Cancer is caused by the abnormal cell growth in an uncontrolled manner. Chemother- apy and radiation are the usual methods to treat cancer. These methods have only
Chapter 1. Introduction 26
partial success rates, in large part due to their lack of specificity [69]. They target rapidly dividing cells with relatively small discrimination between the cancer cells and the normal proliferating cells. Moreover, cancer cells can develop resistance to anticancer agents and pump drug out from the cell. To control the mortality and morbidity rate due to cancer, it is necessary to develop more efficient and selective anti-cancer therapeutics that can be used in place of or in combination with conventional anti-cancer therapeutics.
Cationic AMPs are under consideration as alternative chemotherapeutic agents due to their selective toxicity towards cancer cells, ability to avoid resistance and additive effects in combination therapy [70,71]. AMPs have been primarily studied to develop new therapeutics to fight various infections but it has been reported that certain AMPs like Cecropin A, Cecropin B, BMAP-27, BMAP-28, LL-37, Magainin, Melittin, Defensins, Lactoferricin, Tachyplesin, Buforin II, and Buforin IIb, have the ability to kill cancer cells. The cancer cell membranes are different from normal cells in many ways. Firstly, cancer cell membranes have an anionic character that is similar to bacterial membranes. For this reason, electrostatic interactions might be the core of the selectivity mechanism of AMPs towards cancer cells instead of normal cells. Another major difference between cancer and normal cells is their acidic environment. The pH level of the extracellular space surrounding solid tumors is significantly lower than the pH around normal cells. Therefore, many attempts have been made to develop new pH-dependent anti- cancer therapies which can be active in the lower pH space of solid tumors [72]. There are few AMPs (Histatin, Clavanin, and Chrysophsin, LAH4) which have histidine residues in their sequences. These histidine-rich AMPs are potentially pH sensitive and have an ability to become more active at acidic pH values, as histidine side chain has a pKa value of 6. Thus at neutral pH its side chain is uncharged but at acidic pH values, it has charge of +1. So the presence of histidine
Chapter 1. Introduction 27
can make the overall charge of a peptide sensitive to pH which, in turn, can affect the peptide-membrane interaction [73]. Furthermore, cancer cell membranes are more fluid than healthy cells. This might help AMPs to cause destabilization of the cell membrane. In addition to this, cancer cells have a larger surface area than normal cells and this can result in AMP-mediated cytotoxicity due to larger numbers of AMP molecules interacting with the membrane [74].
Even though AMPs are expected to be selective towards cancer cells without reduced toxicity to normal body cells, the development of AMP-based anti-cancer drugs is very challenging because their selectivity is poorly understood and it is not possible to predict the anti-cancer activity based on AMP structure. A detailed knowledge of membrane-peptide interactions is needed in order to use AMPs as anticancer drugs [69].
1.2.5.1 Gad peptides
Gad peptides, Gad-1 and Gad-2, belong to the Piscidin family of AMP from fish. Piscidins fight against various pathogens in aquatic environments as part of the innate immune system [25]. Gad peptides are found in Atlantic cod (Gadus
morhua) and are paralogs (coded by related genes). Gad-1 and Gad-2 are 21 and
19 residues long respectively and have tendencies to form α helical structure in a membrane environment [26]. The primary amino acid sequences of C-terminus amidated Gad peptides are:
Gad-1:FIHHIIGWISHGVRAIHRAIH-NH2 Gad-2:FLHHIVGLIHHGLSLFGDR-NH2
Gad peptides are natural histidine-rich peptides. Gad-1 has 5 histidines and Gad- 2 has 4. The charge of Gad peptides at pH 7.0 and pH 5.0 is listed in Table 1.4.
Chapter 1. Introduction 28
peptide pH 7.0 pH 5.0
Gad-1 +3 +8
Gad-2 +1 +5
Table 1.4: Expected overall peptide charge of Gad-1 and Gad-2 at pH 7.0 and pH 5.0.
There are other histidine-rich peptides such as LAH4 that are artificial, as opposed to the Gads which are sequences found in nature. Gad peptides provide a means to understand how nature tunes a peptide’s pH-dependent activity. It has been shown that there is a relationship between the pH sensitivity of antimicrobial activity and the number of histidine residues present in the sequence of a peptide [75–77]. In order to test their pH sensitivity, MIC assay experiments with E.coli were done in the presence of Gad-1 and Gad-2 at pH 7.0 and pH 5.0 shown in Figure 1.6. MIC stands for Minimal Inhibitory Concentration and it represents the minimum concentration of peptide that is sufficient to stop bacterial growth. For Gad-1, MIC of 5.1 µM (12.5 µg/mL) was found in 4 out of 6 repeated experiments at pH 5, and in 5 out of 6 experiments at pH 7. For Gad-2, MIC of 11.5 µM (25
µg/mL) was found in 4 out of 6 experiments at pH 5, but the MIC found was
greater than 23.0 µM (50 µg/mL) in 4 out of 6 experiments at neutral pH. This is interesting considering that Gad-1 has five histidine residues and Gad-2 only four. At both pHs, Gad-1 was more active than Gad-2 [26].
Chapter 1. Introduction 29
Figure 1.6: Minimal Inhibitory Concentration (MIC) results for Gad-1 and Gad-2 at pH 7.0 and pH 5.0. Each bar represents one of six replicate experiments and the length of the bars indicates the minimum peptide concentration required to inhibit bacterial growth. No bar indicates an MIC greater than 50 µg/mL (20.2
µM for Gad-1 and 23.0 µM for Gad-2). This figure is taken from McDonald et al.
[26] and reprinted with permission from Elsevier.