# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 5 Mechanisms of Bioactivities
### Immunological Effects of d-AA-Containing HDPs
#### 5.5.2. Apoptotic Activity  
HDPs are capable of inducing apoptosis in neoplasm cells (Figure S5). For example, lactoferricin B has been demonstrated to induce apoptosis in human cancer cells by depolarization of the mitochondrial membrane [90]. The membrane lysis has been proven to occur independently of the apoptotic effects because treatment of cancer cells with anti-apoptotic agents did not inhibit the peptide activity [85]. One reminder here is that this review discusses the apoptotic effect of HDPs, peptides that bear the hallmarks (i.e., positive charge and amphipability) and thus are capable of neutralizing pathogens via cell membrane depolarization. It should be noted that other types of apoptotic peptides were not considered.  
d-AA-containing HDPs are also able to induce apoptosis in cancer cells. [PERSON] et al. [91] tested an all-d analogue of the insect defensin, coprisin, and observed chromosomal DNA fragmentation and the expression of transcripts of inflammatory cytokines (e.g., TNF-\(\alpha\) and IL-1\(\beta\)) in human leukemia cells. Simultaneously, the d-coprisin analogue maintained antimicrobial activity against a range of multi-drug-resistant bacteria. However, the study provided no insight into how the d-coprisin entered the cancer cells to initiate the apoptosis. Compared to the l-form, the d-analogue provided considerably higher selectivity in its direct antimicrobial lysis activity.  
Aside from causing apoptosis in cancer cells, the all-d analogue of the AMP, bovine myeloid antimicrobial peptide (BMAP-28), was able to induce apoptosis in the protozoan parasite _Leishman_ in both the free-living promastigotesote and the intracellular amastigote form [4]. Mouse macrophages infected with _Leishman_ treated with BMAP-28 had up to 68% reduced amastigote burden. The d-form of BMAP-28 was more effective against both promastigotes and amastigotes of _Leishman_. BMAP-28 induces apoptosis in _Leishman_ in a caspase-independent manner. Initially, parasite membrane disruption is observed, with a significant sign of apoptosis as detected by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay only 24 h after the initial peptide exposure. This finding indicated that the peptide also relied to a significant extent on direct parasite lysis. However, this study also did not discuss how the peptide entered the cytoplasm of the pathogen to initiate apoptosis.  
As of now, all-d HDPs have been shown to induce apoptosis, but no report has been found for an apoptotic diastereomer. However, it is very likely that a diastereomer HDP will be able to induce mitochondria-dependent apoptosis should it cross the cell membrane because the membrane structure of mitochondria is quite similar to that of the prokaryotes from which they are believed to have been evolved from [92] and because mitochondrial disruption can result in the release of apoptotic factors that are normally sequestered within the mitochondria [93,94]. An interesting way to provide AMPs with the capacity to disrupt mitochondrial function is by conjugating them to an internalization motif. [PERSON] et al. [95] conjugated the horseshoe crab AMP, tachyplesin, to the integrin homing domain Arg-Gly-Asp (RGD). The resulting construct was effective at inducing apoptosis in tumor cells. JC-1 staining confirmed the disruption of the mitochondrial membrane, and Western blotting revealed the presence of caspase and Fas apoptotic factors.  
Thus in summary, in addition to their strong, selective membrane disruption activities, d-AA-containing HDPs possess notable immune modulation activity and are capable apoptotic agents. A thorough elucidation of the mechanisms responsible for these activities remains to be carried out.