HYD1 was previously identified using a combinatorial peptide synthesis in combination with an adhesion based screen (15
). HYD1 has been previously shown to block random haptotactic migration and inhibit invasion of epithelial prostate carcinoma cells on laminin-5 (laminin 332) (8
). These studies demonstrated that HYD1 interacted with α6 and α3 integrin (laminin receptors) on the tumor cell surface and blocked adhesion while inducing an elevation of laminin-5 (laminin 332)-dependent intracellular signaling, including focal adhesion kinase, mitogen-activated protein kinase kinase and extracellular signal-regulated kinase (8
). Currently it is unclear if cell death associated with HYD1 treatment is due to direct binding of the α subunit or whether HYD1 interacts with an integrin binding death inducing partner, associated with a specific α subunits, in a multi-protein complex. It must be noted that a β1 blocking antibody does not induce cell death in MM cells (data not shown) indicating that HYD1 has unique binding properties and may indeed act as an agonist for inducing cell death. Further studies are warranted to validate HYD1 target(s) associated with the induction of cell death.
In addition to blocking cell adhesion and reversing resistance associated with the HS-5 bone marrow stroma co-culture model, we have demonstrated in this report for the first time that HYD1, a D-amino acid peptide, induces cell death as a single agent in MM cells grown in vitro. More importantly, HYD1 treatment was found to be less-toxic to CD34+ progenitor cells and PBMC compared to MM cells. We further show that HYD1-induced cell death in MM cells demonstrated markers that are characteristic of necrosis, as cell death was accompanied by loss of Δψm, decrease in total cellular ATP and a significant increase in ROS. Also, HYD1-induced cell death could be partially reversed by pre-treating cells with the ROS scavenger, NAC. Another interesting finding in our study is that the oxidative stress induced by HYD1 resulted in the initiation of an autophagic pathway which paradoxically protected the cells against HYD1-induced cell death.
Caspase-dependent apoptosis is induced through two major pathways; one involves the death receptor, called the extrinsic pathway, and utilizes caspase-8 and another is the intrinsic pathway, involving the mitochondria and mediated by caspase-9 (27
). Both of these pathways converge on caspase-3 activation, eventually resulting in DNA fragmentation (29
). In our study HYD1 did not activate either the intrinsic or the extrinsic pathway of apoptosis as confirmed by measuring three different end points; (a) absence of cleaved caspases in HYD1 treated cells by western blotting, (b) absence of caspase activity in HYD1 treated cells by substrate cleavage and (c) failure of zVAD-fmk, a pan caspase inhibitor to block HYD1-induced cell death as ascertained by annexin v-FITC/PI binding studies.
DNA fragmentation can also occur through the mitochondrial death effector proteins AIF and Endo G. Under some cytotoxic stimuli, both AIF and Endo G are released from the mitochondria and translocate to the nucleus where they cause large scale DNA fragmentation (29
). In our study, HYD1 did not trigger the translocation of AIF and Endo G to the nucleus. This observation was consistent with the finding that HYD1 treatment did not activate Bax or caspases. Importantly these studies were performed in cell lines capable of activating caspases. Thus in the cell lines tested, HYD1 activates caspase independent cell death not as a default pathway but rather as a primary mode of cell death.
Necrosis is the consequence of extensive crosstalk between several biochemical and molecular events. Currently there is no single well-described signaling cascade to define necrotic cell death (for review see, (31
)). Similarly in our study, we show three different biochemical events, namely loss of Δψm
, decrease in cellular ATP and increased production of ROS as evidence for the HYD1-induced necrotic cell death. Further studies are warranted to determine the mechanism of HYD1 induced loss of Δψm
without activation of Bax, release of AIF and Endo-G or activation of capsases. Likely candidates for the cause of HYD1-induced loss of Δψm
may include activated Bcl-2 family member BNIP3, cyclophilin D or calcium overload, all of which either alone or in concert are reported to cause a decrease in mitochondrial membrane potential without release of intermembrane space proteins (6
). Loss of Δψm
can lead to mitochondrial dysfunction resulting in a breakdown of the respiratory chain and the overproduction of ROS concomitant with uncontrolled hydrolysis of ATP, ultimately leading to necrotic cell death (34
). Interestingly, in our study ROS seems to be a major player in the induction of necrotic cell death, as we could partially reverse HYD1-induced cell death by pre-treating the cells with a ROS scavenger, NAC.
In the present study, in addition to necrotic cell death, we show that HYD1 treatment resulted in a manifestation of morphological and biochemical markers that are indicative of autophagy. These markers included the extensive formation of double membrane containing vesicles and lipidation of LC3. Even though autophagy is a ubiquitous process in mammalian cells that contributes to the routine turnover of cytoplasmic components in case of cellular stress, autophagy can act as a defense mechanism involving the removal and recycling of damaged proteins and organelles by delivering them to lysosomes (for review see, (35
)). In the present study we show that an increase in ROS, in HYD1 treated cells results in the induction of autophagy. This observation is consistent with previous reports showing the elevated ROS levels-induces autophagy via the oxidation of a cysteine residue on Atg4 (36
In summary, these studies show that while HYD1 can block α4β1 integrin dependent binding of MM cells to FN, HYD1 also induces cell death in vitro. Although the in vivo activity was modest, we are currently pursuing strategies such as PEGylation and cyclization which will increase the bioavailability of the peptide in vivo. Importantly HYD1 increased the levels of melphalan specific cell death and reversed resistance to melphalan associated cell death when co-culturing myeloma cells with the bone marrow stroma cell line HS-5. Together these data suggest that HYD1 may be an attractive agent to combine with agents that a) target the apoptotic machinery and b) are resistant in a bone marrow stroma co-culture model system. Mechanistically HYD1 treatment leads to the loss of Δψm and a decrease in cellular ATP resulting in an increase in ROS production. All of these three biochemical events ultimately lead to a necrotic cell death in MM cells. Moreover, we anticipate that HYD1 will prove to be an important tool to dissect the molecular pathway of caspase independent cell death and allow for investigations for deciphering crosstalk between apoptotic and non-apoptotic cell death pathways. Finally, drug resistance is often associated with an imbalance in pro and anti-apoptotic mediators with the net result favoring cell survival. Thus, agents such as HYD1 that preferentially target alternative cell death pathways may provide an ideal strategy for developing combination therapies to eliminate sub-population of cells resistant to apoptotic cell death.