Characterization of the Pt(II)-Induced Nucleolar Stress Response Pathway

Abstract

There are three FDA-approved platinum chemotherapeutics: cisplatin, carboplatin, and oxaliplatin. Until recently, it was thought that all three Pt(II) drugs work through the same mechanism of action, in which the Pt(II) compounds form inter- and intra-strand DNA crosslinks, leading to the activation of the DNA Damage Response (DDR) pathway. However, recent studies have demonstrated that while cisplatin and carboplatin primarily act through the DDR pathway, oxaliplatin may instead elicit its anticancer effects through disruptions to the cellular nucleolus and ribosome biogenesis inhibition. The nucleolus is a membraneless organelle found within the nucleus and primarily functions as the house of ribosome biogenesis. Any disturbances to the nucleolus or ribosome biogenesis causes a cellular response referred to as nucleolar stress. While the DDR pathway is primarily activated by DNA damage, there are a variety of different stimuli that can cause nucleolar stress induction. Examples of nucleolar stress-inducing agents include small molecule compounds Actinomycin D (ActD), BMH-21, and CX-5461, which were used in this research as comparative tools to elucidate characteristics of the Pt(II)-induced nucleolar stress response pathway. Results from these studies demonstrate that Pt(II)-induced nucleolar stress is irreversible, while stress induction by ActD, BMH-21, and CX-5461 is reversible upon drug removal. This finding indicates potential differences in mechanisms between these nucleolar stress-inducing small molecules and Pt(II) compounds. Furthermore, the cell cycle plays important roles in the nucleolus and is involved in both the nucleolar stress response and DDR pathways. Checkpoint kinase 1 (Chk1) is a regulatory protein involved in various cellular pathways including the cell cycle. Inhibiting Chk1 causes cell cycle arrest in the G1 phase and prevents activation of the DDR pathway, which primarily occurs in the S phase of the cell cycle. Observing the effect of platinum chemotherapeutic treatments with a Chk1 inhibition provides further insight into the relationship between cell cycle progression, DDR and nucleolar stress. Results from these studies indicate an increase in nucleolar stress induction with cisplatin treatment when Chk1 is inhibited and cells are arrested in the G1 phase. This is not observed with cisplatin treatment under regular cell cycle progression and indicates that the Pt(II)-induced nucleolar stress response pathway may be cell cycle dependent and involve Chk1 and G1 cell cycle arrest. Together, this research contributes to the further characterization of the Pt(II)-induced nucleolar stress response pathway, but future works are required to fully understand this unique pathway and the mechanisms of platinum chemotherapeutics.