**5.2.7 Inhibitor of apoptosis**

#### **5.2.7.1 Caspase3 inhibitor**

Various chemotherapeutic agents induce apoptosis of hair follicle cells and cause CIA, although the underlying mechanisms are unclear. Caspase-3 is a key executor of apoptosis and its activation is normally used as an indicator of caspase-dependent apoptosis (Porter and Janicke, 1999). M50054, 2,2'-methylenebis, is an inhibitor of caspase-3 activation that was shown to inhibit etoposide-induced apoptosis in human monocytes. In neonatal rats, topical administration of M50054 reduces CIA induced by etoposide (Tsuda et al., 2001).

Chemotherapy-Induced Alopecia 65

p53-deficient mice show strongly down-regulated Fas in the hair follicle keratinocytes and highly up-regulated Bcl-2 in the dermal papilla as compared to wild-type mice. The role of Fas in the control of cyclophosphamide-induced apoptosis in keratinocytes was also investigated using Fas-deficient mice (Sharov et al., 2004). These mice show significantly reduced CIA and a parallel decrease in apoptotic keratinocytes and FADD and caspase-8 expression. Similarly, anti-Fas ligand neutralizing antibody inhibits cyclophosphamideinduced keratinocyte apoptosis. These studies indicate that Fas signaling is an important pathway in mediating the apoptosis induced by cyclophosphamide and suggest the crosstalk between p53 and Fas death signaling. However, the eventual hair loss observed in Fasdeficient mice points to the lower resistance of hair follicles to cyclophosphamide as compared to p53-deficient mice. Thus, it is likely that Fas signaling represents only a component of the p53-dependent apoptosis machinery in the hair follicles and that other p53 targets are also involved. Cyclophosphamide treatment also alters the expression of melanogenic proteins and causes apoptosis of hair follicle melanocytes (Sharov et al., 2003). In contrast to matrix keratinocytes, the melanocytes undergo apoptosis primarily through

The observation that antioxidants such as NAC protect against CIA in animals suggest the involvement of reactive oxygen species (ROS) in CIA. Various chemotherapeutic agents induce oxidative stress through multiple mechanisms, i.e., activation of NADPH oxidase system and mitochondrial respiration chain. Agents that induce a high level of ROS include anthracyclines (e.g., doxorubicin, epirubicin, and daunorubicin), alkylating agents (e.g., cyclophosphamide), platinum coordination complexes (e.g., cisplatin, carboplatin, and oxaliplatin), and epipodophyllotoxins (e.g., etoposide) (Conklin, 2004). Interestingly, anthracyclines, alkylating agents, platinum complexes, and epipodophyllotoxins also induce CIA more frequently and more severely than most other agents, suggesting a relationship between ROS generation and CIA. The exact mechanism of how ROS induces or promotes CIA is unclear, but likely involves apoptosis regulation since apoptosis of hair follicles is a hallmark of CIA and since ROS generation is generally required for the induction of

CIA is a major side effect that compromises patient quality of life, particularly for females and children. Overcoming CIA remains a major challenge in the management of cancer patients. Significant progresses in the pathobiology and molecular mechanisms of CIA have been made during the past decade, and several physical and pharmacological approaches to treat CIA have been attempted. However, effective treatment strategies have yet to be developed. A key to this success is a better understanding of the human CIA mechanisms which requires the development of more predictive experimental models. Animal models have been useful but have limitations and may not be predictive of human CIA. The newly developed organ culture system using human hair follicles is promising and could lead to the development of more effective treatment strategies for CIA. The recent success in combination chemotherapy also provides mechanistic insights to combating CIA through

Fas signaling but not p53 signaling.

apoptosis by chemotherapeutic agents (Simon et al., 2000).

the use of different combination strategies.

**6.3 Reactive oxygen species** 

**7. Perspectives** 

#### **5.2.7.2 Anti-death FNK protein**

FNK protein constructed from rat Bcl-xL by site-directed mutagenesis (Y22F/Q26N/R165K) localizes to mitochondria and functions to maintain mitochondrial membrane potential (Aosh et al., 2000). Mitochondrial membrane potential regulates the release of cytochrome C, which once binds to caspase-activating proteins such as Apaf-1 initiates the intrinsic caspase cascade and apoptosis (Li et al., 1997). Recently, FNK protein has been fused to protein transduction domain (PTD) to improve its cellular entry. Subcutaneous injection of PTD-FNK protects against CIA induced by etoposide in the neonatal rat model. The fusion protein helps retain hair follicle structures, prevent hair follicle regression and maintain the anagen duration upon etoposide treatment (Nakashima-Kamimura et al., 2008). Indeed, its protective effect on CIA suggests that it could penetrate the epidermis and reach the dermal hair follicles. Localized administration of FNK fusion protein has been suggested as a potential protein therapy for CIA without affecting the chemotherapy efficacy.
