CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition

Abstract

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is known to play a pivotal role in the regulation of tumor progression and the development of resistance to anti-cancer therapies. This study aims to investigate the anti-tumor properties and underlying mechanisms of action of CCT128930, a novel small-molecule inhibitor specifically targeting Akt, in hepatocellular carcinoma cells, particularly the HepG2 cell line.

Our findings reveal that at lower concentrations, CCT128930 paradoxically promotes Akt phosphorylation in both HepG2 and A549 cells rather than suppressing it. Despite this unexpected effect, CCT128930 significantly inhibits cell proliferation by inducing a G1 phase cell cycle arrest. This arrest is associated with a reduction in the expression of cyclin D1 and the cell cycle phosphatase Cdc25A, along with increased levels of the cell cycle inhibitors p21 and p27 and the tumor suppressor protein p53.

At higher concentrations (20 μM), CCT128930 induces apoptotic cell death, as evidenced by the activation of key apoptotic markers including caspase-3, caspase-9, and the cleavage of PARP. Moreover, treatment with CCT128930 leads to enhanced phosphorylation of the MAPK pathway members ERK and JNK in HepG2 cells, suggesting their involvement in the drug response.

Importantly, CCT128930 also activates a DNA damage response, as shown by the phosphorylation of the histone variant H2AX (γ-H2AX), ATM (ataxia-telangiectasia mutated), Chk1, and Chk2 proteins. In addition to apoptosis, CCT128930 induces autophagy, demonstrated by elevated levels of autophagy markers LC3-II and Beclin-1. Interestingly, the pharmacological inhibition of autophagy using chloroquine intensifies CCT128930-induced apoptosis and further enhances γ-H2AX phosphorylation, indicating that autophagy may serve a cytoprotective role in this context.

Collectively, this study enhances our understanding of the multifaceted anti-cancer mechanisms of CCT128930. It demonstrates that this Akt inhibitor exerts its anti-tumor effects through a combination of cell cycle arrest, apoptosis, DNA damage response, and autophagy modulation, highlighting its potential as a therapeutic agent in cancer treatment.

Keywords: CCT128930; Akt; Cell cycle; DNA damage; Autophagy.

Introduction

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling cascade is recognized as a critical regulatory pathway that governs fundamental cellular processes such as cell growth, programmed cell death (apoptosis), and cellular differentiation. This pathway is often disrupted in a wide array of human malignancies due to mutations or genetic alterations affecting its various components. A pivotal function of this pathway is carried out through the activation of the serine/threonine kinase Akt, also referred to as protein kinase B (PKB), which is stimulated upon PI3K activation. Akt facilitates cell cycle progression by inhibiting the transcription of cell cycle inhibitors mediated by FOXO transcription factors. This suppression leads to the accumulation and activation of the mTOR-raptor kinase complex, which subsequently activates p70S6K1, a kinase involved in promoting protein synthesis and cell proliferation.

mTOR acts as a central node within this intricate signaling network, coordinating several cellular responses including proliferation, survival, and autophagy. Autophagy, a conserved cellular mechanism that enables the degradation and recycling of cytoplasmic components through the lysosomal pathway, plays a vital role in maintaining cellular homeostasis, especially under conditions of metabolic or environmental stress. By facilitating the removal of damaged organelles and misfolded proteins, autophagy allows cells to adapt and survive various forms of stress, highlighting its importance in cellular health and disease.

Extensive research has demonstrated that DNA damage or replication errors can lead to the suppression of cyclin-dependent kinases (Cdks) through the activation of cell cycle checkpoints, which act as critical control mechanisms to prevent mitotic entry when genomic integrity is compromised. Among the central players in the DNA damage response are the ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) protein kinases. These kinases become activated in response to genotoxic insults such as ionizing radiation, ultraviolet light, or replication stress. Once activated, ATM and ATR initiate downstream signaling cascades by phosphorylating checkpoint kinases Chk1 and Chk2, which in turn inactivate the phosphatase Cdc25. This inactivation halts cell cycle progression, allowing time for DNA repair. Additionally, both Chk1 and Chk2 contribute to the activation of the tumor suppressor protein p53 via phosphorylation, linking these checkpoint responses to G1 phase arrest as well.

