Induction of Caspase-2 Activation by a DNA Enzyme Evokes Tumor Cell Apoptosis

Abstract

Caspase-2 is an enigmatic caspase that is now increasingly being associated with certain types of cell death in cells exposed to cytotoxic agents. It is now known that in some cases of cell stress, such as DNA damage, activation of this caspase is triggered, sometimes in the absence of activation of both the intrinsic and extrinsic pathways of apoptosis. Part of the reason for this enigma has been lack of a suitable stimulus for this caspase, and with the discovery of DNAzyme 13 (Dz13), a potent oligonucleotide-based caspase-2 activator, much more can now be elucidated. For instance, one thing that could be unraveled is whether caspase-8 and Fas (CD95)-associated protein with death domain are indeed involved in caspase-2 activation as part of the death-inducing signaling complex. It is also becoming apparent that this enigmatic caspase may be important in the mechanisms behind which chemotherapeutic agents inhibit tumor cell growth. A better understanding of the true biological effects of this enzyme may indeed lead to more effective ways of managing tumors clinically. This review article briefly examines the different compounds capable of inducing activation of caspase-2 and proposes Dz13 as one that will be valuable for evaluation of the biological functions of caspase-2.

Introduction

W ith increasing methods for detection, the incidence of cancer is on the rise globally (ACS, 2010). Evasion of apoptosis, a type of cell death, is a hallmark of tumor growth and progression (Evan and Vousden, 2001). Central to apoptosis are a series of cysteine protease proteins known as caspases, responsible for widespread destruction in cells programmed to die. Of these caspases, and despite being one of the most conserved caspases across species, caspase-2 is one about which relatively little is known (Kitevska et al., 2009). Although the absence of an apparent phenotype in caspase-2^−/− mice suggests lack of a critical role for this caspase in cell death (Bergeron et al., 1998), silencing of caspase-2 expression prevents cell death induced by DNA-targeting drugs (Lassus et al., 2002; Robertson et al., 2002). The critical findings to date concerning caspase-2 are summarized in Table 1.

Table 1.

Significant Milestones in Caspase-2 Research with Emphasis on Oncology

Finding	Reference
Caspase-2 (Nedd2/Ich1) identified as a gene highly expressed in embryonic but not in adult brain	Kumar et al. (1992)
Absence of a phenotype in −/− mice suggests lack of a critical role for this caspase in apoptosis	Bergeron et al. (1998)
Is necessary for cell death induced by DNA-targeting drugs	Lassus et al. (2002), Robertson et al. (2002)
Is activated by PIDD and leads to apoptosis	Tinel and Tschopp (2004), Baptiste-Okoh et al. (2008a, 2008b)
A caspase-2-dependent apoptotic program induces apoptosis without interacting with p53 and Bcl-2	Sidi et al. (2008)
Activation occurred in the cytosol and not in nucleus	Bouchier-Hayes et al. (2009)
Loss results in an increased ability of cells to gain malignancy, suggesting that it is a tumor suppressor	Ho et al. (2009)
Represses survivin gene, resulting in apoptosis	Guha et al. (2010)
Dz13 identified as a potent activator in variety of tumor cell lines; apoptosis leads to efficacy	Dass and Choong (2010)

PIDD, p53-induced protein with a death domain; Dz13, DNAzyme 13.

Recently, it has been reported that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and gain malignancy, suggesting that caspase-2 is a tumor suppressor protein (Ho et al., 2009). Also, it has been shown that caspase-2 leads to apoptosis by suppression of the survivin gene, thereby acting as an endogenous inhibitor of nuclear factor-kappa B–dependent cell survival and this mechanism may contribute to tumor suppression in humans (Guha et al., 2010). Caspase-2 activation has been noted to permeabilize mitochondria, and p53-induced protein with a death domain (PIDD) and RIP-associated Ich-1/CED homologous protein with death domain (RAIDD) are important for caspase-2 activation (Tinel and Tschopp, 2004; Baptiste-Okoh et al., 2008a, 2008b), though not in all cases (Vakifahmetoglu et al., 2008; Manzl et al., 2009). PIDD is dispensable for both p53-dependent and -independent apoptosis (Kim et al., 2009). It is notable that caspase-2 activation can be repressed by p21 (Baptiste-Okoh et al., 2008a), and in turn, p21 expression is regulated by caspase-2 at the translational level (Sohn et al., 2011). Figure 1 demonstrates the caspase-2–mediated apoptotic pathway.

