CS-6-Induced p62 Accumulation Exacerbates DNA Damage: A Mechanistic Insight into Colorectal Cancer Suppression

doi:10.21203/rs.3.rs-3548430/v1

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CS-6-Induced p62 Accumulation Exacerbates DNA Damage: A Mechanistic Insight into Colorectal Cancer Suppression

https://doi.org/10.21203/rs.3.rs-3548430/v1

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Gamabufotalin (CS-6), a bufadienolide derived from Chansu, its role in colorectal cancer (CRC) remains unexplored. CS-6 treatment significantly inhibited CRC SW620 and DLD1 cell viability and colony formation in vitro. Furthermore, CS-6 treatment-induced DNA damage and cell cycle arrest in SW620 and DLD1 cells. Proteomics analysis and the western blot assay revealed that CS-6 treatment upregulated p62 expression, an autophagic substrate involved in the regulation of DNA double-strand damage repair. Knockdown of p62 in this study significantly alleviated CS-6-induced DNA damage and the downregulation of cyclin expression in SW620 and DLD1 cells. Additionally, the results indicated increased expression of microtubule-associated protein 1A/1B light chain 3B (LC3B) and reduced binding between B-cell lymphoma-2 (Bcl2) and beclin-1, suggesting that CS-6 treatment activated early-stage autophagy in CRC cells. However, inhibition of late-stage autophagy and autophagy-related protein 5 (ATG5) with chloroquine and si-ATG5, respectively, further indicated that CS-6-induced autophagy defects led to p62 accumulation, exacerbated cell proliferation inhibition, and aggravated DNA damage. In conclusion, this study suggests that CS-6 may exert its anti-tumor effects in CRC by inducing autophagy defects, resulting in p62 accumulation and DNA damage.

Gamabufotalin (CS-6)

Colorectal cancer (CRC)

DNA damage

p62

Autophagy

Colorectal cancer (CRC) ranks as the most prevalent gastrointestinal cancer, characterized by a high mortality rate and poor prognosis[1, 2]. Common treatments for CRC encompass surgery, radiation therapy, targeted therapy, and chemotherapy[3]. Despite recent advancements in treating primary and metastatic CRC, the projected prognosis and mortality rates for 2023 do not indicate significant improvements in cure rates or the long-term survival of CRC patients[4]. Furthermore, chemotherapy and radiotherapy medications can lead to various adverse side effects, including nervous system abnormalities[5], gastrointestinal reactions[6], and myelosuppression[7], negatively impacting the quality of life of patients. An urgent requirement exists for the development of non-toxic, safe, and efficacious alternative drugs to treat colorectal cancer (CRC).

Long-lasting mutations occurring during DNA replication can cause DNA damage, influencing the genetic makeup of an individual[8]. Additionally, the failure to promptly repair DNA damage can result in cell death[9]. Research has shown that uncontrolled mutations and disruptions in genomic stability may lead to tumor heterogeneity and polymorphism, potentially activating anti-tumor immune responses[10]. This includes the mutation of key genes within tumor cells, stalling of the replication fork, and the induction of autophagy and apoptosis[11]. Autophagy is a highly conserved physiological process in eukaryotic cells. It involves the engulfing of faulty proteins, damaged organelles, or other cytoplasmic components by a bilayer autophagosome, which subsequently combines with a lysosome to form an autophagolysosome, resulting in the degradation of its contents. However, excessive autophagy can lead to cell apoptosis and aging, ultimately inhibiting tumor formation[12]. P62, a stress-responsive protein, exerts a regulatory role in several pathways linked to cancer development[13]. Moreover, p62 serves as a key regulator in several cancer types, such as breast cancer[14] and liver cancer[15], playing a pivotal role in the pathogenesis of various diseases. Furthermore, p62 exhibits dynamic associations with various pathways in case of severe DNA damage, associating with several DNA damage repair pathways, including homologous recombination repair, non-homologous recombination repair, and DNA damage response (DDR) pathways, activate autophagy to prevent tumor growth[16–18] DNA damage foci and regulates DNA repair[19]. Positioned as an adaptable protein between the autophagosome and its substrate, p62 is selectively wrapped into the autophagosome, acting as a bridge between microtubule-associated proteins 1A/1B light chain 3B, commonly known as LC3, the protein that plays a significant role in cellular processes, particularly in autophagy and polyubiquitination proteins. Subsequently, it undergoes degradation by proteolytic enzymes within the autophagolysosome.