Autophagy itself is a tightly regulated degradation pathway that relies on the formation of autophagosomes and their fusion with lysosomes. This process maintains a delicate balance between the synthesis and breakdown of cellular components and contributes to the adaptation of cells under stress. In cancer biology, autophagy exhibits a dual role: on one hand, it supports tumor cell survival by mitigating stress and nutrient deprivation, while on the other hand, excessive or dysregulated autophagy can lead to a form of programmed cell death distinct from apoptosis. Interestingly, chemotherapy has been observed to elicit this paradoxical role of autophagy, where it may either protect tumor cells or promote their demise. Some studies suggest that inhibitors targeting the PI3K/Akt/mTOR axis can induce autophagy as a survival mechanism, which in turn may reduce the overall efficacy of these therapeutic agents. This raises the possibility that simultaneous inhibition of autophagy might enhance the anti-cancer potency of PI3K/Akt/mTOR pathway inhibitors.

Given the frequent aberrations in PI3K/Akt/mTOR signaling observed in cancer, targeting this pathway has emerged as a compelling strategy in oncology. Several inhibitors are currently under development and are being evaluated in preclinical and clinical settings. In the present study, we explore the anti-cancer potential of the compound CCT128930 using in vitro cancer models. Our findings reveal that CCT128930 effectively suppresses cell proliferation primarily through the induction of G1 phase cell cycle arrest. At elevated concentrations, the compound also induces apoptotic cell death. Furthermore, our data suggest that CCT128930 provokes a DNA damage response, as evidenced by the activation of γ-H2AX, ATM, Chk1, and Chk2 proteins. In addition, we observed that treatment with CCT128930 leads to the induction of autophagy, marked by increased levels of the autophagic protein LC3-II. Interestingly, when autophagy is pharmacologically inhibited using chloroquine (CQ), the cytotoxic effects of CCT128930 are significantly enhanced. These results indicate that autophagy may act as a resistance mechanism to CCT128930 treatment, and its inhibition may improve therapeutic outcomes. Collectively, our findings support the potential of CCT128930 as a promising anti-cancer agent.

Materials And Methods

Reagents

CCT128930 was obtained from Santa Cruz Biotechnology (Shanghai, China) and was dissolved in dimethyl sulfoxide (DMSO) before use. Several other compounds and reagents were used in this study. Crystal violet, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and chloroquine were purchased from Sigma-Aldrich (St. Louis, USA). PD98059 was acquired from Promega (Madison, USA), while SP600129 was purchased from Calbiochem (California, USA). For detecting apoptosis, the Annexin V-FITC apoptosis detection kit was obtained from KeyGEN Biotech (Nanjing, China). Enhanced chemiluminescence (ECL) reagents for Western blotting were sourced from PerkinElmer (Massachusetts, USA).

Primary antibodies used in the study included those specific for β-actin, Beclin-1, and LC3-II, which were purchased from Sigma-Aldrich. Additional rabbit polyclonal antibodies against key signaling and apoptotic proteins such as total and phosphorylated Akt, p70S6K, caspase-9, caspase-3, PARP, ATM, phospho-ATM, ATR, phospho-ATR, phospho-Chk2, Chk1, phospho-Chk1, γ-H2AX, phospho-p53, phospho-p38, JNK, and Cdc2 were obtained from Cell Signaling Technology (Shanghai, China). Mouse monoclonal antibodies for Chk2, p21, p27, and phospho-JNK were purchased from BD Biosciences. Antibodies targeting cyclin D1, Cdc25A, Cdc25B, Cdc25C, cyclin B1, p53, ERK, and phospho-ERK were acquired from Santa Cruz Biotechnology.

Cells And Cell Culture

Human liver cancer HepG2 cells and lung carcinoma A549 cells were procured from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in RPMI 1640 medium, which was supplemented with 10% fetal calf serum (Gibco, USA), 100 units per milliliter of penicillin, and 100 micrograms per milliliter of streptomycin. All cultures were incubated under standard conditions at 37 degrees Celsius in a humidified atmosphere containing 5% carbon dioxide.

Cell Viability And Colony Formation Assays

To assess cell viability, an MTT assay was performed following a standard procedure. Briefly, cells were seeded in 96-well plates and allowed to adhere for 24 hours. Subsequently, they were exposed to different concentrations of CCT128930. After treatment, 20 microliters of MTT solution (at a concentration of 5 mg/mL) was added to each well and incubated for an additional four hours. Following this, the medium was removed, and the resulting formazan crystals were dissolved in 150 microliters of DMSO. Absorbance was measured at 490 nm to determine cell viability.