FIG. 1.

Caspase-2–mediated apoptotic pathway induced by DNA damage. In this model, central to the cell's fate is whether caspase-2 is upregulated or downregulated. DNA damage induces p53 activation, and in turn, p53 activates either PIDDosome or FADD, which results in caspase-2 activation. Or p53 transactivates p21, which downregulates caspase-2 and leads to cell cycle arrest and, in turn, results in DNA repair instead of apoptosis. Adapted from the works of Olsson et al. (2009) and Baptiste-Okoh et al. (2008a). PIDD, p53-induced protein with a death domain; RAIDD, RIP-associated Ich-1/CED homologous protein with death domain; FADD, Fas(CD95)-associated protein with death domain.

As aforementioned, of all caspases, and despite being one of the most conserved caspases across species, caspase-2 is one about which relatively little is known (Kitevska et al., 2009), and this is partly due to lack of a strong inducer for activation. Against a backdrop of such a deficiency, recent studies demonstrated that some agents, ranging from a widely used anticancer drug to a radiosensitizer, induce caspase-2 activation, and these findings are summarized in Table 2. In particular, a potent inducer of this caspase, an oligonucleotide called DNAzyme 13 (Dz13; a 34mer deoxyribozyme downregulating c-jun expression, Khachigian et al., 2002; Dass 2004), has been recently shown to activate caspase-2 potently in a variety of tumor cell lines (Dass and Choong, 2010). In this review, we discuss recent findings on caspase-2, focusing on chemotherapy-induced activation of this enzyme.

Table 2.

Caspase-2 Inducers in Various Cancer Cells

Agent	Finding	Reference
2-Chloro-2′-deoxy-adenosine/2-Chloro-adenosine	Induces atypical apoptosis in human astrocytoma involving caspase-2.	Ceruti et al. (2003)
PRIMA-1^MET	Induces mitochondrial apoptosis by inducing caspase-2 activation in cells carrying mutant p53.	Shen et al. (2008)
Docetaxel	Induces apoptosis in melanoma cells by activation of caspase-2, which activates Bax and thereby inactivates Bcl-2. Induces apoptosis in melanoma cells by activation of caspase-2, and activation of Bax and inactivation of Bcl-2. Induces apoptosis in prostate cancer cells by activation of caspase-2 and -3, and increased p21 expression, with channeling of cells into mitotic catastrophe. Induces apoptosis in prostate cancer cells by activating caspase-2, which leads to activation of caspase-3 and release of cytochrome c.	Mhaidat et al. (2007a) Mhaidat et al. (2007b) Fabbri et al. (2008) Luo et al. (2010)
DCQ	Increases cell death by enhancing caspase-2 level under hypoxia in HCT-116 cancer cells.	El-Khatib et al. (2010)
Dz13	Is a potential inducer of caspase-2 activation, resulting in apoptosis in breast cancer, prostate cancer, osteosarcoma, and liposarcoma cell lines.	Dass et al. (2010a)
Doxorubicin	Nonpegylated liposomal drug increased Fas expression, accumulate in the Golgi, and activated both caspase-2 and -8, before -3.	Fabbri et al. (2011)

Induction of Caspase-2 Activation in Tumor Cells by Dz13

Previously, the deoxyribozyme Dz13 has been shown to reduce the growth of melanoma indirectly via antiangiogenesis (Zhang et al., 2004) and squamous cell carcinoma directly (Zhang et al., 2006). Dz13 downregulates c-jun (its specific target) levels in rapidly proliferating cells, for instance, when cells are stimulated postquiescence or when c-Jun is elevated (Bhindi et al., 2007). More recently, Dz13 has been shown to inhibit the growth of both osteosarcoma (OS) (Dass et al., 2008b, 2008c, 2008e) and liposarcoma (LS) (Dass et al., 2008d) in orthotopic models in our lab. Tumor growth is inhibited in orthotopic models of prostate cancer, breast cancer, and OS in bone of mice by Dz13 when tumor cells are mixed with Dz13 preimplantation (Tan et al., 2010c). Normal cells in bone such as osteoblasts and chondrocytes are left relatively unharmed, indicating a tumor cell–selective mode of action for Dz13 (Tan et al., 2010b). This finding was quite intriguing and suggested that a fundamental difference between tumor cells and normal cells was being exploited for such a differential cell kill response.