Animals, plants, and microorganisms serve as abundant sources of natural products[20–22]. Naturally-derived compounds possess complex structures and a wide array of pharmacological activities, contributing significantly to the development of numerous chemotherapeutic drugs, such as vincristine[23] and taxol[24]. Several animal-derived substances, including Cinobufagin[25] and Lytta vesicatoria[26], have exhibited potential anti-tumor properties. Among them, Gamabufotalin (CS-6) is a bufodienolactone comprising 1.76–4.97% of toad venom, a traditional Chinese medicine derived from dried toad skin and parotid venom[27, 28]. Studies have unveiled the efficacy of CS-6 against various malignant tumors, including glioblastoma[18], non-small cell lung cancer[29], and breast cancer[30]. Thus, the effects and underlying mechanisms of CS-6 on CRC are yet to be fully elucidated. There are currently studies reporting that the accumulation of p62 can cause autophagic flow blockage, thereby exacerbating DNA damage, but there are no reports on the mechanism of action of related drugs. Our research findings indicate that CS-6 concurrently induces DNA damage and p62 accumulation in CRC cells, thus triggering autophagy to mediate its anti-tumor activity.

2.1 Cell culture and drug therapy

The human colorectal cancer (CRC) cell lines, SW620 (ECACC identifier: 87051203) and DLD1 (ECACC identifier: 90102540) were obtained from the European Collection of Authenticated Cell Cultures (ECACC). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) from Sigma-Aldrich, USA, supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Solarbio, Beijing, China). Cells were maintained in a humidified environment at 37°C with 5% CO2. The experimental compound, CS-6, was supplied by Shandong Luye Pharma LTD (Yantai, Shandong, China). CS-6 for experiments was prepared by dissolving it in dimethyl sulfoxide (DMSO) obtained from MedChemExpress, USA. In some experiments aimed at inhibiting autophagic flux, cells were treated with 10 µM chloroquine (CQ) sourced from MedChemExpress, USA, for a duration of 24 hours.

2.2 Cell viability assay

To evaluate the short-term impact of CS-6 on cell growth, a Cell Counting Kit-8 (CCK-8) detection assay was executed (MedChemExpress). In this assay, cell suspensions were meticulously distributed into individual wells of a 96-well plate, with each well receiving 10 μL of the suspension. Subsequently, the plates were placed inside an incubator set to maintain a temperature of 37°C in an atmosphere containing 5% CO2. Following cell incubation, 5 µL of the CCK solution was added to each well[31], following standard protocols. The plates were once again incubated under the same conditions (37°C, 5% CO2) for a duration of 1 to 4 hours. Finally, the absorbance at 450 nm was measured using an enzymatic marker, thereby enabling the assessment of cell growth in response to CS-6 treatment.

2.3 Colony formation assay (CFA)

SW620 and DLD1 cells were initially seeded in 6-well plates at a density of 500 cells per well and subjected to culturing for 24 hours. Once adherent, the cells underwent exposure to various concentrations of CS-6 (5, 10, 20, 40, 80, 160, 320, and 640 nM) for an additional 24 hours, while untreated cells served as the Control group. Following this 24-hour treatment period, the cells were subjected to incubation for an additional 12 days, during which colonies formed from both the treated and untreated cells. The assessment of these colonies involved initial fixation using a 4% paraformaldehyde solution (Beyotime, Shanghai) for 15minutes [32], followed by washing with phosphate-buffered saline (PBS). Subsequently, the colonies were stained with a 0.1% crystal violet (Solarbio, Beijing, China) solution for 10 minutes. Lastly, the colonies were observed and photographed using an inverted microscope (Leica DMI3000B, Leica Microsystems CMS GmbH, Wetzlar, Germany), and the number of colonies with a diameter exceeding 0.5 mm was counted for further analysis.

