**2.2. Cancer cells reducing anti-apoptosis signals**

(FADD) combines with procaspases 8 and 10, leading to the formation of death-inducing signaling complex (DISC), which activates the downstream signal cascade [9]. The activated caspase 8 modifies Bid into tBid. tBid binds with Bak and Bax, which are pro-apoptosis proteins that control the permeability of mitochondrial outer membrane, to form mitochondrial apoptosis-induced channel (MAC). The intrinsic pathway of apoptosis, also known as the mitochondrial pathway, is initiated by cytochrome C. Cytochrome C is a key protein for electron transfer in mitochondria. Mitochondria releases cytochrome C into the cytosol through MAC in response to stresses of apoptosis-inducing factors [9]. In the presence of ATP/dATP, cytochrome C interacts with the apoptotic protease-activating factor (Apaf-1) in the cytosol to form a complex and promotes the formation of apoptosome that activates procaspase 9 [9].

Notably, a class of proteins exerts anti-apoptosis and pro-apoptosis effects in the apoptosis pathway. These proteins include the Bcl-2 family and inhibitors of apoptosis proteins (IAPs). The Bcl-2 protein family can be classified into two functional groups—one of which has an inhibitory effect on apoptosis through inhibition of MAC formation, such as Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Ced-9, while the other has a promoting effect on apoptosis by promotion of MAC formation, such as Bax, Bak, Bik, Bid, and Harakiri [10]. IAPs are the family of caspase inhibitors, including survivin, livin, Bruce (Apollon), cIAP1, cIAP2, IAP-like protein 2 (ILP-2), the X-linked inhibitor of apoptosis protein (XIAP), and neuronal apoptosis inhibitory protein (NAIP) [11]. Obviously, the homeostasis between anti-apoptosis proteins and pro-apoptosis

In addition to the extrinsic pathway and the intrinsic pathway, there also exists caspaseindependent pathway. This pathway relies on apoptosis-inducing factors (AIFs). AIFs are flavoproteins present in the inner mitochondrial membrane [12], and exhibit the pro-apoptosis effect. AIFs are released into cytoplasm along with the increased permeability or the cleavage of mitochondria. Then, AIFs enter the nucleus and lead to chromatin condensation and break into fragments. Polster has studied the relationship of AIFs and mitochondrial ROS production [13]. Insufficient AIF would reduce the electron transport chain complex I, which relates

The cancer cells evade apoptosis via various mechanisms. Theoretically, in order to resist apoptosis, cancer cells would upregulate anti-apoptotic signals (e.g. Bcl-2, Akt, Mcl-1, etc.) and downregulate pro-apoptotic signals (e.g. Bax, Bak, Bad, etc.), initiate and implicate faulty

In human cancer cells, the downregulation of pro-apoptotic proteins (e.g. Bax, Bak, Bad, Bim, etc.) and the upregulation of anti-apoptotic proteins (e.g. Bcl-2, Akt, Mcl-1, etc.) hinder the formation of MAC, inhibiting the release of cytochrome C from mitochondria and leading to the immortal character of the cancer cells. For example, the increased ubiquitination level of Bax has been found to be positively correlated to tumor malignant degree [15]. The decreased expression of Bad has been observed in small-cell lung cancers (SCLC), breast carcinoma, and gastric cancer. Furthermore, cancer cells regulate Bim in the pro-transcriptional,

The activated caspase 9 further activates the downstream caspases.

proteins is essential for cell survival.

128 Current Understanding of Apoptosis - Programmed Cell Death

to chronic neurodegeneration [14].

apoptosis, etc. The detail is discussed below.

**2.1. Cancer cells resisting pro-apoptotic signals**

A variety of signal pathways are involved in the anti-apoptosis process. The TNF family is associated with apoptosis and malignant tumorigenesis. It has been reported that the translational level of Fas is downregulated in prostate cancer and liver cancer. TRAIL, another membrane of TNF family, intrigues wide anticancer effect by exerting pattern-like function of mFas. It has been found that some cancers show primary resistance and even develop multiple-mechanism resistance to TRAIL-induced apoptosis [19]. For example, overexpression of TRAIL receptor 3 (TRAIL-R3/DcR1) and TRAIL receptor 4 (TRAIL-R4/DcR2) is considered to contribute to the TRAIL-mediated apoptosis evasion of cancer cells. TRAIL-R3 and TRAIL-R4 are decoy receptors without intercellular death domain. The incapability of TRAIL-3 and TRAIL-4 to associate with procaspases 8 and 10 to form DISC attenuates the activation of downstream signaling pathway [20]. Furthermore, gene mutation of diverse proteins generates the anti-apoptotic effect. Shlyakhtina Y. and his colleagues [21] have studied TRAIL-R2 (DR5) within isogenic cancer cell populations. The models were pretreated with distinctive inhibitors, and the results showed that apoptosis evasion involves kinase cascades of functional Erk1/2, p38, and Akt.

Alteration of the p53 pathway also contributes to apoptosis evasion. The p53 gene is a human tumor suppressor gene. The p53 protein endows anticancer effect by activating defected gene repair and causing apoptosis of cancer cells if the damage is irreparable. p53 regulates apoptosis through Bax/Bcl-2, Fas/Apol, IGF-BP3, and other proteins. Inactivation, elimination, and abnormal expression of the p53 gene play important roles in tumorigenesis. About 80% of human tumors are caused by dysfunctional p53 signaling and 50% by p53 gene mutation [22]. Abnormal expression of p53 downregulates Bax/Noxa/Puma expression and upregulates Bcl-2. The upregulation of Bcl-2 prevents cytochrome C release from the mitochondria, inhibiting p53-mediated apoptosis. The downregulation of Bax prevents the formation of MAC on the outer membrane of mitochondria, reducing the pro-apoptotic effect [23]. Furthermore, mouse double minute 2 homolog (MDM2) has also been found to play a pivotal role in the inhibition of p53-mediated apoptosis by negative regulation. In cancer cells, the increase of MDM2 transcription and p53 ubiquitination attenuates p53-mediated apoptosis [24].

NF-κB pathway, one of the highly conserved signal pathways of activating gene transcription, takes complicated apoptotic effects in different cells. Activated NF-κB improves the transcription level of survivin, Bcl-2, Bcl-XL, and XIAP, resulting in resistance to the chemotherapeutic pro-apoptotic signals [5]. However, NF-κB renders pro-apoptotic effect through upregulating caspase 4 in Fas-induced neuroblastoma cell apoptosis [25]. In addition, NF-κB upregulates pro-survival genes via Akt activation.

PI3/AKT pathway mediates the survival signals in cancer cells. Akt is correlated to phosphorylation of diverse signal molecules and has a profound effect on cell survival, cell cycle progression, cell growth, and metabolism. The overexpression and overactivation of Akt have been observed in malignant tumors. For example, Zheng [5] has revealed that the paclitaxel-resistance developed in NSCLC can be ascribed to Akt-1 overexpression and Akt-2 gene amplification. In addition, Akt promotes the phosphorylation of Bad on Ser136/Ser112, leading to the suppression of apoptosis [26]. Akt phosphorylates Forkhead-box Class O (FoxO), a protein family governing a line of apoptotic gene transcription in PI3K/Akt pathway. The phosphorylated FoxO binds with 14-3-3, stays in the cytoplasm, and fails to execute transcription in nucleus [27].
