Alantolactone Induced Apoptosis and DNA Damage of Cervical Cancer through ATM/CHK2 Signaling Pathway

Abstract

Traditional Chinese Medicine, known for its minimal side effects and significant clinical efficacy, has attracted considerable interest for its potential in cancer therapy. In particular, Inula helenium L. has demonstrated effectiveness in inhibiting a variety of cancers. This study focuses on alantolactone (ALT), a prominent compound from Inula helenium L., recognized for its anti-cancer capabilities across multiple cancer types. The primary objective of this study is to examine the influence of ALT on the proliferation, apoptosis, cell cycle, and tumor growth of cervical cancer (CC) cells, along with its associated signaling pathways. To determine protein expression alterations, Western blot analysis was conducted. Furthermore, an in vivo model was created by subcutaneously injecting HeLa cells into nude mice to assess the impact of ALT on cervical cancer. Our research thoroughly investigates the anti-tumor potential of ALT in the context of CC. ALT was found to inhibit cell proliferation and induce apoptosis in SiHa and HeLa cell lines, particularly targeting ataxia-telangiectasia mutated (ATM) proteins associated with DNA damage. The suppression of DNA damage and apoptosis induction when ATM was inhibited underscores the crucial role of the ATM/cell cycle checkpoint kinase 2 (CHK2) axis in ALT’s anti-tumor effects. In vivo studies with a xenograft mouse model further validated ALT’s effectiveness in reducing CC tumor growth and promoting apoptosis. This study offers new insights into how ALT combats CC, highlighting its promise as an effective anti-cervical cancer agent and providing hope for improved treatment outcomes for CC patients.

INTRODUCTION

Cervical cancer (CC) currently ranks as the second most common malignancy in female patients, resulting in over 300000 worldwide fatalities annually.¹⁾ The majority of cervical cancer cases, especially in low- and middle-income countries, are attributed to human papillomavirus (HPV) infections, notably high-risk types like HPV16 and HPV18. Despite incremental progress in clinical therapies, including surgery, chemotherapy, and radiotherapy, persistent drug resistance and adverse side effects remain significant clinical hurdles.^2–4) Therefore, it is imperative to enhance the QOL and survival rates for CC patients through the development of novel compounds with superior therapeutic indices.

Traditional Chinese medicine, celebrated for its low incidence of adverse effects and notable clinical effectiveness, often faces challenges due to the unclear nature of its active ingredients, therapeutic targets, and the mechanisms of action. For example, clinical observations have indicated that Inula helenium L., an herbal component often used in these treatments, may play a significant role in hindering the initiation and progression of a variety of cancers.^5,6) In this context, it is crucial to elucidate the anti-cancer compounds within Chinese medicinal herbs, such as Inula helenium L., and their associated targets and signaling pathways. Increasing research focus has centered on alantolactone (ALT), a prominent sesquiterpene component derived from Inula helenium L.’s root. ALT exhibits anti-cancer effects across various cancer types, including breast cancer, colorectal cancer, pancreatic cancer, and osteosarcoma.^7–9) However, comprehensive understanding of the mechanisms underlying ALT’s anti-cancer effects and its appropriate applications in cancer treatment remain elusive.

In the current study, we systematically investigated ALT’s impact on cervical cancers. Notably, we report, for the first time, that ALT exerts its anti-cervical cancer effects by activating the ataxia-telangiectasia mutated (ATM)/cell cycle checkpoint kinase 2 (CHK2) axis signaling pathway, with a specific focus on targeting ATM proteins (ATMs). Our data indicated that ALT induced DNA damage and apoptosis in two cervical cancer cell types, namely HPV16-positive SiHa and HPV18-positive HeLa cells. Importantly, inhibition of ATM attenuates the DNA damage and apoptosis induction initiated by ALT. In vivo experiments further demonstrate significant tumor reduction and induction of apoptosis in cervical cancer following ALT administration. Collectively, these findings suggest ALT’s potential as a potent anti-cervical cancer drug, while also shedding light on novel mechanistic insights into the efficacy of traditional Chinese medicine herbs in tumor treatment and prevention.

MATERIALS AND METHODS

Cell Culture and Reagents

The human cervical cancer cell lines SiHa and HeLa were procured from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic. Alantolactone (purity > 98%) was purchased from MedChemExpress (MO, U.S.A.). Cell counting kit-8 (CCK8), crystal violet solution, annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit and cell cycle detection kit was obtained from Beyotime (Shanghai, China). Primary antibodies γ-H2A.X, phosphorylated (p)-cell division cycle 2 (CDC2), CDC2, cyclin B1, p-ATM, ATM, p-CHK2, CHK2, p-cell division cycle 25c (CDC25c), CDC25c and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (MA, U.S.A.).