2.4 Cell cycle analysis

The process involved in preparing the cells for analysis is as follows: Firstly, the digested cells (Thermo Fisher, trypsin; catalog number 25200056) were centrifuged at 900 rpm for 3 minutes. Thereafter, the cells were washed with phosphate-buffered saline (PBS) and centrifuged once more. They were then fixed by immersing them in pre-cooled 70% ethanol for a duration of 2 hours at 4°C. Subsequently, another round of washing with PBS was carried out, followed by exposure to 100 µL of RNase A (catalog number CA1510; Solarbio). This treatment lasted for 30 minutes at 37°C, in accordance with the given instructions. Next, the cells underwent staining with 400 µL of propidium iodide (PI) staining solution for 30 minutes in a dark environment at 4°C.

Ultimately, the stained cells were analyzed using a fluorescence microscope, and the red fluorescence was captured at an excitation wavelength of 488 nm.

2.5 Immunofluorescence (IF) assay

Following CS-6 treatment, SW620 and DLD1 cells were cultured on 35-mm confocal dishes (BeyoGold™) and underwent a series of steps for immunofluorescence staining. The process of cell staining and preparation for microscopy involved several steps:

Initially, cells were subjected to a triple rinse with PBS to ensure the removal of any residual substances. Subsequently, they were fixed with 4% paraformaldehyde (Beyotime) for a duration of 15 minutes. Following fixation, three PBS washes, each lasting 3 minutes, were performed to eliminate excess paraformaldehyde.

To prevent nonspecific binding, the cells were blocked with bovine serum albumin (BSA) for 30 minutes at room temperature. Following this, the cells were subjected to an overnight incubation at 4°C with primary antibodies.

After the overnight incubation, the cells were subjected to three rounds of PBS washing, each lasting 3 minutes, with subsequent removal of any excess liquid. Following this, the cells were exposed to secondary antibodies for 1 hour at room temperature on a shaker.

Another set of three PBS washes was conducted after the secondary antibody treatment. To visualize cell nuclei, the cells were counter-stained with DAPI for 15 minutes, followed by another PBS wash.

To minimize autofluorescence and enhance image quality, an anti-fluorescence quencher solution was applied. Finally, the stained cells were observed and photographed using a fluorescence microscope.

2.6 Comet assay

SW620 and DLD1 cells were cultured in dishes and subjected to treatment with CS-6 (80 nM) for 24 h. Afterward, the cells were exposed to pancreatic enzymes, rinsed with pre-cooled PBS in an ice bath, centrifuged to collect cell precipitates, and resuspended in PBS (1×10⁶ cells/mL). The first layer of gel was prepared on a glass slide using the Comet Electrophoresis Kit (KeyGEN BioTECH, Jiangsu, China) following the provided protocol. Subsequently, 10 µL of cell suspension was evenly mixed with 0.7% low melting point agarose, and 70 µL drops were swiftly added to the initial gel layer. These drops were covered with a cover glass and subjected to incubation at 4 ℃ for 10 min. Next, the glass slide was placed in a 10-cm Petri dish, immersed in 10 mL pre-cooled lysate for 1–2 h at 4 ℃, and rinsed with PBS for 3 min. The slide was then positioned in a horizontal electrophoresis tank, filled with electrophoresis buffer up to 0.25 cm above the rubber surface of the slide, and subjected to electrophoresis at room temperature for 20–60 min under alkaline conditions to facilitate DNA uncoiling. Electrophoresis was conducted for 30 min at 25 V. The slide was placed onto a plate and subjected to 1–3 treatments with a neutral buffer at 4°C for 5–10 minutes each. Subsequently, the slides were exposed to 20 µL of PI solution for 10–20 minutes in a dark environment and rinsed three times with ultrapure water. Finally, the sides were covered with a cover glass and viewed under a fluorescence microscope.

2.7 Western blot (WB) assay

The protein analysis process for SW620 and DLD1 cells followed the given instructions. Cells were lysed using a buffer containing protein phosphatase inhibitors (ThermoFisher, America). The protein content was quantified according to the instructions provided by a Bicinchoninic Acid Protein Assay Kit. In the next step, equal quantities of protein samples were subjected to separation through sodium dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis, followed by their subsequent transfer onto polyvinylidene difluoride membranes. To block the membranes, exposure to 5% skim milk in 1× tris-buffered saline with Tween 20 (TBST) was done for over 2 hours at room temperature. Following this process, the membranes were exposed overnight to primary antibodies in 1% BSA at 4°C. Afterward, after three TBST washes, the membranes were incubated with the appropriate secondary antibodies for 40 minutes at room temperature. Finally, the membranes were visualized using an automatic chemiluminescence image analysis system (Tanon-5200), and optical density measurements were performed using ImageJ v1.41 software (Bethesda, Maryland, United States).

