinase inhibitors (serpins) which may function in leucocytes to prevent autolysis induced by the granule serine proteinases.     

Induction of rapid histone degradation by the cytotoxic T lymphocyte protease Granzyme A

The cytotoxic T lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-strand nicking is observed instead of oligonucleosomal fragmentation. Granzyme A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the caspase activator granzyme B. Here we show that granzyme A treatment of isolated nuclei enhances DNA accessibility to exogenous endonucleases. In vitro and after cell loading with perforin, GrnA completely degrades histone H1 and cleaves core histones into approximately 16-kDa fragments. Histone digestion provides a mechanism for unfolding compacted chromatin and facilitating endogenous DNase access to DNA during T cell and natural killer cell granule-mediated apoptosis.     

Fenretinide induces autophagic cell death in caspase-defective breast cancer cells

The elimination of tumour cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumour cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment.