**6.5. NF-kB**

136 Apoptosis and Medicine

**6.2. cFLIP** 

**6.3. IAP** 

**6.4. Bcl-2** 

chemo-sensitivity of CSCs [230].

clinical trials of TRAIL apoptotic pathway targeted therapies may consider genomic analysis of tumour tissue to identify the genomic status of TRAIL apoptotic genes such as *caspase 8* and use it as a genomic marker to predict tumour resistance to TRAIL apoptotic pathwaytargeted therapies [224]. In addition, recent studies are focused on combining mesenchymal stem cells (MSCs) expressing TRAIL and chemotherapy. These MSCs migrated to tumours and reduced the growth of primary cancers and metastases by induction of apoptosis, death and reduced colony formation of the SP and were synergistic when combined with

cFLIP is overexpressed in many types of cancers like melanoma, colon lymphoma and thyroid cancer [226]. The CD133+ populations within the T-cell acute leukemia cell line Jurkat and the breast cancer cell line MCF7 were reported to express higher levels of cFLIP, which was associated with TRAIL resistance. The down-regulation of cFLIP using siRNA restored TRAIL signalling in both cell lines resulting in a dramatic reduction in experimental metastases and the loss of CSC self-renewal [227]. This suggests that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in cancers.

In CD133 + cells isolated from glioblastoma an increased mRNA expression of livin, survivin and the multidrug resistance-associated protein 1 (MRP1) was detected. Therefore, the effects of etoposide, a pro-apoptosis agent, on these associated protein genes in glioblastoma stem-like cells have been studied. Results showed that after etoposide treatment, glioblastoma CSCs displayed a stronger resistance to apoptosis and death. The anti-apoptotic gene livinβ was more related with the high survival rate and MRP1 was more related with transporting chemotherapeutic agent out of glioblastoma stem-like cells [228]. In pancreatic cancer it has been demonstrated that targeting XIAP by RNAi inside the cancer cells, the combination of TRAIL with MSCs suppressed metastatic growth in these tumours [229]. Recently, it has been demonstrated that survivin is regulated by the interleukin-4 (IL-4) pathway in colon CSCs. Blockage of IL-4-mediated signaling pathway with leflunomide, Stat6 inhibitor, increased the nuclear survivin pool suggesting that the IL-4/STAT-6 pathway could escape cell death. IL-4 neutralization, mediated by STAT-6, could down-regulate survivin expression and localization, increasing the nuclear pool and in this way inducing

The proteins Bcl-2 family members are anti-apoptotic molecules known to be overexpressed in most cancers, and are associated directly with the CSCs [231]. Therefore, recent studies are aimed to target these proteins. A representative example is found in pancreatic cancer, against which the most potent and clinically acceptable Bcl-2 inhibitor AT-101 is currently in 20 different clinical trials around the world [232]. In glioblastoma, high

traditional chemotherapy in apoptosis induction [225].

The transcription factor NF-κB has been connected to multiple aspects of oncogenesis, including inhibition of apoptosis by increasing the expression of survival factor. In fact, aberrant regulation of NF-κB has been observed in many cancers, including both solid and hematopoietic tumours [235]. A fairly representative example is found in pancreatic cancer, where there is a clear correlation between the basal activity of NF-kB and the ability to generate angiogenesis and metastasis of pancreatic tumour cells [236] and, more recently it has been found that not only in this way, but also in the non-canonical NF-kB is also activated and functional [237]. A very interesting study by [238] showed that in CD44+ breast CSCs the expression of CD24 potentiated DNA-induced apoptosis by suppressing anti-apoptotic NF-κB signaling. Several therapies are being developed to inhibit this factor because there are many tumours which relate the decrease in the activity of NF-kB with a decrease in the size and tumor growth [239,240,241,242,243].

## **6.6. DNA repair capacity**

A classic mechanism involved in the induction of apoptosis is in response to DNA damage by p53 action. This gene is mutated in most human cancers and inactivated in about 50% of cancers [244]. p53 was found to repress the CSC marker gene CD44 in an experimental breast tumour model and the over-expression of CD44 blocked p53-dependent apoptosis, leading to expansion of tumour-initiating cells [245]. Moreover, glioma CSCs resist radiation through preferential activation of the retainer DNA damage response and an increase in DNA repair capacity. In addition, the radioresistance of CD133+ glioma stem cells could be reversed with a specific inhibitor of Chk1 and Chk2 checkpoint kinases [208]. Another report reinforces the tumour-promoting effect of DNA damage response activation in leukemia stem cells by demonstrating that cell-cycle inhibitor p21 was indispensable for maintaining self-renewal of these CSCs [246].