The primary and secondary antibodies used in the study were as follows:

- Anti-phosphorylated-ataxia telangiectasia mutated kinase (P-ATM; 1:1000; #13050; Cell Signaling Technology)

- Anti-ATM (1:1000; #2873; Cell Signaling Technology)

- Anti-beclin-1 (BECN1; 1:5000; 11306-1-AP; Proteintech)

- Anti-p62/SQSTM1 (1:8000; T55546; Abmart)

- Anti-LC3A/B (1:1000; #12741; Cell Signaling Technology)

- Anti-γH2AX (1:8000; T56572; Abmart)

- Anti-autophagy-related protein 5 (ATG5; 1:1000; #12994; Cell Signaling Technology)

- Anti-B-cell lymphoma-2 (BcL2; 1:5000; 68103-1-Ig; Proteintech)

- Anti-cyclin-dependent kinase 1 (CDK1; 1:5000; 19532-1-AP; Proteintech)

- Anti-cyclin B1 (1:4000; 28603-1-AP; Proteintech)

- Anti-β-actin (1:2000; K101527P; Solarbio)

- Anti-rabbit secondary antibodies (1:10000; 31460; Thermo Fisher)

- Anti-mouse secondary antibodies (1:10000; 31430; Thermo Fisher).

2.8 siRNA transfection

SW620 and DLD1 cells were seeded in 6-well plates and incubated with p62 and ATG5 siRNAs (5'-GAGGAUCCGAGUGUGAAUUUCdTdT-3' and 5'-AGGUACUUUCCUCAAUCACATT-3', respectively (GenePharma, Shanghai, China) to suppress p62 and ATG5 expression. Following transfection with Lipofectamine 3000 (Invitrogen, USA), the cells were cultured in Opti-MEM reduced serum medium (31985070; ThermoFisher) and exposed to 100 nM ATG5 and p62 siRNA for 8 h. Subsequent to the described procedures, the cells were incubated in a complete medium for 48 hours and subsequently exposed to CS-6 treatment for 24 hours. Finally, WB analysis and CCK-8 assays were carried out to evaluate protein expression levels and the viability of CRC cells.

2.9 Co-immunoprecipitation (Co-IP) assay

SW620 and DLD1 cells were treated with an immunoprecipitation lysate buffer (ThermoFisher, USA) containing protein phosphatase inhibitors (Solarbio, Beijing) and then centrifuged to obtain cell lysate. Protein A/G magnetic beads (MedChemExpress, USA) were combined with IgG antibodies (Beyotime, Shanghai) and BECN1 antibodies (ProteinTech, USA). The cell lysate was prepared following the specified instructions. Subsequently, the cell lysate was incubated with the antibody-magnetic bead complex at 4 °C overnight with gentle agitation. Afterward, the beads were washed thrice to eliminate any detergent residue, and they were then treated with 20 μL SDS buffer and boiled for 5 minutes. Finally, the bound proteins were determined by using the WB assay.

2.10 Proteome profiling and protein-protein interaction (PPI) network

Proteins were extracted from CS-6-treated SW620 cells for proteomic analysis (conducted by Shanghai Ouai Biotechnology Co. Ltd., Shanghai, China). Key genes and signaling pathways were identified via the protein interaction network of the differentially expressed genes (DEGs) using the GeNets database.

2.11 Statistical analysis

Statistical analysis was conducted using GraphPad Prism 9.0 software. The data in this study were gathered from a minimum of three independent experiments. To compare two groups, a Student's t-test was utilized, and for analyzing statistical differences among multiple groups, a one-way analysis of variance (ANOVA) was utilized. The statistical significance level was established at *P < 0.05.

3.1 Inhibition of SW620 and DLD1 cell proliferation by CS-6 in vitro

Herein, SW620 and DLD1 cells were subjected to varying concentrations of CS-6 (5, 10, 20, 40, 80, 160, 320, and 640 nM) for 24 hours. Their cell viability and proliferative capacity were assessed using the CCK8 assay and colony formation assay (CFA).