When Dz13-treated cells were observed with electron microscopy, death of cells occurred, exhibiting the classical signs of apoptosis (Tinari et al., 2008)—nuclear fragmentation, chromatin condensation, and membrane blebbing. In addition, mitochondrial structure was perturbed, with septae destroyed. Apoptosis was confirmed with poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, terminal deoxynucleotidyl transferase dUTP nick and labelling (TUNEL) assay, annexin-5 staining, and nuclear chromatin condensation as evidenced by 4′,6-diamidino-2-phenylindole staining (Dass et al., 2010). Structures resembling transfection complexes were observed within cells, specifically within endosome-like organelles. Death occurred via the mitochondrial pathway as shown by cytochrome c release into the cytosol.

Dz13-mediated tumor cell death via apoptosis was determined to be due to caspase-2 activity, as chemical inhibitors to other caspases failed to abrogate Dz13-mediated cell kill (Dass et al., 2010a). The pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone [z-VAD-FMK]) and caspase-2 inhibitor (benzyloxycarbonyl-Val-Asp (OMe)-Val-Ala-Asp (OMe) fluoromethyl ketone [z-VDVAD-FMK]) inhibited Dz13-mediated cell death, once again confirming that the mode of cell death was apoptosis. These results for caspase-2 were then confirmed when siRNA to caspase-2 also abrogated Dz13-mediated apoptosis. Finally, western blotting of cell lysates revealed a clear activation of caspase-2 from its 51 kDa band to its 33 kDa band.

To confirm that only caspase-2 was activated by Dz13 in tumor cells, western blotting was used to screen other effector caspases in the cell lines (Dass et al., 2010a), but none was activated. In addition, PIDD and RAIDD are closely associated with caspase-2 activation (Tinel and Tschopp, 2004). However, previous studies have shown that both PIDD and RAIDD are dispensable for caspase-2 activation (Vakifahmetoglu et al., 2008; Manzl et al., 2009), although, so far, only one report has demonstrated the significance of DNA-protein kinase catalytic subunit (PKcs) in caspase-2 activation. Hence, overall, research is very preliminary in this area of caspase-2-piddosome interaction. Although PIDD and RAIDD were activated or upregulated, respectively, when cells were treated with Dz13, all were deemed to be nonessential for Dz13 activity (Dass et al., 2010a, 2010b) when downregulation with their respective siRNAs failed to abrogate Dz13-mediated cell death.

As Dz13 activation of caspase-2 caused significant tumor cell death in vitro (Dass et al., 2010a), Dz13 was evaluated in orthotopic models of various tumors. Tumors (in this case, of the bone) were allowed to establish before treatment was initiated. These pre-established tumors resemble those observed when patients present to the clinic with initial complaints such as joint tenderness and/or swelling and are scheduled for an initial X-ray and/or magnetic resonance imaging (MRI). Both primary and secondary tumors are inhibited with chitosan-based nanoparticles encapsulating Dz13 (Dz13-NP; Dass et al., 2008a; Tan et al., 2010b), but are further inhibited when Dz13 is delivered with a nanoparticle combined with intraperitoneally administered doxorubicin (Dox) solution (clinically used against bone tumors), highlighting the usefulness of such combination therapy for tumor death (Dass et al., 2008d).

However, one problem encountered was a typical toxicity to both the heart and skin when free Dox is administered systemically at even these relatively low doses to mice (Ta et al., 2009a,b,c; Tan et al., 2010a), mimicking the human response to this cytotoxic agent. Thus, a method for limiting Dox activity to the lesion site and avoiding normal tissue exposure was deemed necessary. To this end, a drug delivery system (DDS) for Dox was developed. The nanoparticle DDS for Dz13 was developed to overcome reliance on commercial transfection reagents, which have been used for more than a decade (DeCruz et al., 1996; Fahmy et al., 2003; Mitchell et al., 2004). These commercial reagents are not recommended for in vivo use mainly because of their cytotoxicity, in general, against mammalian cells (Dass and Burton, 1999; Dass et al., 2002). One other DDS commonly used for oligonucleotide administration in vivo is the commercial osmotic pump (Alzet^®), which can deliver such potentially therapeutic agents such as antisense molecules (Walker et al., 1998, 2002), but when used to administer Dz13, slight hepatotoxicity in mice is noted (Dass and Choong, 2010).