For evaluating long-term effects on cellular proliferation, a colony formation assay was conducted. HepG2 cells were plated at a density of 1500 cells per well in six-well plates and treated with varying concentrations of CCT128930 for a period of two weeks. After the incubation period, colonies were fixed with 1% glutaraldehyde and stained using 0.5% crystal violet. Only those colonies consisting of more than 30 cells were counted using an inverted microscope.

Cell Cycle Analysis

To analyze cell cycle distribution, cells were treated with CCT128930 for 24 hours. At the end of the treatment, the cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and fixed in 70% ethanol at 4 degrees Celsius overnight. After fixation, cells were washed again with PBS, treated with RNase A to eliminate RNA contamination, and stained with propidium iodide (PI) to label DNA. The DNA content was then analyzed using flow cytometry (FACSCalibur, Becton Dickinson, USA) and evaluated with CellQuest software.

Apoptosis Detection Using Annexin V-FITC/PI Staining

To determine the extent of apoptosis, both adherent and floating cells were collected and subjected to staining using the Annexin V-FITC/PI apoptosis detection kit, following the manufacturer’s instructions. The stained cells were analyzed by flow cytometry using the FACScan system in conjunction with CellQuest software (Becton Dickinson), allowing quantification of apoptotic populations.

Western Blot Analysis

To examine the protein expression and post-translational modifications induced by CCT128930, western blot analysis was performed following standard procedures. Cells were first lysed using a cold lysis buffer containing 150 mM sodium chloride, 20 mM Tris-HCl, 1% NP-40, 0.5% sodium deoxycholate (Na-DOC), 0.1% sodium dodecyl sulfate (SDS), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and a cocktail of protease inhibitors. The lysis was carried out on ice for 20 minutes to ensure minimal protein degradation. Cell lysates were clarified by centrifugation, and protein concentrations were quantified. For each sample, 30 micrograms of total protein was loaded onto polyacrylamide gels for electrophoresis. Following separation, proteins were transferred onto membranes, and specific target proteins were detected using enhanced chemiluminescence (ECL) detection reagents.

Immunofluorescence Assay

To further assess molecular responses at the cellular level, an immunofluorescence assay was employed. Cultured cells were initially fixed with ice-cold methanol for 10 minutes at 4 degrees Celsius to preserve cellular structures. Permeabilization was carried out using phosphate-buffered saline (PBS) containing 0.1% Triton X-100 to allow antibody access to intracellular proteins. Following permeabilization, cells were blocked with 3% bovine serum albumin (BSA) to reduce non-specific antibody binding. They were then incubated with a primary antibody specific to γ-H2AX, a marker for DNA damage. After thorough washing, cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:200 dilution, Santa Cruz Biotechnology). To visualize nuclei, cells were counterstained with DAPI. Fluorescence images were captured using a fluorescence microscope equipped with the appropriate filters, allowing for the observation of subcellular protein localization and DNA damage foci.

Statistical Analysis

All experimental data are presented as the mean value ± standard deviation (SD). For comparisons between experimental groups, statistical significance was determined using Student’s t-test. A p-value of less than 0.05 was considered to indicate a statistically significant difference between groups.

Results

CCT128930 Inhibits Cell Proliferation in a Concentration-Dependent Manner

CCT128930, previously identified as a dual inhibitor of the PI3K and mTOR signaling pathways, was evaluated for its effects on phosphorylation events associated with pathway activity. Specifically, we assessed its impact on the phosphorylation status of Akt at serine 473 and p70S6 kinase at threonine 389 in HepG2 and A549 cancer cell lines. Surprisingly, treatment with CCT128930 did not result in decreased phosphorylation of either protein. In fact, at lower concentrations, CCT128930 unexpectedly led to increased phosphorylation of Akt, suggesting a possible compensatory activation or feedback mechanism within the signaling network.

To assess the functional consequences of CCT128930 treatment on cancer cell growth, MTT assays were performed on HepG2 and A549 cells. The results clearly demonstrated a dose-dependent reduction in cell viability, with half-maximal inhibitory concentrations (IC50) determined to be approximately 30.03 µM for HepG2 cells and 32.94 µM for A549 cells. Further, a colony formation assay confirmed the compound’s long-term anti-proliferative effect. Exposure to 10 µM of CCT128930 completely suppressed the ability of HepG2 cells to form colonies, underscoring its potential as a potent antiproliferative agent. Microscopic examination of treated HepG2 cells revealed significant morphological alterations, including reduced cell size and a more compact, rounded shape. These changes are indicative of cellular stress responses and may suggest the initiation of apoptosis or growth arrest mechanisms.

