**2. Novel apoptotic markers/targets in cancer**

The main goal in cancer therapy is the abrogation of tumour cell growth and proliferation, and ultimately the complete elimination of tumour cells. It is commonly accepted that tumour cells treated with anticancer agents undergo apoptosis, and that cells resistant to apoptosis often do not respond to anticancer therapy [34]. Moreover, it is widely demonstrated that some oncogenic mutations suppressing apoptosis may lead to tumour

initiation, progression or metastasis [11]. Apoptosis plays a major control role in cell death when DNA damage is irreparable and multiple stress-inducible molecules have been implied in transmitting the apoptotic signal [35]. Because of the potential detrimental effects on cell survival in case of inappropriate activation of apoptosis programs, apoptosis pathways have to be tightly controlled. However, the concept that apoptosis represents the major mechanism by which cancer cells are eliminated may not universally apply and caspase-independent apoptosis or other modes of cell death have also to be considered as cellular response to anticancer therapy [36].

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 115

identified as an innate immune anti-viral protein approximately 35 years ago [52,53]. Since then, PKR has been linked to normal cell growth and differentiation, inflammation, cytokine signalling, and apoptosis [54]. PKR is a serine/threonine kinase, characterized by two distinct kinase activities: autophosphorylation, which represents the activation reaction, and phosphorylation of eIF-2α [55,56], which impairs eIF-2 activity, resulting in inhibition of protein synthesis [57]. In addition to its translational regulatory function, PKR has a role in signal transduction and transcriptional control through the IkB/NF-kB pathway [58,59]. PKR is activated in response to dsRNA of cellular, viral, or synthetic origin. PKR can also be activated by polyanions such as heparin, dextran sulfate, chondroitin sulfate, and poly-Lglutamine [60]. A range of cellular stresses can also activate PKR, such as arsenite, thapsigargin, H2O2, ethanol and ceramide [61,62,63] presumably through the PKRassociated activator (PACT)/RAX protein [64]. Moreover, it is induced by interferon type I and mediates in part, several functions of these cytokines. Altered PKR activity has been shown to play a role in neurodegenerative diseases (Alzheimer's, Creutzfeldt–Jakob,

Over-expression or continued activation of PKR leads to apoptosis [75,76,77]. PKR mediates the apoptosis induced by several viruses and cellular stresses [78] by activation of intrinsic and extrinsic apoptosis pathways through the FADD/caspase 8 and mitochondrial APAF/caspase 9 activation pathways [79,80]. Recently, PKR has been shown to play an important role in apoptotic cancer cell death induced by 5-FU, doxorubicin and etoposide [15,81,82] and the antitumour activity of tumour suppressors like p53 and PTEN [81,83]. Preclinical studies in mice have shown than in tumours which do not express sufficient levels of PKR are more resistant to doxorubicin and etoposide that tumour expressing higher PKR levels. We have demonstrated that PKR is up-regulated and activated in colon and breast cancer cell lines by inducing apoptotic cell death in response to 5-FU treatment. In addition, cancer cell lines deficient in PKR expression were more resistant to the cytotoxic effect of 5-FU with an IC50 being 2-3 fold higher than cells expressing an active PKR protein. Moreover, apoptosis mediated by PKR in response to 5-FU occurred independently on p53 status highlighted the importance that both p53 and PKR play in the 5-FU-induced cancer cell death, and the relevance acquired by PKR in tumour cells where p53 is mutated. Such results raise the importance of determining PKR status in tumours from patients treated

3- One pathway being targeted for antineoplastic therapy is the Bcl-2 family of proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that bind to and inactivate BH3-domain proapoptotic proteins. It is controversial whether some BH3-domain proteins (Bim or tBid) directly activate multidomain pro-apoptotic proteins (e.g., Bax and Bak) or act via inhibition of those anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that stabilize pro-apoptotic proteins [84]. Since the anti-apoptotic properties of Bcl-2 were discovered, the over-expression of Bcl-2 conferring chemoresistance was reported, and the 3-D protein structure of Bcl-XL was determined. These properties have contributed to the development of protein inhibitors. The first agent targeting Bcl-2 that entered clinical trials was a Bcl-2 antisense (oblimersen sodium), which showed chemosensitizing effects when

Huntington's, and Parkinson's) and cancer [65,66,67,68,69,70,71,72,73,74].

with 5-FU-based chemotherapy [15].