The results from the CCK8 assay showed a concentration-dependent reduction in cell viability, yielding IC50 values of 65.58 and 121.1 nM for SW620 and DLD1 cells, respectively (Fig. 1A, B). Additionally, the CFA results demonstrated a significant decrease in the number of colonies formed by SW620 and DLD1 cells at concentrations of 80 nM and 160 nM (Fig. 1C, D). These findings collectively highlight the inhibitory effect of CS-6 on the proliferation of CRC cells in vitro

Fig. 1. CS-6 inhibited the proliferation of SW620 and DLD1 cells in vitro. SW620 and DLD1 cells were exposed to different concentrations of CS-6 (5, 10, 20, 40, 80, 160, 320, and 640 nM) for 24 h. The untreated cells served as the Control.(A, B) Cell viability of Control and CS-6-treated SW620 and DLD1 cells was assessed using the CCK8 assay.(C, D) The colony formation ability of Control and CS-6-treated SW620 and DLD1 cells was measured using the colony formation assay. Values represent the mean ± standard deviation from three independent experiments. **P<0.01 and ***P<0.001.

3.2: Induction of G2/M phase arrest and modulation of cell cycle-related proteins by CS-6 in SW620 and DLD1 Cells

To unravel the mechanism underlying CS-6-related inhibitory impact on CRC cell proliferation, PI staining was employed to scrutinize the distribution of cell cycle phases in CS-6-treated SW620 and DLD1 cells. The results revealed a significant induction of G2/M phase arrest in both SW620 and DLD1 cells when compared to the control treatment, as illustrated in Figure 2A.

The formation of the CDK1/cyclin B1 complex assumes a pivotal role in initiating the cyclic phosphorylation of various target substrates[33]. This process is indispensable for advancing through the cell cycle. Therefore, WB assays were conducted to delve into the expression of cell cycle-related proteins, aiming to shed light on the mechanism behind CS-6-induced cell cycle arrest in CRC cells, as visually presented in Figure 2B.

The results indicated that CS-6 treatment led to a reduction in the expression levels of cyclin B1 and CDK1 relative to the control treatment in SW620 and DLD1 cells. In summary, these findings suggest that CS-6 exerts its inhibitory effect on CRC cell proliferation by promoting G2/M phase arrest

Figure 2 illustrates the elicitation of G2/M phase arrest and alterations in the expression of cell cycle-related proteins in response to CS-6 treatment in SW620 and DLD1 cells. SW620 and DLD1 cells were exposed to varying concentrations of CS-6 (40, 80, and 160 nM) for a 24-hour duration, while untreated cells served as the Control group.(A) Representative histogram of the cell cycle distribution in the Control and CS-6-treated SW620 and DLD1 cells analyzed using flow cytometry.(B) Western blot analysis reveals the levels of cyclin B1 and cyclin-dependent kinase 1 in both the Control and CS-6-treated SW620 and DLD1 cells. The presented values represent the mean ± standard deviation derived from three independent experiments. Statistical significance is established at *P < 0.05, **P < 0.01, and ***P < 0.001.

3.3 CS-6 induced DNA damage in SW620 and DLD1 cells

To determine whether CS-6-mediated cell cycle arrest in CRC cells is due to DNA damage, the expression of DNA damage markers in CS-6-treated SW620 and DLD1 cells was examined. Phosphorylation is a significant post-translational modification of proteins, and DNA double-strand breaks (DSBs) activate ATM kinase. [34] The WB assay results revealed that the expression levels of DNA damage markers, γH2AX and p-ATM, were significantly upregulated after CS-6 treatment in both SW620 and DLD1 cells (Fig. 3A-D). Furthermore, the IF assay showed increased fluorescence of FITC-labelled γH2AX foci after CS-6 treatment, consistent with the WB results (Fig. 3E-H). The Comet assay further confirmed that CS-6 induced DNA damage in both SW620 and DLD1 cells (Fig. 3I, J).