CCT128930 Induces G1 Phase Arrest in HepG2 and A549 Cells

To explore whether the observed growth inhibition was related to cell cycle disruption, we conducted flow cytometric analyses to determine the cell cycle distribution following CCT128930 treatment. The data revealed a notable accumulation of cells in the G1 phase, with a corresponding decrease in the proportion of cells in S and G2/M phases in both HepG2 and A549 cells. This shift suggests that CCT128930 causes a blockade at the G1 checkpoint, effectively halting cell cycle progression.

To further elucidate the molecular mechanisms underlying G1 arrest, we examined the expression of key regulatory proteins. Western blot analysis showed that treatment with CCT128930 led to an increase in the levels of p21 and p27, two cyclin-dependent kinase inhibitors that function to suppress cell cycle progression. Additionally, an upregulation of p53, a well-known tumor suppressor and regulator of p21, was observed. These findings collectively support the conclusion that CCT128930 mediates its antiproliferative effects, at least in part, by enforcing a G1 phase cell cycle arrest through the activation of p53-dependent and -independent pathways.

High Doses of CCT128930 Induce Apoptosis in HepG2 Cells

In order to determine whether the reduction in cell viability was associated with the induction of apoptosis, we evaluated the apoptotic response in HepG2 cells exposed to increasing concentrations of CCT128930 for 24 hours. Using Annexin V-FITC and propidium iodide (PI) staining followed by flow cytometric analysis, we observed a significant increase in apoptotic cells upon treatment with 20 µM of the compound. The percentage of apoptotic cells rose from 5.33% in the control group to 12.62% in the treated group, indicating a clear dose-dependent induction of programmed cell death.

To confirm the activation of apoptotic pathways, we examined the cleavage of key apoptotic markers by western blotting. Our analysis revealed that treatment with 20 µM of CCT128930 led to the proteolytic cleavage of pro-caspase-3 and pro-caspase-9, two essential components of the intrinsic apoptosis pathway. Moreover, we detected the cleavage of poly (ADP-ribose) polymerase (PARP), a substrate of activated caspase-3 and a hallmark of apoptosis. These findings demonstrate that high concentrations of CCT128930 not only suppress proliferation but also actively induce apoptosis in HepG2 cells through caspase-mediated mechanisms.

ERK And JNK Are Required For CCT128930-Induced Cytotoxicity In HepG2 Cells

The mitogen-activated protein kinase (MAPK) signaling pathway is known for its critical involvement in regulating a range of cellular responses, including the inhibition of cell proliferation and the promotion of apoptosis, particularly in reaction to diverse extracellular stressors. Based on this understanding, an investigation was carried out to determine whether the MAPK family members—specifically ERK, JNK, and p38—exhibit increased activity in HepG2 liver cancer cells upon exposure to the compound CCT128930.

To explore this, western blotting analyses were performed to monitor the phosphorylation status of these kinases following treatment. The results showed a clear elevation in the phosphorylation levels of ERK and JNK proteins after 24 hours of exposure to CCT128930, indicating their activation. In contrast, no notable changes were observed in the phosphorylation state of p38 MAPK, suggesting that p38 may not be significantly involved in the cellular response to this compound.

To further determine whether the observed reduction in cell viability following CCT128930 treatment is dependent on the activation of ERK and JNK pathways, HepG2 cells were co-treated with the compound and specific inhibitors targeting these kinases. The inhibitors used were PD98059, which suppresses ERK activation, and SP600125, which targets JNK1/2. After 24 hours, cell viability was assessed using an MTT assay. The results revealed that the inhibitory effects of CCT128930 on cell viability were noticeably reduced in the presence of either inhibitor. This partial reversal of cytotoxicity suggests that the ERK and JNK signaling pathways play a contributory role in mediating the anti-proliferative and pro-apoptotic effects of CCT128930 in HepG2 cells.

CCT128930 Activates DNA Damage Sensor Kinases And Engages Cellular Checkpoints In HepG2 Cells

To gain deeper insights into the mechanisms underlying the cell cycle arrest and apoptosis induced by CCT128930, the study examined its potential to cause DNA damage in HepG2 cells. A widely recognized marker of DNA damage is the phosphorylation of histone H2AX at serine 139 (referred to as γ-H2AX). Western blotting and immunofluorescence analyses demonstrated a significant increase in γ-H2AX levels following treatment with CCT128930, indicating the presence of DNA double-strand breaks and the activation of a DNA damage response.