Different determinants of drug resistance exist, including loss of cell surface receptors or transporters, altered metabolism, or mutation of specific apoptotic target [37]. The apoptotic signalling pathways are regulated by numerous hub proteins such as p53, Bcl-2, NFkB and MAPKs which function in common. In the following sections, we will summarize some of the cellular proteins considered as potential apoptotic biomarkers in cancer.

1- The tumour suppressor p53 is an important pro-apoptotic factor and tumour inhibitor, and numerous anti-tumour drugs would exert their functions through targeting p53-related signalling pathways. Some clinical investigations indicated that under abnormal situations such as chemotherapy and UV or DNA damage may occur and activate the expression of p53. Activated p53 protein binds to the regulatory sequences of a number of target genes to initiate a program of cell cycle arrest, DNA repair, apoptosis, and angiogenesis [38]. If the damage cannot be repaired completely, over-activation of p53 leads to the tumour growth stagnation or even apoptosis [39,40]. Loss of p53 function is critical in tumourigenesis, and alterations to the *p53* gene (mutations, often resulting in protein over-expression) are frequent events in cancer. Associations of *p53* tumour alterations with patient prognosis and response to adjuvant chemotherapy have been widely studied, and findings are contradictory. The fluoropyrimidine 5-FU is widely used in the treatment of a range of cancers including colorectal cancer and breast cancer [41,42,43], but resistance to the drug remains a major clinical problem. P53 was the first target described for 5-FU- induced apoptosis, however, although several reports have demonstrated that 5-FU-induced apoptosis is dependent on the tumour suppressor p53 protein, apoptosis can also occur in mutant p53 cell lines [44,45,46,47]. Moreover, the relationship between p53 status and sensitivity to chemotherapeutic drugs, including 5- FU, is still controversial. In clinical studies in which adjuvant chemotherapy– treated and non-treated groups could be analyzed, stage III colorectal patients whose tumours demonstrated no *p53* alterations experienced significantly longer survival following 5-FU–based chemotherapy than patients whose tumours over-expressed p53 [48,49]. However, other studies in colon cancer patients failed to demonstrate correlations between *p53* alterations and benefit from adjuvant therapy [50,51]. The identification of new targets involved in 5-FU-induced apoptosis could contribute to clarify the controversy results obtaining in clinic.

2- Recently, we have identified the interferon-induced protein kinase PKR, as a molecular target of 5-FU with an interesting role in the apoptosis induced by this chemotherapeutic drug [15]. The double-stranded RNA (dsRNA)-dependent kinase PKR was initially

cellular response to anticancer therapy [36].

initiation, progression or metastasis [11]. Apoptosis plays a major control role in cell death when DNA damage is irreparable and multiple stress-inducible molecules have been implied in transmitting the apoptotic signal [35]. Because of the potential detrimental effects on cell survival in case of inappropriate activation of apoptosis programs, apoptosis pathways have to be tightly controlled. However, the concept that apoptosis represents the major mechanism by which cancer cells are eliminated may not universally apply and caspase-independent apoptosis or other modes of cell death have also to be considered as

Different determinants of drug resistance exist, including loss of cell surface receptors or transporters, altered metabolism, or mutation of specific apoptotic target [37]. The apoptotic signalling pathways are regulated by numerous hub proteins such as p53, Bcl-2, NFkB and MAPKs which function in common. In the following sections, we will summarize some of

