Abstract
BackgroundRegulated cell death (RCD) is a mechanism by which the cell activates its own machinery to self-destruct. RCD is important for the maintenance of tissue homeostasis and its deregulation is involved in diseases such as cervical cancer. IMMUNEPOTENT CRP (I-CRP) is a dialyzable bovine leukocyte extract that contains transfer factors and acts as an immunomodulator, and can be cytotoxic to cancer cell lines and reduce tumor burden in vivo. Although I-CRP has shown to improve or modulate immune response in inflammation, infectious diseases and cancer, its widespread use has been limited by the absence of conclusive data on the molecular mechanism of its action.MethodsIn this study we analyzed the mechanism by which I-CRP induces cytotoxicity in HeLa cells. We assessed cell viability, cell death, cell cycle, nuclear morphology and DNA integrity, caspase dependence and activity, mitochondrial membrane potential, and reactive oxygen species production.ResultsI-CRP diminishes cell viability in HeLa cells through a RCD pathway and induces cell cycle arrest in the G2/M phase. We show that the I-CRP induces caspase activation but cell death induction is independent of caspases, as observed by the use of a pan-caspase inhibitor, which blocked caspase activity but not cell death. Moreover, we show that I-CRP induces DNA alterations, loss of mitochondrial membrane potential, and production of reactive-oxygen species. Finally, pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented both ROS generation and cell death induced by I-CRP.ConclusionsOur data indicate that I-CRP treatment induced cell cycle arrest in G2/M phase, mitochondrial damage, and ROS-mediated caspase-independent cell death in HeLa cells. This work opens the way to the elucidation of a more detailed cell death pathway that could potentially work in conjunction with caspase-dependent cell death induced by classical chemotherapies.
Highlights
Regulated cell death (RCD) is a mechanism by which the cell activates its own machinery to self-destruct
IMMUNEPOTENT CRP (I-CRP) induces slight caspase activation, as determined by the detection of caspase-3 activation (A). To determine if this type of cell death was dependent on caspase activity we used the pan-caspase inhibitor QVD.oph [19] and we found that, contrary to etoposide treatment, I-CRPmediated cell death was independent of caspase-activation (Fig. 6b), the use of this pan-caspase inhibitor blocked caspase activation and etoposide-induced apoptosis but it did not inhibit the cell death induced by I-CRP (Fig. 6b)
As we found that caspases are dispensable for this type of cell death, we assessed whether the ICRP was able to induce loss of mitochondrial membrane potential and reactive-oxygen species (ROS) production, through tetramethylrhodamine ethyl ester (TMRE) and 2′,7′-dichlorofluorescin diacetate (DCFDA) staining, followed by fluorescence microscopy and flow cytometric analysis
Summary
Regulated cell death (RCD) is a mechanism by which the cell activates its own machinery to self-destruct. Regulated cell death (RCD) is a physiological mechanism by which the cell activates its own machinery to self-destruct It is important for the maintenance of tissue homeostasis and its deregulation induces diseases such as cancer. Naturalderived products have recently attained a lot of interest for their ability to modulate the signaling pathways involved in cancer proliferation or for their protective potential in radiotherapy and chemotherapy [4] Some such natural-derived products are dialyzable leukocyte extracts (DLE), which are mixtures of low molecular weight substances (
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