Interestingly, E2F1 (a tumor suppressor) is in fact upregulated in melanoma cells treated with Dox (Hao et al., 2009), as is activation of caspase-2 by this anthracycline in cervical cancer (Tinel and Tschopp, 2004) and leukemic cells (Panaretakis et al., 2005). However, in our hands, for breast cancer and prostate cancer cell lines, free (nonencapsulated) Dox is not active unless micromolar concentrations are used in vitro, whereas for most OS cells, the EC₅₀ is low at ∼50 nM (unpublished data). In contrast, regardless of cancer cell type, Dz13 has an EC₅₀ almost consistently at 400 nM, and this is increased when an encapsulated form of the oligonucleotide is used for treating cells to 250 nM. The former finding has been the case for 14 cancer cell lines (OS, LS, prostate cancer, breast cancer, squamous cell carcinoma, melanoma) that have been tested (Zhang et al., 2004, 2006; Dass and Choong, 2008) and attests to the latent potential of Dz13 as a potent anticancer agent.

Other Agents That Activate Caspase-2

Apart from Dz13, there are other inducers of caspase-2 activation (summarized in Table 2). However, these have only been tested in a very limited number of cancer cell types, which is why we present Dz13 as a molecule that could become important to understanding the role of caspase-2 in cell biology. Not surprisingly, some of these agents are being used clinically to alleviate the burden of disease in patients suffering from a variety of cancers, although the exact mechanism(s) at play are largely unknown. Amongst these, docetaxel is one compound that has been used most frequently for caspase-2 activation.

The initial study linking docetaxel with activation of caspase-2 was performed by Mhaidat et al. (2007a). In their study, apoptotic cell death could be inhibited by overexpression of Bcl-2, even though docetaxel-induced changes in Bax were not inhibited by such overexpression. Caspase-2 activation seemed to be the initiating event of Bax expression, because inhibition of caspase-2 inhibited changes in Bax and mitochondrial membrane potential. Activation of caspase-8 and then Bid seemed to be a late event, although docetaxel was able to induce apoptosis in cells deficient in caspase-8 and Bid. p53 did not seem to be involved as a p53^−/− cell line was sensitive to docetaxel and an inhibitor of p53 did not inhibit apoptosis.

In a second study, docetaxel treatment caused activation of both c-Jun N-terminal kinase (JNK) and extracelluar signal-regulated kinases (ERK1/2) but not p38 mitogen-activated protein kinase or Akt kinases (Mhaidat et al., 2007b). Apoptosis was dependent on activation of caspase-2 and downstream changes in Bax and Bak by this caspase, leading to subsequent mitochondrial release of apoptosis-inducing factor and cytochrome c.

In a recent study, docetaxel-loaded [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)]2000-stabilized oleic acid-coated hydroxyapatite nanoparticles (Dtxl-NPs) were more cytotoxic against prostate cancer cell lines compared with docetaxel in vitro (Luo et al., 2010). This was linked to increased docetaxel-induced apoptosis in these cells. Prostate cancer cells treated with Dtxl-NPs exhibited significant arrest in the G₂-M phase but a higher sub-G₀/G₁ population when compared with Dtxl. The enhanced apoptosis induced by Dtxl-NPs in the cells was associated with the activation of caspase-2. The study demonstrated an early activation of caspase-2 in Dtxl-NPs–induced apoptosis in cells, which differed from Dtxl-induced apoptosis. When caspase-2 activation was inhibited by small interfering RNA (siRNA) knockdown, a significant inhibition of Dtxl-NPs–induced disruption of MMP and Dtxl-NPs–induced apoptosis was noted.

Dox is an anthracycline anticancer drug molecule that is used against a variety of cancers clinically. A recent study illuminates how this molecule performs its anticancer activities—activation of the initiator caspases-2 and −8 and then of -3 (Fabbri et al., 2011). In a prostate cancer cell line, anthracyclines, in particular a nonpegylated liposomal Dox, were highly concentrated in the Golgi apparatus. The authors hypothesized that the Golgi apparatus, probably acting as a stress sensor, intensified the conventional apoptotic mechanism induced by Dox. They also proposed that apart from the nucleus and the mitochondria, the Golgi apparatus could become an additional new target for anticancer therapy.

