Different anticancer therapies including cytotoxic drugs, γ-irradiation, suicide gene therapy, or immunotherapy appear to induce tumor cell death by activating key elements of apoptosis, the cell's intrinsic death program. Activation of the cascade of proteolytic enzymes known as caspases is a critical component of the execution phase of cell death in most forms of apoptosis. Two main caspase cascades, one triggered by death receptor stimulation and the other one initiated at the mitochondria, have been identified in response to various inducers of cellular stress such as DNA damage. Activation of caspases and apoptosis is tightly regulated at several levels, e.g., by Bcl-2 family members, by inhibitor of apoptosis proteins (IAPs) and upstream inhibitors such as FLIP. Failure to activate apoptotic pathways in response to drug treatment may lead to resistance of tumors cells to anticancer therapies. Therefore, factors affecting caspase activation might be important determinants of drug sensitivity. In addition to caspase-dependent apoptosis, caspase-independent forms of cell death may also play a role for treatment response. Insights into the mechanisms regulating caspase activation as well as other forms of cell death pathways provide a molecular basis for novel strategies targeting resistance of tumor cells.

Introduction

Killing of tumor cells by diverse cytotoxic approaches such as anticancer drugs, γ-irradiation, suicide genes, or immunotherapy has been shown to be mediated through induction of apoptosis in target cells.^1–7 Apoptosis or programmed cell death occurs upon the activation of distinct, intrinsic cell death programs under certain physiological and pathological situations.⁸ The underlying mechanism for initiation of an apoptosis response upon cytotoxic therapy may be different for various stimuli and is only partially understood. However, damage to DNA or to other critical molecules and/or subcellular structures appears to be a common early hit by some inducers which is then propagated by the cellular stress response.⁹ Multiple stress-inducible molecules, e.g., JNK, MAPK/ERK, NF-κB or ceramide may have a profound impact on apoptosis pathways.^10–12 On the other hand, cytotoxic T cells or NK cells may release compounds such as granzyme B which directly activates downstream apoptosis effector mechanisms inside the cell.⁸ Apoptosis is characterized by typical morphological and biochemical hallmarks including cell shrinkage, nuclear DNA fragmentation and membrane blebbing.⁸ Proteolytic enzymes such as caspases play an important role as effector molecules in apoptosis including cytotoxic therapy-induced cell death.^13–19 Because of the potential detrimental effects on cell survival in case of inappropriate caspase activity, activation of caspases has to be tightly controlled. The antiapoptotic mechanisms regulating activation of caspases have also been postulated to be involved in drug resistance of tumor cells.^20–22 However, the concept that anticancer therapies primarily act by triggering apoptosis has also been challenged, since a consistent link between the ability of tumor cells to undergo apoptosis in vitro and their susceptibility to anticancer therapy in vivo has not always been observed.²³ Therefore, nonapoptotic modes of cell death, e.g., necrosis or some forms of cell death that cannot be easily classified, may mediate the cell death response to cytotoxic therapy.^24,25 Also, non-caspase-dependent apoptosis has been found to be induced by anticancer drugs in some cells.^26–28 Thus, a better understanding of these diverse modes of tumor cell death following cytotoxic therapies will provide a molecular basis for new strategies targeting caspase-dependent and independent death pathways in apoptosis-resistant forms of cancer.

Caspases as Central Death Effector Molecules

Most signaling pathways activated by anticancer drugs ultimately result in activation of caspases, a family of cysteine proteases that act as common death effector molecules in various forms of cell death (Fig. 1).^8,13–18 12 human caspases with different substrate specificity have so far been identified that cleave next to aspartate residues.^13–18 Caspases are involved in apoptosis signaling and also in cytokine processing.^13–18 Caspases are synthesized as inactive zymogens and they are activated by proteolytic cleavage.^13–18 Upon activation, each caspase forms a tetramer of the two large and the two small subunits.^13–18 The hierarchy and partial substrate redundance allows to a form proteolytic, signalling cascade with positive feedback properties.^13–18

Figure 1

Activation of apoptosis pathways by anticancer therapy. Anticancer therapy-induced apoptotic pathways can be initiated through different entry sites, e.g., at the plasma membrane by death receptor-mediated signaling (receptor pathway) or at the mitochondria (more...)

