Cytotoxic activity of IMMUNEPOTENT CRP against non-small cell lung cancer cell lines

PeerJ. 2019 Sep 27:7:e7759. doi: 10.7717/peerj.7759. eCollection 2019.

Abstract

Background: IMMUNEPOTENT-CRP® (I-CRP) is a bovine dialyzable leukocyte extract containing transfer factor. It is a cost-effective, unspecific active immunotherapy that has been used in patients with non-small cell lung cancer (NSCLC) as an adjuvant to reduce the side-effects of chemotherapy and radiotherapy, and has shown cytotoxic activity in vitro on different cancer cell lines. However, its mechanism of action against lung cancer cells has not been assessed. Therefore, the objective of this work was to assess the cytotoxic mechanism of I-CRP on lung cancer cell lines.

Methods: We assessed cell viability through MTT assay on the NSCLC cell lines A549, A427, Calu-1, and INER-51 after treatment with I-CRP. To further understand the mechanisms of cell viability diminution we used fluorescence-activated cell sorting to evaluate cell death (annexin-V and propidium iodide [PI] staining), cell cycle and DNA degradation (PI staining), mitochondrial alterations (TMRE staining), and reactive oxygen species (ROS) production (DCFDA staining). Additionally, we evaluated caspase and ROS dependence of cell death by pretreating the cells with the pan-caspase inhibitor Q-VD-OPH and the antioxidant N-acetylcysteine (NAC), respectively.

Results: Our data shows that I-CRP is cytotoxic to NSCLC cell lines in a dose and time dependent manner, without substantial differences between the four cell lines tested (A549, A427, Calu-1, and INER-51). Cytotoxicity is induced through regulated cell death and cell cycle arrest induction. I-CRP-induced cell death in NSCLC cell lines is characterized by DNA degradation, mitochondrial damage, and ROS production. Moreover, cell death is independent of caspases but relies on ROS production, as it is abrogated with NAC.

Conclusion: Altogether, these results improve the knowledge about the cytotoxic activity of I-CRP on NSCLC cells, indicating that cell death, cell cycle arrest, DNA degradation and mitochondrial damage are important features, while ROS play the main role for I-CRP mediated cytotoxicity in lung cancer cells.

Keywords: Apoptosis; Cell cycle; Cell death; Mitochondrial damage; NSCLC; Non small cell lung cancer; ROS; Reactive oxygen species; Transfer factor.

Grants and funding

This work was supported by the Laboratorio de Inmunología y Virología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, in collaboration with CONACYT, ‘Red Temática de Inmunología en Cancer y Enfermedades Infecciosas’ [grant number 253053]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.