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.

IntroductionMammalian cell division, mitotic transfer of genome to new daughter cells, occurs when the cell has enough cytoplasmic content and DNA replication is finished correctly. The regulatory mechanisms during interphase (G1, S, G2 phases) control the cell division decision by the presence of various time-dependent and regularly activated molecules such as cyclin and cyclin-dependent kinases (CDKs) (Morgan et al., 1998). Each interphase phase has its own activated cyclin-CDK complexes that permit cell cycle progression during surveillance mechanisms (Arellano et al., 1997). Growth promoting signals due to growth factors change the cell cycle regulation and promote neoplastic transformation (Paulovich et al., 1997). Recently, new approaches to antineoplastic therapy are focused on cyclin-CDK complex inhibition.Thus far, 6 classes of chemical CDK inhibitors have been characterized, including purine-derived CDK inhibitors (Meijer, 1996). Olomoucine, roscovitine, and purvalanol are the most commonly investigated CDK inhibitors that cause cell cycle arrest and trigger apoptotic cell death. Roscovitine (CYC202, Seliciclib) or purvalanol induce apoptosis by causing cell cycle arrest in the G1 and G2/M phases in prostate (Ringer et al., 2010), lung (Zhang et al., 2010, multiple myeloma (Komina et al., 2011), colon, and breast cancer cell lines (Wesierska-Gadek et al., 2004;Arisan et al., 2012). Although CDK inhibitor-induced apoptotic cell death has been determined in breast cancer, the exact underlying molecular targets in cell death and survival mechanism have not been clarified yet.MAPKs are regulated by various cellular arrangements such as cell growth, differentiation, and apoptotic cell Abstract: Roscovitine and purvalanol are specific cyclin-dependent kinase (CDK) inhibitors, which induce apoptosis by triggering cell cycle arrest in various cancer cells such as colon, prostate, and breast cancer cells. Although the apoptotic action of roscovitine was clarified at the molecular level, the exact mechanism of purvalanol-induced apoptosis is still under investigation. The mitogenactivated protein kinase (MAPK) signaling cascade is activated by different inducers related to growth, proliferation, differentiation processes, or environmental stress factors. Recent reports showed that modulation of MAPKs might lead to regulation of c-Myc, which is a transcription factor for the polyamine (PA) biosynthesis enzyme, ornithine decarboxylase (ODC). PAs are amine-derived cationic molecules that play crucial roles in cell proliferation, growth, and differentiation. In this study, we investigated the potential role of the MAPK signaling cascade in the purvalanol-induced apoptosis mechanism by comparing the results of roscovitine in MCF-7 and MDA-MB-231 breast cancer cells. We found that CDK inhibitors decreased the cell viability in a dose-and time-dependent manner in MCF-7 and MDA-MB-231 cancer cells. Although both CDK inhibitors induced cell cycle arrest, which led to apoptosis by activating caspases and PARP cleavage in ...