Cell Viability Assay

Cell viability was assessed using the CCK8. Cells were seeded in 96-well plates and treated with varying concentrations of ALT for a 24 h. CCK8 solution (10 + 90 µL medium) was added to each well and incubated for 1.5 h. Subsequently, the absorbance (optical density, OD value) at 450 nm was measured.

Clonogenic Assay

Cells were plated in 12-well plates at a density of 1000 cells and incubated for 24 h. After treatment with ALT for 24 h, the cells were fixed using 4% paraformaldehyde and then stained with a crystal violet solution for 20 min.

Cell Apoptosis Analysis

The Annexin V-FITC apoptosis detection kit was used to examine cell apoptosis following the manufacturer’s instructions. Cells were seeded in six-well plates at a density of 3 × 10⁵ cells per well. After washing with phosphate buffer saline (PBS), cells were harvested and suspended in 100 µL of 1 × binding buffer. Cells were then stained with 5 µL Annexin V-FITC and 10 µL propidium iodide (PI). The results were immediately analyzed using flow cytometry.

Cell Cycle Analysis

PI staining was performed to analyze the cell cycle according to the manufacturer’s instructions. Cells were treated with varying concentrations of ALT and incubated for 24 h. Subsequently, cells were harvested, fixed using 70% cold ethanol overnight, and then stained with 500 µL propidium iodide staining solution (PI + ribonuclease (RNase) A) per tube. This was followed by incubation at 37 °C for 30 min in the dark before analysis by flow cytometry.

Western Blotting

Cells were collected and lysed with radioimmunoprecipitation assay (RIPA) buffer for 30 min on ice. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit. Equal amounts (20 µg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature. Subsequently, membranes were incubated with specific primary antibodies (γ-H2A.X, pCDC2, CDC2, cyclin B1, p-ATM, ATM, p-CHK2, CHK2, pCDC25c, and CDC25c) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, the membranes were exposed to enhanced chemiluminescence (ECL) substrate and visualized using a chemiluminescence detection system.

Comet Assay

The oxiselect comet assay kit was used to detect DNA double-strand breaks (DSBs). Cells were washed with PBS, mixed with low melting point agarose, and dispensed onto agarose gel-coated glass slides. Electrophoresis was carried out at 25 V and 300 milliamps for 25 min. Following electrophoresis, the samples underwent neutralization using trihydrochloric acid. Cell nuclei were stained with Hoechst, and images were captured with a fluorescence microscope for analysis of comet tails.

Tumor Xenograft Experiment

Female BALB/c-nude mice were obtained from Shanghai University of Chinese Traditional Medicine. HeLa cells suspended in PBS at a concentration of 5 × 10⁶ cells in 200 µL were injected into the flank of the mice. After one week of implantation, ALT at doses of 10 mg/kg (ALT-low dose, ALT-L), 20 mg/kg (ALT-high dose, ALT-H), or 5-fluorouracil (5-Fu) at 48 mg/kg (dissolved in saline) was administered orally to the mice every three days for 21 d. In the ALT + AZD0156 group, 10 mg/kg AZD0156 was administered by gavage three times a week. Tumor growth was recorded every three days, and tumor volume was calculated using the formula V = (Length × Width²)/2. At the end of the experiments, the mice were euthanized, and tumors were harvested for further analysis. All animal experimental protocols were approved by the Animal Care Committee of Shanghai University of Traditional Chinese medicine.

Immunohistochemical Staining

Tumor tissues from mice were excised, fixed with 4% paraformaldehyde overnight, and subjected to tissue embedding the following day. The sliced tissue was subjected to antigen retrieval by immersion in ethylenediaminetetraacetic acid (EDTA) citrate buffer and microwave treatment. Following this, primary antibodies targeting γ-H2A.X were left to incubate overnight at 4 °C, and subsequently, they were subjected to a suitable secondary antibody at room temperature for 1 h. Finally, the sections were scrutinized utilizing a 3,3′-diaminobenzidine (DAB) substrate kit and subsequently counterstained with hematoxylin. The staining images were captured utilizing a digital slide scanner.