The anticancer agent 2-chloro-2′-deoxy-adenosine (Cladribine) and its derivative 2-chloro-adenosine have been shown to induce apoptosis of human astrocytoma cells (Ceruti et al., 2000). The exact pathway involved was not known until more recently (Ceruti et al., 2003), wherein both compounds produced a gradual, time-dependent activation of caspase-3. When caspase-3 activation was suppressed with a chemical inhibitor, apoptosis induction was also inhibited. The initiator caspases-9 and -8 were only marginally activated at later times of apoptosis induction. Intriguingly, neither of the adenosine analogs had any effect on mitochondrial membrane potential, which helped rule out a role for intrinsic apoptotic pathway involving caspase-9 and cytochrome c. As confirmation, no cytosolic accumulation of cytochrome c was detected with either of the adenosine analogs. Interestingly, at early time points after addition of either of the adenosine analogs and preceding the activation of caspase-3, caspase-2 activity was markedly increased. Caspase-2 inhibition with N-benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fmk significantly reduced both adenosine analog-induced caspase-2 activation and the associated apoptosis. The authors concluded that the adenosine analogs induced apoptosis in these cells by activating an atypical apoptotic cascade involving caspase-2 as the initiator caspase and an effector caspase (caspase-3), although a direct link between the two caspases was not demonstrated.

PRIMA-1^MET, a mutant p53-targeting compound, activates caspase-2 in a human lung cancer cell (Shen et al., 2008). As mentioned earlier, it has been known for years that p53 is essential for caspase-2 activation (Vakifahmetoglu et al., 2006). Moreover, the early activation of caspase-2 post-PRIMA-1^MET treatment supports the initiator caspase nature of this enzyme. This assumption was further strengthened by recent data demonstrating that both caspase-2 inhibition and siRNA knockdown blocked PRIMA-1^MET-induced cytochrome c release and downstream caspase activation (Shen et al., 2008). PRIMA-1^MET treatment caused increased levels of the small PIDD-CC fragment in treated lung cancer cells but not in the parental p53^−/− cells. The formation of this fragment is strongly correlated with the apoptotic response of cells and is preceded by activation of caspase-2 (Tinel and Tschopp, 2004).

Future Directions

In addition to caspase-2, in some cases (Dass et al., 2008b, 2008e), caspase-8 is also involved in Dz13-mediated apoptotic death of tumor cells. Further, it has been demonstrated that the piddosomal components of PIDD, RAIDD, and DNA-PKcs are all not necessary for caspase-2 activation by Dz13 (Dass and Choong, 2010). Dz13 can then be used to examine whether the death-inducing signaling complex (DISC), which is required for caspase-8 activation, is responsible for caspase-2 activation in tumor cells (Fig. 2), as has been shown in cells with p53 (Olsson et al., 2009). Dz13 may be acting through the death receptor pathway of apoptosis, which involves extracellular ligands interacting with members of the TNF receptor family to recruit the adaptor molecule Fas (CD95)-associated protein with death domain (FADD). These findings will shed light on the interactions between caspase-2 and caspase-8 via FADD. This will be an important basic biology question to answer, especially whether it occurs in the absence of the tumor suppressor p53 protein, which is thought to induce this interaction (Olsson et al., 2009). The latter has not been reported to date and would lend valuable information to this field of research. The study will also elucidate the mechanisms behind Dz13's proapoptotic activity as it pertains to caspase-2 and -8 and FADD.

FIG. 2.

Dz13 induces FADD/caspase-2 apoptotic pathway in mammalian cells. Caspase-2–dependent death can be initiated in membrane-based activation platforms (DISC). Subsequent mitochondrial membrane permeabilization, involving BH3-interacting domain (Bid) processing, then ensues. Caspase-2 is also reported to contribute to a mitochondrial amplification loop downstream of caspase-3 activation. Adapted from the works of Hengartner (2000), Green and Kroemer (1998), and Krumschnabel et al. (2009). Dz13, DNAzyme 13; DISC, death-inducing signaling complex.

Targeted therapy using biological agents in combination with conventional chemotherapy has the potential to increase the efficacy of treatment while reducing morbidity. As caspase-2 is central to Dz13 proapoptotic activity, formalin-fixed, paraffin-embedded clinical specimens of OS were analyzed for caspase-2 protein immunohistochemically (Dass and Choong, 2010). Importantly, all the tumors expressed caspase-2, with expression being sporadic and localized to various aspects of the tumors, in areas exhibiting growth spurts and more mature sites such as osteoid tissue. Such widespread distribution of caspase-2 in tumor tissue will assist in a wider cell death response with an agent capable of activating caspase-2, such as Dz13.