Since the DNA damage response is orchestrated primarily by the kinases ATM and ATR, the effects of CCT128930 on these proteins were evaluated. The treatment resulted in a marked increase in the phosphorylation of ATM at serine 1981, which is indicative of its activation, while no significant changes were observed in the phosphorylation status of ATR at serine 428. This suggests a predominant involvement of the ATM pathway in response to CCT128930-induced DNA damage.

Moreover, because checkpoint kinases Chk1 and Chk2 are direct downstream targets of ATM and ATR and serve as critical regulators of cell cycle progression and apoptosis in response to genotoxic stress, their activation status was also assessed. The analysis revealed that both Chk1 and Chk2 underwent increased phosphorylation following exposure to CCT128930, further supporting the notion that this compound activates the ATM-Chk1/Chk2 axis, thereby enforcing cell cycle checkpoints and promoting apoptotic pathways in HepG2 cells.

Inhibition Of Autophagy Exacerbates The Cytotoxic Activity Of CCT128930

Autophagy is a fundamental cellular process that enables the degradation and recycling of intracellular components, thereby maintaining cellular homeostasis. To determine whether CCT128930 induces autophagy in HepG2 cells, researchers evaluated the formation of autophagosomes by monitoring the levels and distribution of LC3, a key autophagy marker. Cells were transfected with a tandem fluorescent-tagged LC3 construct (RFP-GFP-LC3) and subsequently treated with CCT128930. After 24 hours, a clear shift from diffuse to punctate LC3 localization was observed, indicating autophagosome formation. Additionally, western blot analysis confirmed an increase in LC3-II levels, the lipidated form of LC3 associated with autophagosome membranes. The expression of Beclin1, another critical regulator of autophagy, was also found to be elevated, further confirming the induction of autophagy in response to CCT128930.

To discern whether autophagy plays a protective or detrimental role in CCT128930-treated cells, autophagy was pharmacologically inhibited using chloroquine (CQ), a lysosomal protease inhibitor that blocks the final steps of autophagic degradation. The presence of CQ significantly increased the levels of apoptosis in cells exposed to CCT128930, as evidenced by enhanced activation and cleavage of apoptosis-related proteins such as caspase-3 and PARP. These findings suggest that autophagy acts as a cellular survival mechanism in this context, and its inhibition shifts the balance toward apoptotic cell death. Therefore, blocking autophagy enhances the cytotoxic effects of CCT128930, highlighting a potential therapeutic strategy for increasing its efficacy.

Inhibition Of Autophagy Enhances The CCT128930-Induced Phosphorylation Of H2AX

To explore the interplay between autophagy and DNA damage responses in the presence of CCT128930, the study investigated whether autophagy inhibition affects the extent of DNA damage induced by this compound. Specifically, researchers assessed the phosphorylation levels of H2AX in cells co-treated with CCT128930 and CQ. The results showed that inhibiting autophagy led to a marked increase in γ-H2AX levels compared to treatment with CCT128930 alone. This indicates that autophagy may mitigate DNA damage, acting as a protective buffer against genotoxic stress. Conversely, its inhibition amplifies the DNA damage response, potentially compounding the stress inflicted by CCT128930 and further promoting apoptotic pathways.

Collectively, these findings provide compelling evidence that CCT128930 exerts its anticancer effects in HepG2 cells through a multifaceted mechanism involving activation of MAPK signaling, induction of DNA damage and checkpoint responses, and stimulation of autophagy. Moreover, autophagy appears to function as a survival response that counteracts the cytotoxic effects of CCT128930, and its inhibition may offer a promising avenue to enhance the therapeutic potential of this compound in liver cancer treatment.

Discussion

The unsatisfactory outcomes associated with conventional chemotherapy in hepatocellular carcinoma (HCC), contrasted with the clinical success of sorafenib, have galvanized efforts within the research community to identify novel molecular targets that could improve therapeutic options for HCC patients. Within this context, CCT128930 emerges as a promising candidate. This compound is a newly developed ATP-competitive inhibitor of Akt, discovered through fragment-based in silico screening combined with structure-guided drug design. Its molecular specificity is notable, as it selectively inhibits Akt over protein kinase A (PKA) by exploiting a subtle but critical single amino acid difference between these kinases. Prior research has demonstrated the antitumor efficacy of CCT128930 in human breast cancer xenograft models, specifically in U87MG and BT474 cell lines. Building upon these findings, the present study elucidates the mechanisms by which CCT128930 suppresses cell cycle progression, induces DNA damage, and triggers autophagy in the human hepatoma cell line HepG2.