Caspases involved in apoptosis signaling are currently categorized into initiator and effector caspases, respectively.^13–18 Initiator caspases transduce various signals into protease activity and are directly linked to death inducing signaling complexes (DISCs): caspase-8 or caspase-10 via their death effector domain (DED) interact with adaptor proteins (FADD) recruited and bound to activated death receptors while caspase-9 is recruited to the apoptosome via its CARD domain.¹³ Effector caspases cleave various cytoplasmatic or nuclear substrates prompting the occurrence of morphologic features of apoptosis.^13–18 For example, polynucleosomal DNA fragmentation is initiated by cleavage of ICAD (inhibitor of caspase-activated DNase), the inhibitor of the endonuclease CAD (caspase-activated DNase) that cleaves DNA into the characteristic oligomeric fragments.⁸ DNA condensation is caused by AIF, a mitochondrial protein that translocates to the nucleus upon death triggering, and by Acinus, which stands for “apoptotic chromation condensation inducer in the nucleus”.^29,30 AIF may also mediate caspase-independent cleavage of DNA into larger fragments.^29,30 Likewise, loss of overal cell shape is due to proteolysis of cytoskeletal proteins including fodrin, gelsolin, actin, plectrin, cytokeratin, while nuclear shrinking and budding occurs after degradation of lamin.⁸

Pathways of Caspase Activation

Activation of caspases can principially be triggered by two different mechanisms: according to the induced proximity model initiator caspases such as caspase-8 or 9 are activated in a multimeric complex, e.g., caspase-8 in the death inducing signaling complex (DISC) and caspase-9 at the apoptosome.^8,13,31–33 Alternatively, caspases are activated by catalytic processing of the zymogens at specific cleavage sites.¹³ Caspase activation can be initiated through different entry sites, e.g., at the plasma membrane by death receptor-mediated signaling (receptor pathway) or at the mitochondria (mitochondrial pathway).^8,13 Stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily such as CD95 (APO-1/Fas) or TRAIL receptors results in receptor aggregation and recruitment of the adaptor molecule Fas-associated death domain (FADD) and caspase-8.^31–33 Upon recruitment caspase-8 becomes activated and initiates apoptosis by direct cleavage of downstream effector caspases.^31–33 The mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis inducing factor (AIF), Smac/DIABLO, Omi/HtrA2, endonuclease G, caspase-2 or caspase-9 from the mitochondrial intermembrane space.^34–39 The release of cytochrome c into the cytosol triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex.^40,41 Smac/DIABLO and Omi/HtrA2 promote caspase activation through neutralizing the inhibitory effects to IAPs, while AIF and endonuclease G cause DNA condensation.^{30,38,39,42,43}

The receptor and the mitochondrial pathway can be interconnected at different levels.⁴⁴ Following death receptor stimulation activation of caspase-8 may result in cleavage of Bid, a BH3 domain containing protein of the Bcl-2 family which assumes cytochrome c-releasing activity upon cleavage thereby initiating a mitochondrial amplification loop.^43,44 In addition, mitochondria-triggered caspase-6 cleavage may feed back to the receptor pathway by cleaving caspase-8.¹⁶