Statistical Analysis

Data are presented as the mean ± standard deviation of more than three independent assays. One-way ANOVA was used for multiple comparisons. A p-value of <0.05 was considered statistically significant.

RESULTS

Alantolactone Initiated Cytotoxicity in CC Cells

To investigate the potential cytotoxicity of ALT, we initially examined its impact on cell viability in two human CC cell lines, namely HPV16-positive SiHa and HPV18-positive HeLa. As illustrated in Figs. 1A and B, ALT (0.5–8 µM) led to a concentration-dependent reduction in the viability of both SiHa and HeLa cells. Subsequently, the colony formation assay provided additional evidence of ALT’s potent anti-CC effects in vitro, consistent with the findings from the CCK8 assay (Figs. 1C, D). Flow cytometry was then employed to examine the influence of ALT on cell apoptosis. The experimental findings demonstrated that ALT promoted apoptosis in a dose-dependent manner in both SiHa and HeLa cells (Fig. 1E).

Fig. 1. Alantolactone Initiated Cytotoxicity in CC Cells

(A, B) SiHa and HeLa cells were treated with various concentrations of ATL, and cell viability was detected by the CCK8 assay. (C, D) Representative images of colony formation in SiHa and HeLa cells. (E) Cell apoptosis in SiHa and HeLa cells following 24 h of ATL treatment was assessed using flow cytometry. Each experiment was repeated five times. * p < 0.05, ** p < 0.01.

Alantolactone Arrested Cervical Cancer Cells at G2/M Phase

5-Ethynil-2’-deoxyuridine (EdU) incorporation assays were utilized to evaluate the effect of ALT on DNA synthesis in two cervical cancer cell lines, SiHa and HeLa. Figure 2A clearly demonstrated that with escalating concentrations of ALT, there was a notable decrease in EdU incorporation, signifying a reduction in DNA synthesis attributed to ALT treatment. To corroborate ALT’s efficacy in inducing cell cycle arrest in these cervical cancer cells, flow cytometry assays were performed. As shown in Fig. 2B, an increased concentration of ALT corresponded to a higher percentage of cells in both SiHa and HeLa lines being arrested in the G2/M phase. This finding was further substantiated by Western blot analysis, which showed changes in the expression levels of proteins associated with the G2/M phase, such as CDC2 and cyclin B1, as indicated in Fig. 2C.

Fig. 2. Alantolactone Arrested Cervical Cancer Cells at G2/M Phase

(A) The EdU assay was employed to assess the effect of a 24 h ALT intervention on DNA synthesis. (B) The cell cycle phase distribution of SiHa and HeLa cells was determined after treatment with ALT for 24 h using flow cytometry. (C) Expression and quantification of CDC2 and cyclin B1 protein in SiHa and HeLa cell treated with ALT for 24 h. Each experiment was repeated five times. * p < 0.05, ** p < 0.01.

Our data strongly suggested that the presence of ALT significantly downregulated the expression of CDC2 and cyclin B1, both of which was associated with G2/M phase cell cycle arrest. In summary, ALT played a pivotal role in inhibiting the proliferation of cervical cancer cells by inducing G2/M phase accumulation.

Alantolactone Induced DNA Damage in CC Cells

G2/M arrest is typically triggered by DNA damage, and therefore, we postulated that ALT treatment might induce DNA damage.¹⁰⁾ As anticipated, exposure to ALT resulted in the elongation of comet tails (Fig. 3A). Furthermore, the levels of γ-H2A.X showed a dose-dependent increase upon ALT incubation, as demonstrated by both Western blot and immunofluorescence assay (Figs. 3B, C).

Fig. 3. Alantolactone Induced DNA Damage in CC Cells

(A) The comet assay was utilized to assess DNA double-strand breaks in SiHa and HeLa cells following treatment with ALT for 24 h. (B, C) Immunofluorescent staining and Western blot were performed to analyze the expression of the γ-H2A.X protein in SiHa and HeLa cells following a 24 h treatment with ALT. Each experiment was repeated five times. * p < 0.05, ** p < 0.01.

Alantolactone Activated DNA Damage-Related Signaling Pathway

Next, we aimed to delve into the mechanism through which ALT induced DNA damage in CC cells. As widely acknowledged, p-ATM and p-CHK2 serve as well-established molecular markers of DNA damage.^10,11) Through Western blot analysis, we noted elevated expression levels of both p-ATM and p-CHK2 in SiHa and HeLa cells, correlating with ALT dosage (Fig. 4A). Furthermore, the downstream protein p-CDC25c exhibited decreasing expression in response to ALT treatment in a dose-dependent fashion (Fig. 4B). These findings collectively suggested that the DNA damage-related signaling pathway is activated by ALT treatment.

