Lung cancer is a very common cancer diagnosed and the leading cause of cancer death. Among them, the treatment options for non-small cell lung cancer cell (NSCLC) are very limited, mainly due to the frequent development of anti-cancer drug resistance. Targeted DNA repair mechanisms have reached clinical application, and new therapeutic strategies including targeting cell cycle checkpoint sites, such as ATR and CHK1, are currently under clinical development. In this study, NSCLC A549 cells and gemcitabine-resistant strain A549/G+ cells were used to explore the synergistic effect of CHK1 inhibition and gemcitabine, and to evaluate the potential of overcoming drug resistance and related molecular mechanisms. Our results showed that inhibition of CHK1 and gemcitabine combination significantly reduced the proliferation ability of the two cell lines. We also revealed the effect of full-length PARP degradation and reduced Bcl-2/Bax ratio on increased apoptosis. Inhibition of CHK1 expression leads to DNA damage, induces phosphorylation of γ-H2AX, and affects the repair of homologous recombination ability through Rad51. Mechanistically, gemcitabine increased phosphorylation-ATR (pATR) and phosphorylation-CHK1 (pCHK1), indicating activation of the DNA repair system and ATR-CHK1-CDC25A pathway. Inhibition of CHK1 resulted in increased synthesis of CDK2/Cyclin A2 and CDK2/Cyclin E1 complexes, and more cells entered the subsequent cell cycle, leading to S phase arrest and mitotic catastrophe. In conclusion, in our study, we identified inhibition of CHK1 as a potential treatment for NSCLC and confirmed that inhibition of this kinase can overcome acquired gemcitabine resistance.
Research Article
CHK1 Inhibition Overcomes Gemcitabine Resistance in Non-Small Cell Lung Cancer Cell A549
https://doi.org/10.21203/rs.3.rs-1990572/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted
You are reading this older preprint version
Lung cancer is a very common diagnosed cancer and the main cause of cancer death, among which non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer [1]. According to estimates of global cancer incidence and mortality in 2020, among men, lung cancer is the most common cancer (14.3% of all cancer cases) and the leading cause of cancer deaths (21.5% of all cancer deaths), among women, lung cancer is the most common cause of cancer death after breast cancer (13.7% of total cancer deaths) [2]. Compared with most countries, China has a relatively high death rate from lung cancer. It is predicted that the death rate of lung cancer in China may increase by about 40% by 2030 [3]. Due to inadequate screening programs and late onset of clinical symptoms, most patients with lung cancer are already in the middle and late stages when they are diagnosed with poor prognosis. At this time, the best period of surgical treatment has been missed, and drug therapy is the last major means [4]. In recent years, although conventional treatments such as surgery, chemotherapy and radiotherapy have made great progress, the overall 5-year survival rate of lung cancer is only 15%. At present, several driver gene mutations have been found to be used for treatment, but the emergence of acquired drug resistance still inevitably hinders the efficacy of treatment [5].
Gemcitabine (Gem) is a pyrimidine nucleotide analogue, which belongs to anti-metabolic anti-cancer drugs, and is also a specific anti-metabolic drug of cell cycle. It mainly interferes with the process of cycle by acting on the DNA synthesis stage of cell cycle, namely S phase, so as to prevent the self-replication of DNA. Under certain conditions, it can also block progression from G1 phase to S phase. Gemcitabine works by being transported into cells via a special nucleoside transporter. After entering the cell membrane via the nucleoside transporter, gemcitabine underwent complex intracellular conversion to difluorodeoxycytidine diphosphate (dFdCDP) and difluorodeoxycytidine triphosphate (dFdCTP) [6]. DFdCDP is an effective inhibitor of ribonucleotide reductase (RR), which mainly consumes deoxyribonucleotide required for DNA synthesis by inhibiting the activity of RR, and reduces the amount of deoxycytidine diphosphate (dCDP) generated, thus inhibiting the synthesis of DNA [7]. In addition, dFdCTP can bind to DNA, terminating the DNA strand when the extended strand binds to a nucleotide. Because the binding of dFdCTP to DNA seems to be resistant to normal DNA repair mechanisms, this extra nucleotide may be an important reason for dFdCTP not to be detected by repair enzymes, resulting in DNA double-strand breaks and ultimately apoptosis [8, 9]. Despite the success of gemcitabine, its antitumor activity and toxicity vary from person to person, and the development of drug resistance remains an important reason for the low response rate, poor efficacy, and severe toxicity of recurrent tumors.
The normal operation of cell cycle is the result of precise and orderly regulation of various proteins. There is a precise monitoring mechanism in cells to ensure the faithfulness of cell replication, which is called cell cycle checkpoint. When DNA damage or DNA replication is blocked, cell cycle checkpoint sites are activated, stopping the operation of the cell cycle in time, until DNA repair or troubleshooting, the cell cycle can be moved to the next phase. If DNA damage cannot be repaired, apoptosis is initiated. Tumor is a kind of disease in which the regulation mechanism of cell cycle is destroyed. In the whole monitoring system of cell cycle progress, cell cycle checkpoints play a core role. Checkpoint kinase 1 (CHK1) and checkpoint kinase 2 (CHK2) are important protein kinases in cell cycle checkpoint sites, which regulate downstream target protein activity and expression through signal transduction and leading to cell cycle arrest [10, 11]. Chemotherapeutic drug resistance is a major cause of tumor relapse, increased treatment difficulty, failure of therapy, and poor prognosis. And DNA damage response (DDR) is a leading cause to affect tumor chemotherapeutic drug resistance. In recent years, CHK1 has attracted extensive attention in regulating DDR from the whole globe. People expect to cause mitotic catastrophe and promote cell death, and CHK1 is a promising target for cancer therapy [12].