1- The tumour suppressor p53 is an important pro-apoptotic factor and tumour inhibitor, and numerous anti-tumour drugs would exert their functions through targeting p53-related signalling pathways. Some clinical investigations indicated that under abnormal situations such as chemotherapy and UV or DNA damage may occur and activate the expression of p53. Activated p53 protein binds to the regulatory sequences of a number of target genes to initiate a program of cell cycle arrest, DNA repair, apoptosis, and angiogenesis [38]. If the damage cannot be repaired completely, over-activation of p53 leads to the tumour growth stagnation or even apoptosis [39,40]. Loss of p53 function is critical in tumourigenesis, and alterations to the *p53* gene (mutations, often resulting in protein over-expression) are frequent events in cancer. Associations of *p53* tumour alterations with patient prognosis and response to adjuvant chemotherapy have been widely studied, and findings are contradictory. The fluoropyrimidine 5-FU is widely used in the treatment of a range of cancers including colorectal cancer and breast cancer [41,42,43], but resistance to the drug remains a major clinical problem. P53 was the first target described for 5-FU- induced apoptosis, however, although several reports have demonstrated that 5-FU-induced apoptosis is dependent on the tumour suppressor p53 protein, apoptosis can also occur in mutant p53 cell lines [44,45,46,47]. Moreover, the relationship between p53 status and sensitivity to chemotherapeutic drugs, including 5- FU, is still controversial. In clinical studies in which adjuvant chemotherapy– treated and non-treated groups could be analyzed, stage III colorectal patients whose tumours demonstrated no *p53* alterations experienced significantly longer survival following 5-FU–based chemotherapy than patients whose tumours over-expressed p53 [48,49]. However, other studies in colon cancer patients failed to demonstrate correlations between *p53* alterations and benefit from adjuvant therapy [50,51]. The identification of new targets involved in 5-FU-induced apoptosis could

2- Recently, we have identified the interferon-induced protein kinase PKR, as a molecular target of 5-FU with an interesting role in the apoptosis induced by this chemotherapeutic drug [15]. The double-stranded RNA (dsRNA)-dependent kinase PKR was initially

the cellular proteins considered as potential apoptotic biomarkers in cancer.

contribute to clarify the controversy results obtaining in clinic.

identified as an innate immune anti-viral protein approximately 35 years ago [52,53]. Since then, PKR has been linked to normal cell growth and differentiation, inflammation, cytokine signalling, and apoptosis [54]. PKR is a serine/threonine kinase, characterized by two distinct kinase activities: autophosphorylation, which represents the activation reaction, and phosphorylation of eIF-2α [55,56], which impairs eIF-2 activity, resulting in inhibition of protein synthesis [57]. In addition to its translational regulatory function, PKR has a role in signal transduction and transcriptional control through the IkB/NF-kB pathway [58,59]. PKR is activated in response to dsRNA of cellular, viral, or synthetic origin. PKR can also be activated by polyanions such as heparin, dextran sulfate, chondroitin sulfate, and poly-Lglutamine [60]. A range of cellular stresses can also activate PKR, such as arsenite, thapsigargin, H2O2, ethanol and ceramide [61,62,63] presumably through the PKRassociated activator (PACT)/RAX protein [64]. Moreover, it is induced by interferon type I and mediates in part, several functions of these cytokines. Altered PKR activity has been shown to play a role in neurodegenerative diseases (Alzheimer's, Creutzfeldt–Jakob, Huntington's, and Parkinson's) and cancer [65,66,67,68,69,70,71,72,73,74].

Over-expression or continued activation of PKR leads to apoptosis [75,76,77]. PKR mediates the apoptosis induced by several viruses and cellular stresses [78] by activation of intrinsic and extrinsic apoptosis pathways through the FADD/caspase 8 and mitochondrial APAF/caspase 9 activation pathways [79,80]. Recently, PKR has been shown to play an important role in apoptotic cancer cell death induced by 5-FU, doxorubicin and etoposide [15,81,82] and the antitumour activity of tumour suppressors like p53 and PTEN [81,83]. Preclinical studies in mice have shown than in tumours which do not express sufficient levels of PKR are more resistant to doxorubicin and etoposide that tumour expressing higher PKR levels. We have demonstrated that PKR is up-regulated and activated in colon and breast cancer cell lines by inducing apoptotic cell death in response to 5-FU treatment. In addition, cancer cell lines deficient in PKR expression were more resistant to the cytotoxic effect of 5-FU with an IC50 being 2-3 fold higher than cells expressing an active PKR protein. Moreover, apoptosis mediated by PKR in response to 5-FU occurred independently on p53 status highlighted the importance that both p53 and PKR play in the 5-FU-induced cancer cell death, and the relevance acquired by PKR in tumour cells where p53 is mutated. Such results raise the importance of determining PKR status in tumours from patients treated with 5-FU-based chemotherapy [15].