Fig. 4. Alantolactone Activated DNA Damage-Related Signaling Pathway

(A, B) After 24 h of treatment with ALT, the protein expression levels of p-ATM, ATM, p-CHK2, CHK2, p-CDC25c, and CDC25c were measured using Western blot. Each experiment was repeated five times. * p < 0.05, ** p < 0.01.

ATM/CHK2 Axis Contributed to ALT-Induced Apoptosis, Cell Cycle Arrest and DNA Damage of Cervical Cancer Cells

This preliminary finding strongly suggested that the ATM/CHK2 pathway played a significant role when ALT exerted its therapeutic effects. To further validate this hypothesis, we investigated whether the ATM inhibitor AZD0156 affected the therapeutic effects of ALT, including CC cell apoptosis, cell cycle arrest, and DNA damage. As depicted in Fig. 5A, the inhibition of ATM resulted in the reversal of ALT-induced apoptosis in both SiHa and HeLa cells. Conversely, inhibiting ATM alone had no discernible effect on cell apoptosis compared to the control group receiving no treatment. Similar results were obtained in cell cycle assessments, where ATM inhibition rescued cells that had undergone ALT-induced cell cycle arrest in the G2/M phase (Fig. 5B). Additionally, examination of DNA damage, as indicated by γ-H2A.X expression, revealed consistent patterns (Fig. 5C). Further exploration of the ATM/CHK2 signaling pathway revealed that while the key proteins p-ATM and p-CHK2 were upregulated during ALT treatment, the expression of p-CDC25c showed a contrasting trend, returning to baseline levels upon ATM inhibitionn (Fig. 5D). These collective findings strongly supported the notion that ALT induces apoptosis, cell cycle arrest, and DNA damage through the ATM/CHK2 axis.

Fig. 5. ATM/CHK2 Axis Contributed to ALT-Induced Apoptosis, Cell Cycle Arrest and DNA Damage of Cervical Cancer Cells

(A) SiHa and HeLa cells were incubated for 24 h with ALT, AZD0156 or combination of ALT and AZD0156, and cell apoptosis were determined using flow cytometry. (B) SiHa and HeLa cells were cultured with ALT, AZD0156, or a combination of both for 24 h, the cell cycle phase distribution was determined by flow cytometry. (C, D) The expression of γ-H2Ax, p-ATM, ATM, p-CHK2, CHK2, p-CDC25c, and CDC25c in SiHa and HeLa cell treated with ALT, AZD0156 or combination of ALT and AZD0156 were detected using Western blot. Each experiment was repeated five times. * p < 0.05, ** p < 0.01 and *** p < 0.001.

In summary, our study provided compelling evidence that ALT exerts its effects on apoptosis, cell cycle regulation, and DNA damage in CC cells via the ATM/CHK2 signaling pathway.

Alantolactone Inhibited the Tumor Growth of CC In Vivo

To validate the in vivo effectiveness of ALT, we established a xenograft mouse model using HeLa cells. As depicted in Fig. 6A, the administration of ALT led to a substantial inhibition of tumor growth in comparison to the control group. Moreover, ALT treatment resulted in a significant reduction in the volume of CC tumors in mice (Fig. 6A). Immunohistochemical staining of Ki67 in tumor specimens revealed that ALT decreased the proportion of actively proliferating cells (Fig. 6B). Immunohistochemical staining analysis was performed to detect the expression of γ-H2A.X. In line with the in vitro results, ALT treatment elevated the expression of γ-H2A.X (Fig. 6C). Subsequently, Western blot experiments showed that ALT treatment significantly promoted the expression levels of p-ATM and p-CHK2 proteins in tumor tissue (Fig. 6D). To further determine which apoptosis was induced by ALT, Western blotting was conducted. We found that the protein levels of Bax were significantly increased, and Bcl-2 were down-regulated by ALT treatment (Fig. 6E).