Summary

As methods for detection become more sensitive and widespread, the incidence of cancer has been noted to be on the rise globally. Evasion of apoptosis, a type of cell death, is a hallmark of tumor growth and progression. Central to apoptosis are a series of cysteine protease proteins known as caspases, responsible for widespread destruction in cells programmed to die. Caspase-2, an enigmatic caspase increasingly being associated with certain types of cell death in cells exposed to cytotoxic agents, is one that little is known of compared with its counterpart caspases. Part of the reason for this enigma has been lack of a suitable stimulus for this caspase, and with the discovery of Dz13, a potent inducer, much more can now be elucidated. For instance, one thing that could be unraveled is whether caspase-8 and FADD are indeed involved in caspase-2 activation as part of the DISC. Finally, a better understanding of caspase-2 biology may lead to more effective ways of treating tumors. Indeed, most tumor cells tested to date are inhibited by Dz13, a potent inducer of caspase-2 activation.

Footnotes

Acknowledgment

The authors thank the Victorian University Research and Development Grant Scheme (VU-RDGS) for financial support.

Disclosure Statement

The authors declare that there is no conflict of interest in relation to writing this manuscript.

References

ACS. 2010. Cancer Facts and Figures 2010. American Cancer Society, Inc.: Atlanta, GA, USA.

Baptiste-Okoh

, Barsotti

A.M.

, Prives

2008a. Caspase 2 is both required for p53-mediated apoptosis and downregulated by p53 in a p21-dependent manner. Cell Cycle, 7:1133–1138.

Baptiste-Okoh

, Barsotti

A.M.

, Prives

2008b. A role for caspase 2 and PIDD in the process of p53-mediated apoptosis. Proc Natl Acad Sci U S A, 105:1937–1942.

Bergeron

et al. 1998. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev, 12:1304–1314.

Bhindi

, Fahmy

R.G.

, Lowe

H.C.

, Chesterman

C.N.

, Dass

C.R.

, Cairns

M.J.

, Saravolac

E.G.

, Sun

L.Q.

, Khachigian

L.M.

2007. Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies. Am J Pathol, 171:1079–1088.

Bouchier-Hayes

et al. 2009. Characterization of cytoplasmic carpase-2 activation by induced proximity. Mol Cell, 35:830–840.

Ceruti

et al. 2000. Apoptosis induced by 2-chloro-adenosine and 2-chloro-2'-deoxy-adenosine in a human astrocytoma cell line: differential mechanisms and possible clinical relevance. J Neurosci Res, 60:388–400.

Ceruti

et al. 2003. A key role for caspase-2 and caspase-3 in the apoptosis induced by 2-chloro-2'-deoxy-adenosine (cladribine) and 2-chloro-adenosine in human astrocytoma cells. Mol Pharmacol, 63:1437–1447.

Dass

C.R.

2004. DNAzymes: cleaving a path to clinical trials. Trends Pharmacol Sci, 25:395–397.

10.

Dass

C.R.

, Burton

M.A.

1999. Lipoplexes and tumours. J Pharm Pharmacol, 51:755–770.

11.

Dass

C.R.

, Choong

PFM

. 2008. c-jun: a potential therapeutic target for multiple pathologies. Pharmazie, 63:411–414.

12.

Dass

C.R.

, Choong

P.F.M.

2010a. Sequence-related off-target effect of Dz13 against human tumor cells and safety in adult and fetal mice following systemic administration. Oligonucleotides, 20:51–60.

13.

Dass

C.R.

, Friedhuber

A.M.

, Khachigian

L.M.

, Dunstan

D.E.

, Choong

P.F.M.

2008a. Biocompatible chitosan-DNAzyme nanoparticles exhibits enhanced biological activity. J Microencaps, 25:421–425.

14.

Dass

C.R.

, Friedhuber

A.M.

, Khachigian

L.M.

, Dunstan

D.E.

, Choong

P.F.M.

2008b. Downregulation of c-jun results in apoptosis-mediated anti-osteosarcoma activity in an orthotopic model. Cancer Biol Ther, 7:1033–1036.

15.

Dass

C.R.

, Galloway

S.J.

, Choong

P.F.M.

2010a. Dz13, a c-jun DNAzyme, is a potent inducer of caspase-2 activation. Oligonucleotides, 20:137–146.

16.

Dass

C.R.

, Galloway

S.J.

, Clark

J.C.

, Khachigian

L.M.

, Choong

P.F.

2008c. Involvement of c-jun in human liposarcoma growth. Cancer Biol Ther, 7:1297–1301.

17.

Dass

C.R.

, Khachigian

L.M.

, Choong

P.F.

2008d. c-Jun knockdown sensitizes osteosarcoma to doxorubicin. Mol Cancer Ther, 7:1909–1912.