3- One pathway being targeted for antineoplastic therapy is the Bcl-2 family of proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that bind to and inactivate BH3-domain proapoptotic proteins. It is controversial whether some BH3-domain proteins (Bim or tBid) directly activate multidomain pro-apoptotic proteins (e.g., Bax and Bak) or act via inhibition of those anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that stabilize pro-apoptotic proteins [84]. Since the anti-apoptotic properties of Bcl-2 were discovered, the over-expression of Bcl-2 conferring chemoresistance was reported, and the 3-D protein structure of Bcl-XL was determined. These properties have contributed to the development of protein inhibitors. The first agent targeting Bcl-2 that entered clinical trials was a Bcl-2 antisense (oblimersen sodium), which showed chemosensitizing effects when

combined with conventional chemotherapy drugs in chronic lymphocytic leukemia (CLL) patients, leading to improved survival [85,86]. More recent advances include the discovery of small molecule inhibitors of the Bcl-2 family proteins. They are designed to bind the hydrophobic groove of anti-apoptotic Bcl-2 proteins in place of BH3-only proteins (i.e., BH3 mimetics). They can oligomerize Bax or Bak, which can subsequently depolarize mitochondrial membrane potential to release cytochrome *c*. To date, one Bcl-2 antisense and three small molecule Bcl-2 protein inhibitors are being tested in clinical trials. Preclinical studies seem promising, especially in combination with additional chemotherapy agents. Ongoing and planned phase II clinical trials to define the activity of single agents and drug combinations will determine the direction of future clinical development of the Bcl-2 inhibitors [84].

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 117

Because of the importance of this pathway in cancer progression and chemotherapy-induced cell death, apoptosome-associated proteins may be important markers for colorectal cancer prognosis and chemotherapy response. Several studies have examined the immunohistochemical expression of individual proteins associated with apoptosis execution in colorectal cancer [96]. Increased expression of APAF-1 has been shown to be associated with longer patient survival in rectal cancer patients [97] and loss of APAF-1 has been implicated in tumour progression and more aggressive disease [98]. Similarly, longer overall survival has been associated with increased Smac [99] and caspase 9 [100]. The anti-apoptotic XIAP has also been implicated as a potential prognostic marker for colorectal cancer, with increased expression correlating with poor patient outcome [101]. However, no study to date has provided a comprehensive analysis of these key regulatory proteins as markers for colorectal cancer prognosis or chemotherapy response. Recently, the pro-caspase 3 expression in colorectal tumours has been associated with better recurrence-free and overall survival, and serves as an independent prognostic marker in localised Stage II disease [102]. This result is in agreement with previous studies that demonstrated as caspase 3 expression is a positive prognostic marker in hepatocellular carcinoma [103] and diffuse large B-cell lymphoma [104]. 6- The extrinsic apoptosis pathway is triggered by the binding of death ligands of the tumour necrosis factor (TNF) family to their appropriate death receptors (DRs) on the cell surface. One TNF family member, TRAIL or Apo2L, seems to preferentially cause apoptosis of transformed cells and can be systemically administered in the absence of severe toxicity. Therefore, there has been enthusiasm for the use of TRAIL or agonist antibodies to the TRAIL DR4 and DR5 in cancer therapy. Nonetheless, many cancer cells are very resistant to TRAIL apoptosis in vitro. Therefore, there is much interest in identifying compounds that can be combined with TRAIL to amplify its apoptotic effects [105]. The combination of TRIAL DR agonists with numerous conventional and investigational anticancer drugs has been reported. Synergy has been described for the combination of TRAIL with a variety of cytotoxic agents including irinotecan, camptothecin, 5-FU, carboplatin, paclitaxel, doxorubicin, and gemcitabine in diverse preclinical models [106,107]. Many human and mouse cancer cells lines can be sensitized by proteasome inhibitors such as bortezomib (VELCADE) to the apoptotic effects of TRAIL DR agonists. Interestingly, non-transformed cells seem to be much more resistant to the apoptotic effects of bortezomib and TRAIL than are cancer cells. This suggests that a therapeutic window may exist in vivo where this combination may have a therapeutic benefit in the absence of accompanying toxicity. However, the molecular mechanism(s) of action whereby proteasome inhibition in cancer

cells results in sensitization to TRAIL apoptosis remains unclear [108].

