The oncolytic peptide LTX-315 kills cancer cells through Bax/Bak-regulated mitochondrial membrane permeabilization

Abstract

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes.

The oncolytic peptide LTX-315 overcomes resistance of cancers to immunotherapy with CTLA4 checkpoint blockade

“…[19][20][21][22][23][24] The chemical modifications of bovine lactoferricin derivatives allowed for the development of a lead compound, LTX-315 with a shorter chemically modified peptide length and optimal (selective) anticancer properties. [25][26][27][28][29] By creating pores and disintegrating the cytoplasmic membranes, as well as targeting the mitochondria, LTX-315 promotes the release of damage-associated molecular patterns (DAMPs) associated with immunogenic cell death (ICD). 26,27,29 Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce classical 'apoptosis' associated with apoptotic nuclear condensation and caspase-3-dependent cell death.…”

“…[25][26][27][28][29] By creating pores and disintegrating the cytoplasmic membranes, as well as targeting the mitochondria, LTX-315 promotes the release of damage-associated molecular patterns (DAMPs) associated with immunogenic cell death (ICD). 26,27,29 Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce classical 'apoptosis' associated with apoptotic nuclear condensation and caspase-3-dependent cell death. Rather, the oncolytic peptide-mediated cell death exhibited a necrotic phenotype that was not regulated in as much as neither necrostatin-1 nor cyclosporin A compromised cell death.…”

The oncolytic compound LTX-401 targets the Golgi apparatus

“…LTX-401 was preferentially enriched in the cytosolic and Golgi fractions, not in mitochondria (Figure 3c). This strongly contrasts with LTX-315 that preferentially enriches in the mitochondrial fraction, 8 not in the Golgi (Figure 3d). Altogether these results suggest that LTX-401 may disrupt the Golgi via a direct effect, upon reaching high local concentrations.…”

“…Thus, BFA can reduce the LTX-401-induced signs of mitochondrial membrane permeabilization (such as the release of the intermembrane proteins cytochrome c and SMAC), and DKO of the two pro-apoptotic multidomain proteins BAX and BAK (which both supposedly act on mitochondria) reduces LTX-401-induced killing. Moreover, mitophagy-enforced removal of mitochondria from the cells 8,14 reduces their susceptibility to LTX-401-mediated killing, yet does not reduce LTX-401-induced disruption of the Golgi. This observation again pleads in favor of the interpretation that mitochondria are secondary targets of LTX-401, downstream of its action on the Golgi.…”

“…6 LTX-315 has the capacity of causing a necrotic cell death (i.e., cell death without caspase activation and without morphological features of apoptosis such as nuclear fragmentation) that is partially relying on mitochondrial membrane permeabilization by this oncolytic peptide, 4 which actually redistributes specifically towards the mitochondrial compartment. 8 LTX-315 has also been found to stimulate a series of alterations in cancer cells that give rise to immunogenic cell death (ICD), that is, a type of cell death that elicits a potent anticancer immune response. 9 Accordingly, LTX-315 triggers the exposure or release of several danger-associated molecular patterns (DAMPs) associated with ICD, namely the translocation of calreticulin (CALR) from the lumen of the endoplasmic reticulum to the cell surface; the release of ATP from cytoplasmic storage sites into the extracellular space; the exodus of high mobility group box 1 (HMGB1) from the nucleus to the extracellular milieu; and a type-1 interferon response.…”