18.

Dass

C.R.

, Khachigian

L.M.

, Choong

P.F.M.

2008e. c-Jun is critical for the progression of osteosarcoma: proof in an orthotopic spontaneously metastasizing model. Mol Cancer Ther, 6:1289–1292.

19.

Dass

C.R.

, Saravolac

E.G.

, Li

, Sun

L.-Q.

2002. Cellular uptake, distribution and stability of 10–23 deoxyribozymes. Antisense Nucleic Acid Drug Dev, 12:289–299.

20.

Dass

C.R.

, Tan

M.L.

, Galloway

S.J.

, Choong

P.F.M.

2010b. Dz13 induces a cytotoxic stress response with upregulation of E2F1 in tumor cells metastasizing to or from bone. Oligonucleotides, 20:79–91.

21.

DeCruz

E.E.

, Walker

T.L.

, Dass

C.R.

, Burton

M.A.

1996. The basis for somatic gene therapy of cancer. J Exp Ther Oncol, 1:73–83.

22.

El-Khatib

et al. 2010. Cell death by the quinoxaline dioxide DCQ in human colon cancer cells is enhanced under hypoxia and is independent of p53 and p21. Radiat Oncol, 5:107.

23.

Evan

G.I.

, Vousden

K.H.

2001. Proliferation, cell cycle and apoptosis in cancer. Nature, 411:342–348.

24.

Fabbri

et al. 2008. Mitotic catastrophe and apoptosis induced by docetaxel in hormone-refractory prostate cancer cells. J Cell Physiol, 217:494–501.

25.

Fabbri

et al. 2011. Activity of different anthracycline formulations in hormone-refractory prostate cancer cell lines: role of Golgi apparatus. J Cell Physiol[Epub ahead of print]10.1002/jcp.22654.

26.

Fahmy

et al. 2003. Early Growth Response factor-1: A key mediator of tumor angiogenesis and neovascularisation. Nat Med, 9:1026–1032.

27.

Green

, Kroemer

1998. The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol, 8:267–271.

28.

Guha

et al. 2010. Caspase 2-mediated tumor suppression involves survivin gene silencing. Oncogene, 29:1280–1292.

29.

Hao

, Zhou

H.S.

, McMasters

K.M.

2009. Chemosensitization of tumor cells: inactivation of nuclear factor-kappa B associated with chemosensitivity in melanoma cells after combination treatment with E2F-1 and doxorubicin. Methods Mol Biol, 542:301–313.

30.

Hengartner

M.O.

2000. The biochemistry of apoptosis. Nature, 407:770–776.

31.

L.H.

et al. 2009. A tumor suppressor function for caspase-2. Proc Natl Acad Sci U S A, 106:5336–5341.

32.

Khachigian

L.M.

et al. 2002. c-Jun regulates vascular smooth muscle cell growth and neointima formation after arterial injury. Inhibition by a novel DNA enzyme targeting c-Jun. J Biol Chem, 277:22985–22991.

33.

Kim

I.R.

et al. 2009. DNA damage- and stress-induced apoptosis occurs independently of PIDD. Apoptosis, 14:1039–1049.

34.

Kitevska

, Spencer

D.M.

, Hawkins

C.J.

2009. Caspase-2, controversial killer or checkpoint controller? Apoptosis, 14:829–848.

35.

Krumschnabel

et al. 2009. Caspase-2: killer, savior and safeguard - emerging versatile roles for an ill-defined caspase. Oncogene, 28:3093–3096.

36.

Kumar

et al. 1992. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem Biophys Res Commun, 185:1155–1161.

37.

Lassus

, Opitz-Araya

, Lazebnik

2002. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science, 297:1352–1354.

38.

Luo

et al. 2010. Docetaxel loaded oleic acid-coated hydroxyapatite nanoparticles enhance the docetaxel-induced apoptosis through activation of caspase-2 in androgen independent prostate cancer cells. J Control Release, 147:278–288.

39.

Mhaidat

N.M.

et al. 2007a. Docetaxel-induced apoptosis in melanoma cells is dependent on activation of caspase-2. Mol Cancer Ther, 6:752–761.

40.

Mhaidat

N.M.

et al. 2007b. Docetaxel-induced apoptosis of human melanoma is mediated by activation of c-Jun NH2-terminal kinase and inhibited by the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway. Clin Cancer Res, 13:1308–1314.

41.

Manzl

et al. 2009. Caspase-2 activation in the absence of PIDDosome formation. J Cell Biol, 185:291–303.