Fig. 3. CS-6 induced DNA damage in SW620 and DLD1 cells. SW620 and DLD1 cells were treated with 80 nM CS-6 for 24 h. The untreated cells were used as Control. (A–D) Western blot analysis of phosphorylated-ataxia telangiectasia mutated kinase (p-ATM), ATM, and γH2AX levels in Control and CS-6-treated SW620 and DLD1 cells. (E–H) Confocal microscopy images showing γH2AX and nuclear staining in Control and CS-6-treated SW620 and DLD1 cells, presented as single-channel and merged images. Scale bar = 5 µm. (I, J) Comet assay illustrating CS-6-induced DNA damage in CRC cells. The data represents the mean ± standard deviation from three independent experiments. Statistical significance is established at *P < 0.05, **P < 0.01, and ***P < 0.001.

3.4 CS-6 upregulated p62 in SW620 and DLD1 cells

To further investigate the specific mechanisms by which CS-6 mediates DNA damage in CRC cells, proteomic analysis was conducted to identify proteins interacting with CS-6. Studies have revealed that p62 is shuttled into the nucleus to mediate DNA damage and participate in DNA damage repair[35]. Proteomic analysis results indicated that p62 was significantly upregulated in CS-6-treated SW620 cells (Fig. 4A). Additionally, protein-protein interaction (PPI) analysis revealed a significant interaction between ATM and p62 (Fig. 4B), while WB assays demonstrated the upregulation of p62 in both SW620 and DLD1 cells after CS-6 treatment (Fig. 4C, D). To investigate the role of p62 in CS-6-treated CRC cells, transient transfection of SW620 and DLD1 cells with siRNA (si-p62) was performed to silence p62. WB analysis revealed that p62 knockdown increased cyclin expression and reduced the levels of DNA damage-associated proteins, including p-ATM and γH2AX, in CS-6-treated CRC cells (Fig. 4 E–H). In summary, these findings suggest that CS-6 may promote DNA damage in SW620 and DLD1 cells via the p62-regulated ATM/γH2AX pathway.

Fig. 4. CS-6 upregulated p62 expression in SW620 and DLD1 cells. SW620 and DLD1 cells were treated with various concentrations of CS-6 for 24 h. The untreated cells were used as Control. Additionally, SW620 and DLD1 cells were transfected with scrambled siRNA (Control) and p62 siRNA for 36 h, followed by treatment with 80 nM CS-6 for 24 h. (A, B) Volcano map displaying differentially expressed genes and protein interactions in SW620 cells assessed by proteomics analysis. (C, D) Western blot (WB) assay showing p62 and DNA damage-associated proteins (p-ATM, ATM, γH2AX, p62, and β-actin) levels in Control and CS-6-treated SW620 and DLD1 cells. (E–H) WB assay of cell cycle-associated proteins (cyclin B1 and cyclin-dependent kinase 1) and DNA damage-associated proteins in Control and si-p62-transfected/CS-6-treated CRC cells. Values represent the mean ± standard deviation derived from three independent experiments. *P<0.05 and **P<0.01.

3.5 CS-6 induced autophagy and inhibited autophagic flux in SW620 and DLD1 cells

In addition to its role in DNA damage and repair, p62 is also a known substrate for autophagy. Previous studies have demonstrated that upregulation of p62 may be associated with impairment of the downstream autophagic flux pathway and inhibition of autophagosome-lysosome fusion[36]. Therefore, the role of CS-6 in activating autophagy in CRC cells was explored. Autophagy activation requires the release of BECN1 from the BECN1/BcL2 complex[37]. The Co-IP assay showed that CS-6 disrupted the BECN1/BcL2 interaction in SW620 and DLD1 cells (Fig. 5A, B). Additionally, the WB assay revealed that the expression of BECN1 and LC3-B significantly increased in SW620 and DLD1 cells after CS-6 treatment (Fig. 5C, D) In summary, these findings suggest that CS-6 induced autophagy but impaired autophagic flux in SW620 and DLD1 cells.

Fig. 5. CS-6 induced autophagy and inhibited autophagic flux in SW620 and DLD1 cells. SW620 and DLD1 cells underwent treatment with varying concentrations of CS-6 for 24 hours. Untreated cells served as the Control. (A, B) Co-immunoprecipitation analysis was conducted to assess the interaction between Beclin-1 (BECN1) and BcL2 in Control cells and those treated with 80 nM CS-6. (C, D) Western blot analysis was performed to determine the levels of autophagy-related proteins (BECN1 and microtubule-associated proteins 1A/1B light chain 3B) in Control cells and those exposed to 40, 80, and 160 nM CS-6. The presented values indicate the mean ± standard deviation derived from three independent experiments. Statistical significance is established at *P<0.05, **P<0.01, and ***P<0.001.