Improved clinical response may be obtained by identifying therapies that are particularly effective in activating apoptosis and determining how those therapies may be modified to effect maximum apoptosis induction. The cell cycle apparatus and apoptosis have attracted the attention of researchers intent on developing new types of anticancer therapy [109,110].

**3. Selective antitumour-drug inducers of apoptosis** 

4- PUMA (p53 upregulated modulator of apoptosis) is a BH3-only Bcl-2 family member and a critical mediator of p53-dependent and -independent apoptosis induced by a wide variety of stimuli, including genotoxic stress, deregulated oncogene expression, toxins, altered redox status, growth factor/cytokine withdrawal and infection. It serves as a proximal signalling molecule whose expression is regulated by transcription factors in response to these stimuli. PUMA transduces death signals primarily to the mitochondria and acts indirectly on the Bcl-2 family members Bax and/or Bak by relieving the inhibition imposed by anti-apoptotic members. It directly binds and antagonizes all known anti-apoptotic Bcl-2 family members to induce mitochondrial dysfunction and caspase activation [87]. Several lines of evidence suggest that the function of PUMA is compromised in cancer cells. PUMA expression was found to be reduced in malignant cutaneous melanoma, and PUMA expression appears to be an independent predictor of poor prognosis in patients [88]. In addition, approximately 40% of primary human Burkitt's lymphomas do not express detectable levels of PUMA, which is attributable, in part, to DNA methylation [89]. Evidence of PUMA induction by therapeutic agents in patients has just begun to emerge. Analysis of tissue biopsies from breast cancer patients showed that *PUMA* mRNA was induced within 6 h of chemotherapy [90]. Increased expression of *PUMA* and *Bim* is associated with better prognosis in patients receiving 5-FUbased therapy in stage II and III colon cancer, and is an independent prognostic marker for overall and disease-free survival [91]. PUMA ablation or inhibition leads to apoptosis deficiency underlying increased risks for cancer development and therapeutic resistance. Although elevated PUMA expression elicits profound chemo- and radio-sensitization in cancer cells, inhibition of PUMA expression may be useful for curbing excessive cell death associated with tissue injury and degenerative diseases. Therefore, PUMA is a general sensor of cell death stimuli and a promising drug target for cancer.

5- The apoptosome, a complex of cytochrome-c and APAF-1, recruits and activates the initiator pro-caspase 9, leading to the activation of the effector caspases, in particular procaspase 3, culminating in those biochemical and morphological changes associated with apoptotic cell death [92]. This pathway is further regulated by the inhibitor of caspase protein XIAP, which works through the direct inhibition of active caspases 9 and 3 [93] and is also implicated in the ubiquitination of caspases, targeting them for proteasomal degradation [94]. Moreover there is a direct interaction between XIAP and its antagonist Smac [95].

inhibitors [84].

combined with conventional chemotherapy drugs in chronic lymphocytic leukemia (CLL) patients, leading to improved survival [85,86]. More recent advances include the discovery of small molecule inhibitors of the Bcl-2 family proteins. They are designed to bind the hydrophobic groove of anti-apoptotic Bcl-2 proteins in place of BH3-only proteins (i.e., BH3 mimetics). They can oligomerize Bax or Bak, which can subsequently depolarize mitochondrial membrane potential to release cytochrome *c*. To date, one Bcl-2 antisense and three small molecule Bcl-2 protein inhibitors are being tested in clinical trials. Preclinical studies seem promising, especially in combination with additional chemotherapy agents. Ongoing and planned phase II clinical trials to define the activity of single agents and drug combinations will determine the direction of future clinical development of the Bcl-2