42.

Mitchell

, Dass

C.R.

, Sun

L.-Q.

, Khachigian

L.M.

2004. Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumor growth by DNAzymes targeting the zinc finger transcription factor EGR-1. Nucleic Acids Res, 32:3065–3069.

43.

Olsson

et al. 2009. DISC-mediated activation of caspase-2 in DNA damage-induced apoptosis. Oncogene, 28:1949–1959.

44.

Panaretakis

et al. 2005. Doxorubicin requires the sequential activation of caspase-2, protein kinase Cδ, and c-Jun NH2-terminal kinase to induce apoptosis. Mol Biol Cell, 16:3821–3831.

45.

Robertson

J.D.

et al. 2002. Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J Biol Chem, 277:29803–29809.

46.

Shen

et al. 2008. PRIMA-1MET induces mitochondrial apoptosis through activation of caspase-2. Oncogene, 27:6571–6580.

47.

Sidi

et al. 2008. Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3. Cell, 133:864–877.

48.

Sohn

, Budach

, Jänicke

R.U.

2011. Caspase-2 is required for DNA damage-induced expression of the CDK inhibitor p21(WAF1/CIP1) Cell Death Differ, 18:1664–1674.

49.

H.T.

, Dass

C.R.

, Larson

, Choong

P.F.

, Dunstan

D.E.

2009a. A chitosan-dipotassium orthophosphate hydrogel for the delivery of doxorubicin in the treatment of osteosarcoma. Biomaterials, 30:3605–3613.

50.

H.T.

, Dass

C.R.

, Larson

, Choong

P.F.

, Dunstan

D.E.

2009b. A chitosan hydrogel delivery system for osteosarcoma gene therapy with pigment epithelium-derived factor combined with chemotherapy. Biomaterials, 30:4815–4823.

51.

H.T.

, Han

, Larson

, Dass

C.R.

, Dunstan

D.E.

2009c. Novel chitosan-based hydrogel drug delivery system. Int J Pharm, 371:134–141.

52.

Tan

M.L.

, Choong

P.F.M.

, Dass

C.R.

2010a. Direct anti-metastatic efficacy by the DNA enzyme Dz13 and downregulated MMP-2, MMP-9 and MT1-MMP in tumours. Cancer Cell Int, 10:9.

53.

Tan

M.L.

, Dunstan

D.E.

, Friedhuber

A.M.

, Choong

P.F.M.

, Dass

C.R.

2010b. A nanoparticulate system that enhances the efficacy of the tumoricide Dz13 when administered proximal to the lesion site. J Control Release, 144:196–202.

54.

Tan

M.L.

, Friedhuber

A.M.

, Dunstan

D.E.

, Choong

P.F.

, Dass

C.R.

2010c. The performance of doxorubicin encapsulated in chitosan-dextran sulphate microparticles in an osteosarcoma model. Biomaterials, 31:541–551.

55.

Tinari

et al. 2008. Analyzing morphological and ultrastructural features in cell death. Methods Enzymol, 442:1–26.

56.

Tinel

, Tschopp

2004. The piddosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science, 304:843–846.

57.

Vakifahmetoglu

et al. 2006. Functional connection between p53 and caspase-2 is essential for apoptosis induced by DNA damage. Oncogene, 25:5683–5692.

58.

Vakifahmetoglu

et al. 2008. DNA damage induces two distinct modes of cell death in ovarian carcinomas. Cell Death Differ, 15:555–566.

59.

Walker

T.L.

, Dass

C.R.

, DeCruz

E.E.

, Burton

M.A.

1998. A method for intratumoral continuous infusion of antisense oligodeoxynucleotides. J Pharm Sci, 87:387–389.

60.

Walker

T.L.

, Dass

C.R.

, Burton

M.A.

2002. Enhanced in vivo tumour response from combination carboplatin and low dose c-myc antisense oligonucleotides. Anticancer Res, 14:2237–2245.

61.

Zhang

et al. 2006. Squamous cell carcinoma growth in mice and in culture is regulated by c-Jun and its control of matrix metalloproteinase-2 and −9 expression. Oncogene, 25:7260–7266.

62.

Zhang

, Dass

C.R.

, Sumithran

, Di Girolamo

, Sun

L.-Q.

, Khachigian

L.M.

2004. Effect of Dz targeting c-Jun on solid tumor growth and angiogenesis in rodents. J Natl Cancer Inst, 96:683–696.