3.6 CS-6-induced autophagy defects exacerbated DNA damage in SW620 and DLD1 cells

To further explore the crosstalk between CS-6-induced DNA damage and autophagy in CRC cells, SW620 and DLD1 cells were treated with CQ, a late-autophagy flux inhibitor. The results revealed that combined treatment with 10 μM CQ and 80 nM CS-6 significantly reduced the cell viability of CRC cells compared to CS-6 treatment alone (Fig. 6 A, B). Moreover, combined CS-6/CQ treatment significantly increased LC3A/B and p62 levels in CRC cells, indicating a synergistic effect on the inhibition of autophagic flux (Fig. 6C, D). Furthermore, CQ treatment aggravated the CS-6-induced DNA damage in CRC cells (Fig. 6C, D). ATG5 is an autophagy-related protein required for the formation of autophagosomes.[38] Transient transfection of ATG5 siRNA (si-ATG5) to inhibit autophagic activity not only restored the cell viability of SW620 and DLD1 cells but further attenuated the CS-6-induced DNA damage in CRC cells (Fig. 7. A–D). Overall, these results indicate that CS-6-mediated autophagy aggravates DNA damage in CRC cells, and suppression of autophagic activity could enhance the anti-CRC effect of CS-6.

Fig. 6. CS-6-induced autophagy defects exacerbated DNA damage in SW620 and DLD1 cells. SW620 and DLD1 cells were treated with 80 nM CS-6 and/or 10 μM chloroquine (CQ) for 24 h. (A, B) Cell viability of CS-6-treated and CS-6/CQ-treated SW620 and DLD1 cells was measured using the CCK8 assay. (C, D) Western blot assay assessed the levels of phosphorylated-ataxia telangiectasia mutated kinase (p-ATM), ATM, and γH2AX in CS-6-treated and CS-6/CQ-treated SW620 and DLD1 cells. The values presented are the mean ± standard deviation derived from three independent experiments. *P<0.05 and **P<0.01.

Fig. 7. Inhibition of CS-6-induced autophagy reversed DNA damage in SW620 and DLD1 cells.SW620 and DLD1 cells were transfected with scrambled siRNA (Control) and si-ATG5 for 48 hours, followed by treatment with 80 nM CS-6 for 24 hours. (A, B) Cell viability of Control and si-ATG5-transfected/CS-6-treated SW620 and DLD1 cells was measured using the CCK8 assay. (C, D) Western blot assay measured the levels of phosphorylated-ataxia telangiectasia mutated kinase (p-ATM), ATM, and γH2AX in Control and si-ATG5-transfected/CS-6-treated SW620 and DLD1 cells. The data presented represents the mean value along with the standard deviation derived from three separate experiments. Statistical significance is denoted as * for P-values less than 0.05, ** for P-values less than 0.01, and *** for P-values less than 0.001.

The occurrence of DNA damage can stem from various factors, such as disruptions in cellular metabolism, exposure to toxins, or exposure to ultraviolet radiation. When DNA damage remains unaddressed, it can trigger programmed cell death mechanisms like apoptosis, autophagy, and necrosis. The DDR pathway functions as a critical defense mechanism, identifying DNA damage and initializing DNA repair processes to preserve genomic integrity[39]. Key regulators within the DDR signaling pathway encompass Ataxia Telangiectasia Mutated (ATM) Ataxia Telangiectasia and Rad3-Related Protein (ATR). These kinases (ATM and ATR), alongside DNA-PK, work in parallel cascades to deactivate the CDK1 complex, thereby arresting or preventing the progression of cell cycle checkpoints, including the G2/M checkpoint. In the study conducted, it was observed that CS-6 induced G2/M cell cycle arrest and activated the ATM-γH2AX DDR pathway in SW620 and DLD1 cells. Activation of ATM and ATR in response to substantial DSBs has been suggested to promote cell death and suppress cancer progression during the early stages of tumor formation[39].