4- PUMA (p53 upregulated modulator of apoptosis) is a BH3-only Bcl-2 family member and a critical mediator of p53-dependent and -independent apoptosis induced by a wide variety of stimuli, including genotoxic stress, deregulated oncogene expression, toxins, altered redox status, growth factor/cytokine withdrawal and infection. It serves as a proximal signalling molecule whose expression is regulated by transcription factors in response to these stimuli. PUMA transduces death signals primarily to the mitochondria and acts indirectly on the Bcl-2 family members Bax and/or Bak by relieving the inhibition imposed by anti-apoptotic members. It directly binds and antagonizes all known anti-apoptotic Bcl-2 family members to induce mitochondrial dysfunction and caspase activation [87]. Several lines of evidence suggest that the function of PUMA is compromised in cancer cells. PUMA expression was found to be reduced in malignant cutaneous melanoma, and PUMA expression appears to be an independent predictor of poor prognosis in patients [88]. In addition, approximately 40% of primary human Burkitt's lymphomas do not express detectable levels of PUMA, which is attributable, in part, to DNA methylation [89]. Evidence of PUMA induction by therapeutic agents in patients has just begun to emerge. Analysis of tissue biopsies from breast cancer patients showed that *PUMA* mRNA was induced within 6 h of chemotherapy [90]. Increased expression of *PUMA* and *Bim* is associated with better prognosis in patients receiving 5-FUbased therapy in stage II and III colon cancer, and is an independent prognostic marker for overall and disease-free survival [91]. PUMA ablation or inhibition leads to apoptosis deficiency underlying increased risks for cancer development and therapeutic resistance. Although elevated PUMA expression elicits profound chemo- and radio-sensitization in cancer cells, inhibition of PUMA expression may be useful for curbing excessive cell death associated with tissue injury and degenerative diseases. Therefore, PUMA is a general sensor

5- The apoptosome, a complex of cytochrome-c and APAF-1, recruits and activates the initiator pro-caspase 9, leading to the activation of the effector caspases, in particular procaspase 3, culminating in those biochemical and morphological changes associated with apoptotic cell death [92]. This pathway is further regulated by the inhibitor of caspase protein XIAP, which works through the direct inhibition of active caspases 9 and 3 [93] and is also implicated in the ubiquitination of caspases, targeting them for proteasomal degradation [94].

Moreover there is a direct interaction between XIAP and its antagonist Smac [95].

of cell death stimuli and a promising drug target for cancer.

Because of the importance of this pathway in cancer progression and chemotherapy-induced cell death, apoptosome-associated proteins may be important markers for colorectal cancer prognosis and chemotherapy response. Several studies have examined the immunohistochemical expression of individual proteins associated with apoptosis execution in colorectal cancer [96]. Increased expression of APAF-1 has been shown to be associated with longer patient survival in rectal cancer patients [97] and loss of APAF-1 has been implicated in tumour progression and more aggressive disease [98]. Similarly, longer overall survival has been associated with increased Smac [99] and caspase 9 [100]. The anti-apoptotic XIAP has also been implicated as a potential prognostic marker for colorectal cancer, with increased expression correlating with poor patient outcome [101]. However, no study to date has provided a comprehensive analysis of these key regulatory proteins as markers for colorectal cancer prognosis or chemotherapy response. Recently, the pro-caspase 3 expression in colorectal tumours has been associated with better recurrence-free and overall survival, and serves as an independent prognostic marker in localised Stage II disease [102]. This result is in agreement with previous studies that demonstrated as caspase 3 expression is a positive prognostic marker in hepatocellular carcinoma [103] and diffuse large B-cell lymphoma [104].

6- The extrinsic apoptosis pathway is triggered by the binding of death ligands of the tumour necrosis factor (TNF) family to their appropriate death receptors (DRs) on the cell surface. One TNF family member, TRAIL or Apo2L, seems to preferentially cause apoptosis of transformed cells and can be systemically administered in the absence of severe toxicity. Therefore, there has been enthusiasm for the use of TRAIL or agonist antibodies to the TRAIL DR4 and DR5 in cancer therapy. Nonetheless, many cancer cells are very resistant to TRAIL apoptosis in vitro. Therefore, there is much interest in identifying compounds that can be combined with TRAIL to amplify its apoptotic effects [105]. The combination of TRIAL DR agonists with numerous conventional and investigational anticancer drugs has been reported. Synergy has been described for the combination of TRAIL with a variety of cytotoxic agents including irinotecan, camptothecin, 5-FU, carboplatin, paclitaxel, doxorubicin, and gemcitabine in diverse preclinical models [106,107]. Many human and mouse cancer cells lines can be sensitized by proteasome inhibitors such as bortezomib (VELCADE) to the apoptotic effects of TRAIL DR agonists. Interestingly, non-transformed cells seem to be much more resistant to the apoptotic effects of bortezomib and TRAIL than are cancer cells. This suggests that a therapeutic window may exist in vivo where this combination may have a therapeutic benefit in the absence of accompanying toxicity. However, the molecular mechanism(s) of action whereby proteasome inhibition in cancer cells results in sensitization to TRAIL apoptosis remains unclear [108].

