Mechanisms of Cytotoxicity and Resistance by Membrane Interacting Peptide Amphiphiles and Ivermectin Aggregates in Human Cervical Cancer Cells

Abstract

Peptide amphiphiles (PAs) are emerging as versatile biomaterials capable of self-assembling into nanostructures with diverse biological applications. Previous studies have demonstrated that several types of PA can interact with and disrupt the integrity of the plasma membrane, leading to the assumption that the cytotoxicity of these materials is chiefly through accidental cell death (ACD). It is typically harder for cells to attain resistance to cytotoxins that stimulate ACD, making Pas an attractive alternative to the targeted destruction of cancer cells and microbes. In this study, we examined the mechanism of cytotoxicity of a weak beta-sheet forming cationic PA in cervical cancer cells using phase contrast and fluorescent live-cell imaging microscopy and conventional cell viability assays. Rather than causing direct plasma membrane disruption and ACD, we found that this PA-induced cell death in cervical cancer cells was more consistent with a regulated cell death (RCD) pathway that correlated with endocytosis and compromised mitochondrial function. Further investigation revealed that the PA evoked cytosolic Ca2+ oscillations, which are known to influence mitochondrial function and RCD. Surprisingly, suppressing PA-evoked Ca2+ elevations through extracellular chelation with EGTA increased rather than decreased PA cytotoxicity, whereas raising the extracellular Ca2+ concentration rendered the cells less sensitive to the PA. This suggests that Ca2+ elevations provide a moderate cytoprotective effect. Confocal Dynasore played a role in PA-induced cell death. Confocal imaging strongly suggested that PA undergoes internalization into cells, and so we conducted preliminary experiments to see if inhibition of dynamin-dependent endocytosis affected PA cytotoxicity. The dynamin-inhibiting drug Dynasor did significantly reduce PA-induced cell death. These findings shed light on an alternative mechanism by which PAs can induce cell death, suggesting a role for RCD pathways in addition to ACD. Furthermore, they likely explain our observation that a small population of cells were able to obtain significant resistance to PA cytotoxicity, which is not typically observed for ACD-inducing toxins. We next explored resistance development in cervical cancer cells that were exposed to sublethal concentrations of PA for prolonged periods. Interestingly, time-lapse imaging of these resistant cells revealed notable morphological changes, where they transitioned to an elongated, spindle-shaped phenotype, coupled with alterations in cell motility patterns. Furthermore, functional assays using the EdU incorporation assay revealed a significant increase in EdU uptake in the resistant cell population, indicating a compensatory upregulation in cell division. These observations suggest a dynamic cellular response aimed at counteracting the cytotoxic effects of the PA. It is essential to understand the mechanisms underlying the cytotoxicity and development of resistance to PA-induced cytotoxicity in order to devise effective anticancer and antimicrobial therapeutics. By unraveling the cellular adaptations that confer resistance, we can identify novel targets and strategies to overcome resistance mechanisms and enhance the efficacy of PA-based therapies for cervical cancer.

It is interesting to note that our investigation goes beyond peptide amphiphiles and explores the effects of the anthelmintic drug ivermectin on cervical cancer cells. An old drug, ivermectin, has gained much recent attention due to the possibility of it being repurposed as an anticancer and antiviral therapy. However, although ivermectin has been reported to have anticancer and antiviral properties in vitro, the effective concentrations employed are close to or exceed the aqueous solubility limit for this hydrophobic drug. Based on this, we recently made an important observation: ivermectin precipitates in both serum-supplemented and serum-free culture media at concentrations typically employed in in vitro cytotoxicity experiments. Furthermore, the cytotoxic effects of ivermectin precipitation were remarkably mitigated upon filtration of particles greater than 0.2 μm in diameter, highlighting the role of insoluble precipitates in mediating cellular toxicity. Pre-treatment of cells with a filtered ivermectin solution did show a modest growth suppressant effect, but only at concentrations far higher than known to be attainable in vivo. These findings emphasize the critical importance of considering the solubility limits of hydrophobic drugs when assessing their cytotoxicity in vitro. As the search for novel anticancer agents intensifies, our data emphasize the necessity of understanding drug solubility and precipitation kinetics to guide drug development and screening processes.