To elucidate the mechanisms underlying the effects of CS-6 in cancer cells, a proteomics analysis was executed, suggesting a significant increase in p62 levels in CS-6-treated CRC cells. p62 is a stress-responsive protein that modulates the autophagic degradation process and nucleocytoplasmic shuttling. During autophagy, p62 is selectively engulfed into autophagosomes and subsequently degraded by proteolytic enzymes, resulting in reduced p62 levels. Therefore, p62 levels are inversely correlated with autophagic activity. Our findings indicated that CS-6 could activate autophagy in CRC cells by disrupting the BECN1/BcL2 complex. However, the accumulation of p62 in CRC cells suggested that CS-6 treatment disrupted the autophagic flux in CRC cells (Fig. 6). Previous research has linked p62 to DNA DSB repair. Defects in autophagy following DNA damage are associated with histone H2A ubiquitination. The cytoplasmic accumulation of p62 in autophagy-deficient cells impedes the recruitment of DNA repair proteins, including BRCA1, RAP 80, and Rad 51, to the DNA DSB binding sites, thus hindering DNA repair and exacerbating DNA damage.[40] Our results demonstrated that combined treatment with CS-6 and CQ not only synergistically inhibited autophagic flux but also intensified DNA damage in CRC cells. Furthermore, the knockdown of ATG5, an autophagy-related protein, reduced DNA damage in CRC cells. These outcomes suggest that CQ amplifies DNA damage by promoting p62 accumulation, blocking late-stage autophagy exacerbates CS-6-induced DNA damage, and blocking autophagy suppresses CS-6-induced DNA damage. Consequently, CS-6-induced p62 accumulation can lead to severe DNA damage that remains unrepaired, further escalating DNA damage.

CS-6, the primary steroidal compound found in Chansu, exhibits substantial anti-tumor activity with minimal toxicity and side effects. It inhibits cell proliferation, induces apoptosis, and regulates immune responses in various cancer types[41]. Previous research has unveiled several roles of CS-6 in different cancers, including its inhibition of COX2 and Hsp90 in lung cancer[42], its potential in glioblastoma treatment in combination with other drugs[43], its ability to inhibit osteosarcoma stem cells through the TGF-β/periostin/PI3K/AKT pathway[44, 45], and its capacity to degrade c-Myc in multiple myeloma cells[46]. However, studies investigating the underlying anti-cancer effects and mechanisms of CS-6 in CRC have been lacking. Our study demonstrates that CS-6 exerts a potent anti-tumor effect on SW620 and DLD1 cells by inducing DNA damage, causing autophagy defects, promoting p62 accumulation, and impeding the repair of DNA, thereby exacerbating DNA damage (Fig. 8). In conclusion, our findings suggest that CS-6 holds significant anti-cancer potential in treating CRC and may serve as a promising therapeutic agent for managing this condition.

Ackonwledgements

We are grateful to the Medical Research Centre of Binzhou Medical University for providing large scale instrumentation and equipment support. Funding: This work was supported by National Natural Science Foundation of China Youth Fund hosted by Prof. Sixue Bi [grant numbers 82104463]; Yantai School Land Integration Development Project in 2021 [grant number 2021XDRHXMPT15 to D.L.]; Youth Fund Project of Shandong Natural Science Foundation hosted by Prof. Sixue Bi [grant number ZR202103060169]; Taishan Scholars Construction Engineering of Shandong Province [to Defang Li], the Yantai High-End Talent Introduction Plan “Double Hundred” [to Defang Li].

Declarations

This manuscript has not been submitted for publication nor has been published in whole or in part elsewhere. I attest to the fact that all authors listed on the title page have read the manuscript, attest to the validity and legitimacy of the data and its interpretation, and agree to its submission to online Discover Oncology. Yitong Gong chargethe acquisitionn of data and drafted the work. Xuehui Cui made substantial contributions to the conception or design of the work. Menghan Dong and Liushuyue Wang analyzed and interpreted the data. Defang Li revised work critically for important intellectual content. Zhen Shi and Sixue Bi approved the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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CS-6-Induced p62 Accumulation Exacerbates DNA Damage: A Mechanistic Insight into Colorectal Cancer Suppression

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