A hallmark of cancer development is the abnormal and uncontrolled proliferation of tumor cells. Therefore, targeting and inhibiting proliferative signals represent a cornerstone of effective anticancer therapy. In this study, it was observed that treatment of HepG2 cells with CCT128930 led to a pronounced accumulation of cells in the G1 phase of the cell cycle, indicating a cell cycle arrest at this checkpoint. At the molecular level, this was accompanied by a reduction in the expression of cyclin D1 and Cdc25A, both essential for progression through the G1/S transition. Concurrently, levels of the cyclin-dependent kinase inhibitors p21 and p27, as well as the tumor suppressor protein p53, were increased, further reinforcing the blockade of cell cycle progression. Beyond its anti-proliferative effects, high concentrations of CCT128930 were also capable of inducing apoptosis in HepG2 cells. This apoptotic induction was confirmed by significant upregulation of cleaved caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP), key executors and markers of programmed cell death, after 24 hours of treatment.

One biochemical signature often associated with apoptotic cell death is the generation of DNA double-strand breaks (DSBs), which result in oligonucleosomal fragmentation of DNA. Importantly, outside the apoptotic context, DSBs rapidly activate highly conserved DNA damage response (DDR) pathways aimed at repairing the damage and preserving genomic integrity. The biological purpose of the DDR is to safeguard cells from genomic instability; however, if the damage is irreparable, DDR signaling leads cells to undergo apoptosis. Central to the DDR are sensor kinases such as ATM, ATR, and DNA-dependent protein kinase (DNA-PK), which phosphorylate downstream checkpoint proteins including Chk1 and Chk2. In this study, the molecular effects of CCT128930 on DDR activation were examined to better understand the mechanisms driving cell cycle arrest.

A notable chromatin modification during the DDR is the phosphorylation of histone H2AX at serine 139, commonly known as γ-H2AX, which serves as a critical marker for DNA damage and facilitates the recruitment of DNA repair machinery to the sites of DSBs. Following CCT128930 exposure, a marked increase in γ-H2AX phosphorylation and formation of nuclear γ-H2AX foci was detected in HepG2 cells, indicating extensive DNA damage. This was accompanied by enhanced phosphorylation of ATM at Ser-1981, a modification known to activate ATM kinase activity. In contrast, phosphorylation levels of ATR remained unchanged, suggesting that ATM, rather than ATR, is primarily involved in mediating the DNA damage response to CCT128930. Consistent with ATM activation, phosphorylation of downstream checkpoint kinases Chk1 and Chk2, as well as the tumor suppressor p53, were elevated, corroborating the activation of DDR signaling and enforcement of cell cycle checkpoints following treatment.

Recent studies have increasingly recognized the dual role of autophagy in cellular responses to DNA damage, complementing the extensively studied apoptosis pathways. Autophagy is a highly conserved catabolic process that helps maintain cellular homeostasis by degrading and recycling intracellular components, especially under stress conditions such as nutrient deprivation, growth factor withdrawal, and oxidative stress. A hallmark of autophagy activation is the conversion of the microtubule-associated protein LC3 from its cytosolic form (LC3-I) to a lipidated, autophagosome-associated form (LC3-II). In this study, treatment with CCT128930 induced autophagy in HepG2 cells, as evidenced by elevated levels of LC3-II. This finding raises the intriguing question of whether autophagy serves a protective or detrimental role in the context of CCT128930 treatment. To investigate this, the autophagy inhibitor chloroquine (CQ), which impedes lysosomal degradation, was employed. The combination of CCT128930 and CQ significantly enhanced apoptotic cell death compared to CCT128930 alone, indicating that autophagy functions as a survival mechanism in HepG2 cells under CCT128930-induced stress. Thus, targeting autophagy pathways may potentiate the pro-apoptotic and anticancer efficacy of CCT128930.

In summary, the findings of this study suggest that CCT128930 effectively inhibits the growth of cancer cells by inducing cell cycle arrest, triggering DNA damage responses, and promoting autophagy in a dose-dependent manner. Treatment with CCT128930 also stimulates phosphorylation of ERK1/2 and JNK1/2, indicating activation of these MAPK pathways. Furthermore, blocking autophagy with the lysosomal inhibitor CQ enhances the apoptosis-inducing and anticancer activities of CCT128930 in HepG2 cells. These results highlight the therapeutic potential of combining CCT128930 with autophagy inhibitors as a strategy for improving treatment outcomes in hepatocellular carcinoma.