## **3. Selective antitumour-drug inducers of apoptosis**

Improved clinical response may be obtained by identifying therapies that are particularly effective in activating apoptosis and determining how those therapies may be modified to effect maximum apoptosis induction. The cell cycle apparatus and apoptosis have attracted the attention of researchers intent on developing new types of anticancer therapy [109,110]. We will concentrate in this part of the review on the evolution of the chemical structures and on the biological properties, whilst the chemical syntheses will be referred to through the corresponding original references.

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 119

are the only commonly used cytotoxic agents shown to elicit apoptosis in breast cancer cells [111,112]. Quantitation of apoptotic cells was done by monitoring the binding of fluorescein isothiocyanate (FITC)-labelled annexin V (a phosphatidylserine-binding protein) to cells in response to our title compounds as described [113]. The apoptosis study shows that **3**, **4** and **5**, at their IC50 concentrations, provoke early apoptosis in the cells treated for 24 and 48 h. It is worth pointing out that **3** (entry 6) induces greater apoptosis at 48 h (46.73%) than at 24 h (40.08%). The compounds that show the most important apoptotic indexes at 24 h are **4** (57.33%, entry 7) and **5** (54.33%, entry 8), whereas at 48 h is **4** (51.37%, entry 7). These compounds are more potent as apoptosis inductors against the MCF-7 human breast cancer cells than paclitaxel (Taxol®), which induced programmed cell death of up to 43% of the cell population [114]. Accordingly, the early apoptotic inductions and the low IC50 values give

Since the synthesized compounds induce very important apoptosis, we have carried out studies of the expression of some of the genes that intervene in this phenomenon, among which p53 and the family bcl-2 are outstanding. The tumour suppressor gene p53 protects the integrity of the genome so that if the DNA of the cell is damaged by an agent, an overexpression of it is produced inducing the stopping in G1 for the repair of the damage, or if this is not possible, enter apoptosis [115]. On the other hand, the members of the family of proteins Bcl-2 work as regulators of apoptosis, Bcl-2 and Bcl-XL protecting against apoptosis. Bax, Bak and Bad induce such a phenomenon [116]. The treatment of the MCF-7 cells (wild-type p53) with these compounds provoked in general an increase in the protein expression of p53, mainly for 5-FU and **4**, and a marked decrease of the levels of bcl-2 for all of them. These data show that p53 activity is restored with the compounds, allowing the entrance of the tumour cells in apoptosis, which permits their elimination by this mechanism. In the same way bcl-2 inhibition facilitates the entrance of cells into the

**Entry Compound IC50 (μM)**a **Cell Cycle (48 h)**<sup>b</sup> **Apoptosis**<sup>c</sup>

The data indicate the percentage of cells undergoing apoptosis in each sample. All experiments were conducted in

**Table 1.** Anti-proliferative activitiy, cell cycle dysregulation, and apoptosis induction in the MCF-7

duplicate and gave similar results. The data are means ± SEM of three independent determinations.

human breast cancer cell line after treatment for 24 and 48 h for the compounds.

1 Control 68.39 12.04 19.57 1.24 1.24 **5-FU** 2.75 58.07 2.10 39.38 56.75 52.81 **Ftorafur** 3.00 0.11 45.62 0.00 54.38 52.20 58.06 **1** 7.00 0.61 74.41 15.77 9.82 8.45 12.17 **2** 4.50 0.33 73.41 13.15 13.44 1.50 3.50 **3** 22.0 0.93 71.76 10.08 18.16 40.08 46.73 **4** 14.0 1.02 86.14 1.60 12.26 57.33 51.37 **5** 69.0 2.31 68.61 9.60 21.79 54.33 35.49 **6** 5.50 0.58 82.48 5.13 12.40 14.37 19.05

G0/G1 G2/M S 24 h 48 h

Apoptosis was determined using an annexin V-based assay [113].

rise to a significant anti-tumour activity.

programmed cell death.

aSee [117]. bDetermined by flow cytometry: see [16]. c