Fig. 6. Alantolactone Inhibited the Tumor Growth of CC in Vivo

(A) Macroscopic views of xenograft tumors at the endpoint of the experiment and growth curves of HeLa xenograft tumors in BALB/c nude mice (n = 5). (B, C) Ki67 and γ-H2AX expression levels in xenograft tumors as measured by immunohistochemical staining assay (n = 5). (D) The expression of p-ATM, ATM, p-CHK2, and CHK2 in xenograft tumors using Western blot (n = 3). (E) The expression of Bcl-2 and Bax in xenograft tumors using Western blot (n = 3). (F) Growth curves of HeLa xenograft tumors in BALB/c nude mice (n = 5). (G) Ki67 and γ-H2AX expression levels in xenograft tumors as measured by immunohistochemical staining assay (n = 5). (H) The expression of p-ATM, ATM, p-CHK2, and CHK2 in xenograft tumors using Western blot (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001.

To investigate the involvement of the ATM/CHK2 signaling pathway in the anti-CC effects of ALT in vivo, we administered the ATM inhibitor AZD0156 orally to mice to assess whether AZD0156 countered the anti-cancer effects of ALT on HeLa cell xenograft mice. The results revealed a significant increase in tumor volume in the ALT + AZD0156 group compared to the ALT-alone group (Fig. 6F).

Immunohistochemical staining of tumor tissues revealed a notable upregulation in Ki67 expression and a concurrent downregulation in γ-H2A.X expression within the tumors of the ALT + AZD0156 group, as compared to the ALT-alone group (Fig. 6G). Western blot analysis demonstrated significantly reduced expression levels of p-ATM and p-CHK2 proteins in tumors of the ALT + AZD0156 group compared to the ALT-alone group (Fig. 6H).

In summary, our results underscore the promising therapeutic potential of ALT in an in vivo setting, as it effectively restrained tumor growth, inhibited tumor cell proliferation, and activated the ATM/CHK2 signaling pathway in HeLa xenograft mice.

DISCUSSION

Alantolactone (ALT), a sesquiterpene lactone found in the roots of Inula helenium L. as well as several other Asteraceae plants, has been a subject of growing interest due to its versatile traditional medicinal applications and its emerging role in combating cancer. This natural compound has been employed in traditional medicine for centuries, and its applications extend to treating conditions such as asthma, bronchitis, gastroenteritis, and tuberculosis.^12,13)

In recent years, ALT has emerged as a prominent player in cancer research. ALT has garnered substantial attention owing to its anti-inflammatory and anti-tumor activities, largely attributed to its remarkable ability to modulate signaling pathways in cancer cells. ATL’s most remarkable potential in its anti-tumor properties have generated considerable enthusiasm, especially in the realm of colorectal,¹⁴⁾ lung,^15,16) breast,¹⁷⁾ and leukemia cancers.¹⁸⁾ Its anti-cancer effects are multifaceted, encompassing the induction of G2/M cell cycle arrest, the initiation of mitochondrial-dependent apoptosis, and the induction of DNA damage.^19,20) It significantly dampens the activity of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), effectively curbing cancer cell proliferation and promoting programmed cell death in lung cancer cell lines, including NCI-H1299 and Anip973.^21–24) Additionally, a pioneered study has unveiled ALT’s ability to suppress the activation of YAP1/TAZ, crucial regulators of cell growth, further underscoring its anti-cancer potential.²⁵⁾ These effects are associated, in part, with ATL’s ability to inhibit STAT3, suppress NF-kB signaling, tip the balance of Bax/Bcl-2 ratio in favor of apoptosis, and activate caspases, key players in cell death pathways.²⁶⁾ Notably, ATL directly targets thioredoxin reductase (TrxR), a critical enzyme involved in redox regulation, leading to the generation of reactive oxygen species (ROS) and the induction of endoplasmic reticulum (ER) stress, ultimately culminating in apoptosis, particularly in breast cancer cells.^26,27) Furthermore, ALT exerts its inhibitory effects by directly interacting with the Aurora-A protein. By binding to the aurora-A-TPX2 complex, ALT disrupts the complex’s functionality.^27–29) This interference results in a range of significant consequences, including the prevention of centrosome amplification, the reduction of chromosomal instability, and the inhibition of oncogenic transformations. Impressively, ALT demonstrates its therapeutic synergy by enhancing the cytotoxic effects of existing anti-cancer agents, such as oxaliplatin and olaparib, both in animal models and in cell culture, all while displaying minimal side effects.