Signaling Pathways in Cancer Therapy

The relative contribution of the receptor and the mitochondrial pathway to drug-induced apoptosis has been a subject of controversial discussion.^1,2,4 A number of studies suggested that cancer therapy-triggered apoptosis involves the CD95 system by upregulating the expression of CD95L which then binds to its receptor and stimulates the receptor pathway in an autocrine or paracrine manner.^45–64 In support of this model, upregulation of CD95 mRNA and protein was found in a variety of tumor cell lines derived from T cell leukemia cells, neuroblastoma, malignant brain tumors, hepatoma, colon, breast or small lung cell carcinoma upon treatment with cytotoxic drugs such as doxorubicin, etoposide, cisplatin, 5-FU or bleomycin.^45–59 The increase in CD95L transcription and mRNA levels was found to be related to drug-induced activation of the transcription factors AP-1 and NF-κB.^55,60,61 Also, CD95 expression increased upon drug treatment, in particular in cells harboring wild-type p53, as the CD95 promotor contains p53 binding sites.^51,57,58 In addition, soluble antagonistic CD95 receptors, antagonistic CD95L antibodies or DN-FADD reduced drug-induced apoptosis under certain circumstances.^45,49,57,59 Moreover, it was recently demonstrated in vivo that 5-fluorouracil induced apoptosis in mouse thymocytes via activation of the CD95 system, since apoptosis was blocked by neutralizing CD95L antibodies or in lpr mice lacking a functional CD95 receptor.⁶² Also, CD95L-independent activation of the receptor pathway through CD95 receptor oligomerization has been reported, e.g., by UV irradiation, cytotoxic drugs or suicide gene therapy using the herpes simplex thymidine kinase (HSV/TK) system.^65–67

Other reports however challenged the concept that death receptor signaling is involved in drug-mediated cell death.^68–74 Antagonistic antibodies against CD95L or CD95 did not protect from cancer chemotherapeutically induced death in several cell line models.^72–74 Although splenocytes from lpr mice exhibit decreased sensitivity to γ-irradiation, thymocytes of these mice did not show increased proliferation upon γ-irradiation or cytotoxic drugs.⁶⁸ Moreover, overexpression of FLIP, DN-FADD or the serpin CrmA that inhibits caspase-8 did not confer protection.^69,72–74 In addition, targeted disruption of genes involved in death receptor signaling suggested a dispensable role of the CD95 system in drug-induced apoptosis, at least in nontransformed cells. FADD^−/− and caspase-8^−/− fibroblasts are resistant to death receptor stimulation, but equally sensitive to cytotoxic drugs.^75,76 In contrast, caspase-9^−/− embryonic stem cells and Apaf1^−/− thymocytes remain sensitive to death receptor triggering, however, and are resistant to cytotoxic drugs.^77,78 The discrepancies in data may be explained by the relative contribution of the death receptor versus the mitochondrial pathway depending on the cytotoxic drug, dose and kinetics or on differences between certain cell types. For CD95 signaling, 2 different cell types have been identified:⁷⁹ type I cells undergo CD95-triggered apoptosis independent of mitochondria, since caspase-8 is already efficiently activated at the DISC upstream of mitochondria. In contrast, type II cells depend on the mitochondrial pathway, since only little caspase-8 is recruited and activated at the DISC.⁷⁹ A similar cell type dependent signaling has also been identified in response to drug treatment.⁵⁴ Although the CD95 system is involved in anticancer drug-induced apoptosis under certain circumstances, the majority of cytotoxic drugs initiate cell death by triggering the cytochrome c/Apaf-1/caspase-9 dependent pathway through the mitochondria. Collectively, these data point to a crucial role of the mitochondrial pathway in drug-induced apoptosis, while the CD95 system may amplify and accelerate drug-induced apoptosis under certain conditions. Importantly, this amplification of the chemoresponse may be clinically meaningful, since it may critically affect the time required for execution of the death program.⁸⁰

Regulation of Caspase Activation

Given the important role of caspases as effector molecules in various forms of cell death including drug-induced apoptosis, the ability of anticancer agents to trigger caspase activation appears to be a critical determinant of sensitivity or resistance to cytotoxic therapies. As a consequence, inhibition of caspase activation may be an important factor in chemoresistance. Given the central role of caspases for cell death execution one might expect a high frequency of caspase mutations in tumors. Interestingly however, screening for mutations in initiator or executioner caspases in a variety of human tumors has not reveiled a high frequency of genomic aberrations in caspase genes.^81,82 Instead, caspase expression and function appears to be epigenetically downregulated in tumors by mechanisms described below, suggesting that restoration of a functional caspase system may be important to overcome resistance in tumors.^82,83