## **6.7. miRNAs, apoptosis and CSCs**

Recently, miRNAs have emerged as key regulators of "stemness", collaborating in the maintenance of pluripotency, control of self-renewal, and differentiation of stem cells. Moreover, certain miRNAs involved in apoptosis appear to influence the CSC fate by controlling self-renewal. It has been shown that restoration of miR-34 modulates selfrenewal in pancreatic CSCs by directly regulating down-stream target gene Notch and Bcl-2 [247]. Also the restoration of miR-34, inhibit p53-mutant gastric cancer tumourspheres growth in vitro and tumour formation in vivo, which is reported to be correlated to the selfrenewal of CSCs [248]. As miR-34 is a significant tumour suppressor of CSCs by regulation

of both apoptosis and self-renewal properties, restoration of miR-34 may hold significant promise for a novel molecular therapy. Data also suggest that let-7 regulates apoptosis and CSC differentiation, which is considered as a key "keeper" of the differentiated state. In this context decreased expression of these TSmiRs implicated in self-renewal could lead to further cancer progression.

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 139

[3] Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with

[4] Ashkenazi A (2008) Targeting the extrinsic apoptosis pathway in cancer. Cytokine

[5] Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ, et al. (2000) Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death

[6] Kischkel FC, Lawrence DA, Tinel A, LeBlanc H, Virmani A, et al. (2001) Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of

[7] Lavrik I, Golks A, Krammer PH (2005) Death receptor signaling. J Cell Sci 118: 265-267. [8] Fulda S, Galluzzi L, Kroemer G (2010) Targeting mitochondria for cancer therapy. Nat

[9] Wu G, Chai J, Suber TL, Wu JW, Du C, et al. (2000) Structural basis of IAP recognition

[10] 10. Fulda S, Vucic D (2012) Targeting IAP proteins for therapeutic intervention in

[12] Marchal JA, Boulaiz H, Suarez I, Saniger E, Campos J, et al. (2004) Growth inhibition, G(1)-arrest, and apoptosis in MCF-7 human breast cancer cells by novel highly

[13] Grivicich I, Regner A, da Rocha AB, Grass LB, Alves PA, et al. (2005) Irinotecan/5 fluorouracil combination induces alterations in mitochondrial membrane potential and

[14] Johnstone RW, Ruefli AA, Lowe SW (2002) Apoptosis: a link between cancer genetics

[15] Garcia MA, Carrasco E, Aguilera M, Alvarez P, Rivas C, et al. (2011) The chemotherapeutic drug 5-fluorouracil promotes PKR-mediated apoptosis in a p53-

[16] Saniger E, Campos JM, Entrena A, Marchal JA, Suarez I, et al. (2003) Medium benzenefused oxacycles with the 5-fluorouracil moiety: synthesis, antiproliferative activities and

[17] Saniger E, Campos JM, Entrena A, Marchal JA, Boulaiz H, et al. (2003) Neighbouringgroup participation as the key step in the reactivity of acyclic and cyclic salicyl-derived O,O-acetals with 5-fluorouracil. Antiproliferative activity, cell cycle dysregulation and apoptotic induction of new O,N-acetals against breast cancer cells. Tetrahedron 59:

[18] Conejo-Garcia A, Nunez MC, Marchal JA, Rodriguez-Serrano F, Aranega A, et al. (2008) Regiospecific microwave-assisted synthesis and cytotoxic activity against human breast cancer cells of (RS)-6-substituted-7-or 9-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-7H-

independent manner in colon and breast cancer cells. PLoS One 6: e23887.

apoptosis induction in breast cancer cells. Tetrahedron 59: 5457-5467.

or-9H-purines. European Journal of Medicinal Chemistry 43: 1742-1748.

[11] Lowe SW, Lin AW (2000) Apoptosis in cancer. Carcinogenesis 21: 485-495.

lipophilic 5-fluorouracil derivatives. Invest New Drugs 22: 379-389.

caspases on colon cancer cell lines. Oncol Res 15: 385-392.

wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257.

Growth Factor Rev 19: 325-331.

Rev Drug Discov 9: 447-464.

receptors 4 and 5. Immunity 12: 611-620.

caspase-8. J Biol Chem 276: 46639-46646.

by Smac/DIABLO. Nature 408: 1008-1012.

cancer. Nat Rev Drug Discov 11: 109-124.

and chemotherapy. Cell 108: 153-164.

8017-8026.