It is important to emphasize that the disruption of precise cellular cycle control is intricately linked with the progression of CC. Consequently, prompting a pause in the G2/M phase has emerged as a promising strategy to impede the relentless growth of CC cells. This is consistent with Hu’study, in which case ALT inhibited cancer cell proliferation and induced apoptosis with G2/M phase cell cycle arrest.³⁰⁾ Specifically, ALT effectively curtailed cell proliferation, fosters apoptosis, and orchestrates cell cycle arrest at the G2/M phase in CC cells. Additionally, it inhibited CDC2 and cyclin B1 expression. In this research, our primary discovery was that ALT exerts a pronounced anti- CC effect by orchestrating a series of crucial cellular responses.

Activation of DNA damage response pathways is a cornerstone strategy in the development of anti-tumor therapies.³¹⁾ DNA damage not only triggers cell cycle arrest but also sets in motion the intricate process of apoptosis, a programmed cell death mechanism vital for eliminating rogue cancer cells.³²⁾ Many conventional chemotherapeutic agents, such as cisplatin, carboplatin, and doxorubicin, leverage DNA damage as a central mechanism of action.^33,34) Furthermore, natural compounds have gained recognition for their capacity to impede cancer cell proliferation by inducing DNA damage.^35,36) These naturally derived compounds play a pivotal role in the synthesis of anti-tumor drugs and include notable examples like berberine, calprotectin, and paclitaxel.

In light of our compelling findings that ALT leads to an increase in DNA damage markers, including DNA lesions and the phosphorylated protein γ-H2AX,³⁷⁾ we ventured to explore the possibility that ALT actively contributes to the DNA damage response. This revelation opens new avenues for understanding the precise mechanisms through which ALT exerts its anti-CC effects and reinforces its potential as a promising anti-cancer agent. Of note, A study of ALT on colorectal cancer also revealed ATL-induced ROS overload on human colorectal cancer cells (SW480 and SW1116). In particular, a significant accumulation of oxidized guanine (8-oxoG) resulted in an immediate increase in DNA strand breaks following the surge in ROS levels, indicating extensive DNA damage that consistent with our study.³⁸⁾

ATM is a mutated protein associated with ataxia-telangiectasia (AT), identified in 1995, with a phosphatidylinositol 3-kinase (PI3K)-like kinase domain located at its C-terminus.^39,40) AT is an autosomal recessive genetic disorder characterized by immunodeficiency and susceptibility to malignant tumors in affected individuals.⁴¹⁾

Within the DNA damage response (DDR), ATM plays a pivotal role in coordinating cellular responses to double-strand breaks (DSBs), including DNA repair, checkpoint activation, apoptosis, senescence, chromatin structure changes, transcription, and mRNA splicing.⁴²⁾ ATM functions as a master kinase, phosphorylating hundreds of substrates in response to DNA damage.^42,43) Checkpoint kinase 2 (CHK2) represents another pivotal target of ATM activity. Once activated, CHK2 phosphorylates a range of proteins, including CDC25A, thereby regulating cell cycle arrest and triggering apoptosis.⁴⁴⁾ Phosphorylation of CHK2 at T68 is commonly utilized as a marker for ATM activation, even though there are alternative pathways that can activate CHK2 aside from ATM.^45,46) In light of these, we investigated the role of the ATM/CHK2 pathway in ALT-induced effects on apoptosis, cell cycle disturbances, and DNA damage in CC cells. We found that ALT induces apoptosis, regulates cell cycle progression, and causes DNA damage through the ATM/CHK2 axis. All of these changes can be reversed by inhibition of ATM. Actually, it has been reported that blocking the ATM/CHK2 DNA damage checkpoint pathway resulted in the generation of replication stress and the buildup of cytosolic DNA. Consistently, our mouse model indicated that exposure to ALT resulted in a significant decrease in tumor size and Ki67, while upregulating the expression of γ-H2A.X. These studies collectively suggested the efficacy of activating the ATM/CHK2 pathway in in ALT anti-tumor.

CONCLUSION

In summary, the results from this study compellingly demonstrate that ALT effectively inhibits the initiation and progression of cervical cancer by instigating DNA damage through the ATM/CHK2 signaling pathway. This pivotal discovery accentuates the potential of the ATM/CHK2 axis as a promising target for future therapeutic interventions utilizing ALT.

Acknowledgments

This work was supported by the Pudong New District Key Speciality Construction Project under Grant [No. PWZzk2022-14] and Key Specialty Construction Project of Pudong Health and Family Planning Commission of Shanghai [No. 2022-14].

Conflict of Interest

The authors declare no conflict of interest.

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