Caspase-Independent Cell Death

Although a large body of data point to an essential role of caspase-mediated tumor cell death upon cytotoxic therapy, this concept has also been challenged.^1–7,23 Thus, a clear, consistent link between the cells' ability to undergo apoptosis and their susceptibility to anticancer therapy could not be observed.²³ In addition, the p53 status did not always correlate with the ability of a tumor cell to respond to treatment.¹²⁰ Cells harboring wild-type p53 may fail to respond and those lacking functional p53 may even respond better.¹²⁰ Moreover, nonapoptotic modes of cell death, e.g., necrosis or some forms of cell death that cannot be clearly classified, have also been taken into consideration as response to cytotoxic therapy.^23–28 Also, delayed regression of tumors upon e.g., irradiation has been taken as evidence against a predominant apoptotic mode of cell death, since apoptosis appears to be induced fairly rapidly in vitro and in vivo upon appropriate stimulation.²³ Although signaling pathways and molecules involved in these alternative forms of cell death have not yet exactly been defined, non-caspase proteases such as calpains or cathepsins, Bax or Bax-like molecules and AIF or endonuclease G may be involved.^8,23–28 The relative contribution of these different modes of cell death for chemoresponses in vitro and in vivo remains to be defined.

Role of Caspases for Treatment Response in Vivo

What is the clinical impact of caspase expression and/or activity on individual patient's response to anticancer therapy in vivo? Unfortunately, this question is far from being answered yet. Aside from Bcl-2 family proteins, most molecules involved in the regulation of apoptosis including caspases have not extensively been studied in clinical specimens.^22,43 Although the potential relation between expression levels of procaspases in clinical samples and patients' response to chemotherapy has been addressed in several studies, the conclusive answer is still missing.^121–125 Some investigators postulated a correlation between procaspase-3 expression and clinical response, e.g., in leukemia, Hodgkin's disease or NSCLC.^121–124 However, this conclusion was not always based on a direct correlation between procaspase-3 expression levels and individual treatment responses and further studies did not confirm these findings. In bone marrow samples with predominance of leukemic blasts, a wide variation of caspase-2, -3, -7, -8, and -9 was found among different specimens, however, their level did not correlate with prognostic factors or response to induction chemotherapy.¹²⁵ Loss of spontaneous caspase-3 cleavage was reported in ALL samples at relapse compared to those at initial diagnosis.¹⁰⁷ As discussed before, while mutations in caspase genes have only infrequently been found in tumors, inactivation of caspase expression by epigenetic alterations such as promotor hypermethylation appears to be a primary mechanism of disabling of the caspase cascades in tumors.^81–83 How inactivation of caspase expression by DNA hypermethylation will correlate with clinical outcome remains a subject of future studies. The prospective study of clinical samples is further complexed by the necessity of multiparameter analysis. The expression level and activity of caspases is affected in vivo by positive and negative apoptosis regulators, such as Bcl-2 family proteins or IAPs.^100,110 Thus, an assessment of the impact of caspase expression and/or function on chemoresponse in vivo will require multiparamter analysis, e.g., by expression profiling.

Conclusion

Numerous studies over the last years have indicated that anticancer therapies primarily act by activating the apoptosis response pathway in tumor cells.^1–7 However, several points still remain to be addressed: First, most of the apoptosis signaling components have not been studied in clinical samples.^2,4,6,7,22 Second, many experimental studies indicate that alterations in components of the apoptotic machinery have an impact on sensitivity of tumor cells towards cytotoxic therapy, this premise remains to be tested in clinical settings.^1–5,22 Moreover, the biology that determines the individual responses of different tumors to cytotoxic therapies warrants further investigations to provide the basis for more specific therapeutic interventions. Finally, the concept that apoptosis represents the major mechanism by which tumor cells are eliminated by cytotoxic therapies may not universally apply and caspase-independent modes of cell death have also also to be considered.^23–28

Nonetheless, studies on the regulation of apoptosis signaling pathways triggered by anticancer therapies have provided substantial insights into the molecular mechanisms regulating the response of tumor cells towards current therapies. Future studies on the role of apoptosis signaling molecules in individual tumors both in vitro and in vivo in tumor cells of patients under chemotherapy, e.g., by DNA microarrays or proteomic studies, may provide the basis for “tailored” tumor therapy and may identify new targets for therapeutic interventions.

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