Roscovitine-treated HeLa cells finalize autophagy later than apoptosis by downregulating Bcl-2

Abstract

The cell cycle is tightly regulated by the family of cyclin-dependent kinases (CDKs). CDKs act as regulatory factors on serine and threonine residues by phosphorylating their substrates and cyclins. CDK‑targeting drugs have previously demonstrated promising effects as cancer therapeutics both in vitro and in vivo. Roscovitine, a purine‑derivative and specific CDK inhibitor, has been demonstrated to arrest the cell cycle and induce apoptosis in a number of different cancer cell lines, including HeLa cervical cancer cells. In the present study, roscovitine was able to decrease both the cell viability and cell survival as well as induce apoptosis in a dose‑dependent manner in HeLa cells by modulating the mitochondrial membrane potential. The decrease of anti‑apoptotic B-cell lymphoma 2 (Bcl‑2) and Bcl-2 extra large protein expression was accompanied by the increase in pro‑apoptotic Bcl-2-associated X protein and P53-upregulated modulator of apoptosis expression. The marked decrease in Bcl‑2 following exposure to roscovitine (20 µM) for 48 h prompted us to determine the autophagic regulation. The outcome revealed that roscovitine triggered Beclin‑1 downregulation and microtubule-associated light chain 3 cleavage starting from 12 h of incubation. Another biomarker of autophagy, p62, a crucial protein for autophagic vacuole formation, was diminished following 48 h. In addition, monodansyl cadaverin staining of autophagosomes also confirmed the autophagic regulation by roscovitine treatment. The expression levels of different Bcl‑2 family members determined whether apoptosis or autophagy were induced following incubation with roscovitine for different time periods. Downregulation of pro‑apoptotic Bcl‑2 family members indicated induction of apoptosis, while the downregulation of anti‑apoptotic Bcl‑2 family members rapidly induced autophagosome formation in HeLa cells.

Pan Cyclin-Dependent Kinase Inhibitors for the Treatment of Breast Cancer

“…This wide spectrum impact on the cell cycle can disrupt several signaling pathways, including JAK-STAT, 88 ER, 89 p53, 90 NF-κβ, 91 and Ras-MAPK. 92 Consequently, it can reduce the expression of several genes involved in the survival of cancer cells (e.g., XIAP, 93 Mcl-1, 94 survivin, 93 and Bcl-2 95 ) and increase Bcl-x, 96 p53, 97 and p53AIP1. 98 The pro-apoptotic effects of this CDK inhibitor have synergistic impacts when it is combined with chemotherapeutics.…”

“…92,104 Interestingly, roscovitine also enhances the autophagy process. 87,95,97 In addition to cancer, the efficacy of roscovitine as a strong CDK/cyclin E inhibitor has been confirmed in various pathologic conditions such as glomerulonephritis, viral infections, and neurodegenerative disorders. Since it can induce apoptosis in neutrophils, it is proposed that roscovitine could be used as a potential treatment for chronic inflammatory diseases.…”

Therapeutic Targeting of the General RNA Polymerase II Transcription Machinery

“…Unlike flavopiridol, which causes global down regulation of gene expression, roscovitine led to a unique gene expression profile with a smaller subset of sensitive genes [64]. Genes responsive to roscovitine are crucial for the induction of apoptosis in a variety of cancers, and include Bcl-2 [77], survivin [78], and the p53-upregulated modulator of apoptosis (PUMA) [79]. Roscovitine is thought to directly block transcription of some genes by inhibiting CDK7 and CDK9.…”