CHK1 is a serine/threonine (Ser/Thr) protein kinase that controls the G2/M phase of DNA damage in fission yeast. The human CHK1 gene is located on chromosome 11q22-23 and is highly conserved in evolution. At the same time, CHK1 is also a significant regulator of DNA damage and replication detection sites, playing a critical role in regulating the cell cycle, participating in DNA damage repair, and influencing cell survival and apoptosis [13]. When some factors break DNA, the ataxia telangiectasia and Rad3 related kinase (ATR) activated and phosphorylated CHK1. And it induced the replication stress pathway and launched the DNA damage repair system. This is called ATR-CHK1-Cell Division Cyclin 25A (CDC25A) pathway [14, 15]. When kinase ATR detects DNA double-strand breaks or long single-strand breaks, upstream ATR transmits DNA damage signals to CHK1 through mediators for activation [16]. CHK1’s activation and phosphorylation inactivates CDC25A phosphatase, which leads to CDK inactivation and the cell cycle arrest in S/G2 or G2/M phases [17, 18]. At present, high expression of CHK1 has been found in a variety of human tumors, such as breast cancer, lung cancer, liver cancer, gastric cancer, and nasopharyngeal cancer [19]. Its expression is often positively correlated with tumor grade and disease recurrence. It was found that tumor cells with high expression of CHK1 were more resistant to DNA damage reactions caused by radiotherapy, chemotherapy, or other tumor treatments, which promoted the generation of tumor cells with a higher degree of malignancy and may also lead to frequent tumor recurrence and drug resistance.
Therefore, we will explore the relationship between CHK1 and gemcitabine resistance through relevant experiments. And explain the reversal of gemcitabine resistance mediated by ATR-CHK1-CDC25A pathway in this article.
2.1 Cell Lines, Culture Conditions, and Reagent
Lung cancer cell line A549 was kindly provided by Cancer Hospital, Chinese Academy of Medical Sciences. Gemcitabine-resistant A549 cells (A549/G+) were generated by continuous administration of gemcitabine at low concentrations for 10 months in our laboratory as previously reported [20]. They were grown in Dulbecco’s modified Eagle’s (DMEM) (Hyclone, USA) and the media was supplemented with 10% fetal bovine serum (FBS; Sangon Biotech, China), 100 U/mL penicillin and 100 µg/mL streptomycin (Solarbio, China) at 37°C in a 5% CO2 atmosphere. To passage the cell lines, each confluent monolayer was washed with phosphate-buffered saline (PBS; Solarbio, China) and detached with a 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) solution (Gibco, USA). Gemcitabine hydrochloride was purchased from Eli Lilly and Company (USA) and prepared in water. To knock down the expression of target genes, cell lines were transfected with small interfering RNA (siRNA) (10 nmol/L). The siRNA sequences were as follows: siCHK1-1, GTGATGGATTGGAGTTCAA; siCHK1-2, GAAAGAGATCTGTATCAAT; siCHK1-3, CAGTGAAGATTGTAGATAT.
2.2 Real-Time Quantitative Polymerase Chain Reaction (qPCR) Assay
Total RNA was isolated from cells with RNAiso Plus (Takara, Japan) and cDNA was produced by the Prime Script RT Master Mix (Takara, Japan). The TB Green Premix Ex Taq™ II Kit (Takara, Japan) was used to detect relative mRNA expression by Real-time quantitative PCR instrument (Thermo Fisher, USA) with 40 cycles of PCR thermocycling. The primer sequences were as follows: GAPDH forward, 5'-CAG GAG GCA TTG CTG ATG AT -3' and reverse, 5'-GAA GGC TGG GGC TCA TTT-3'; CHK1 forward, 5'-TTC AGG TGG TGT GTC AGA GTC TCC-3' and reverse, 5'-TGT GCG GGG TTC TGG CTG AG-3'. The mRNA expression of the target gene was calculated by the 2−ΔΔT method and normalized against the expression level of GAPDH.
2.3 Western Blot Analysis
Protein samples were prepared by lysing cells in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) containing protease inhibitor (Applygen, China) and phosphatase inhibitor cocktail (Beyotime, China) and were stored at 4°C for 30 min, vibrated with a vibrator every 5 minutes. Lysates were centrifuged to obtain protein extracts for western blotting. The BCA method (Thermo Scientific, USA) was used to detect the protein concentration. SDS-PAGE gels (10% gel) (Beyotime, China) were used for protein electrophoresis (equal amount per protein sample), and the separated proteins were transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk in TBS with 0.2% Tween-20 (TBST) and incubated with the following primary antibodies at 4°C overnight: GAPDH (diluted 1:1000, Beyotime, cat. no. AF1186), Cleaved PARP (diluted 1:1000, CST, cat. no. 5625), Bcl-2 (diluted 1:1000, Bioworld, cat. no. BS70205), Bax (diluted 1:10000, Abcam, cat. no. ab32503), phospho-Chk1-Ser345 (diluted 1:1000, CST, cat. no. 2348), CHK1 (diluted 1:10000, Abcam, cat. no. ab40866), Rad51 (diluted 1:1000, Abcam, cat. no. ab133534), phospho-Histone H2AX-Ser139 (diluted 1:1000, CST, cat. no. 9718), Cyclin A2 (diluted 1:2000, CST, cat. no. 4656), Cyclin E1 (diluted 1:1000, CST, cat. no. 4129), RRM2 (diluted 1:10000, Abcam, cat. no. ab172476), CDK2 (diluted 1:1000, CST, cat. no. 2546), phospho-CDK2-Y15 (diluted 1:10000, Abcam, cat. no. ab76146), ATR (diluted 1:1000, CST, cat. no. 13934), phospho-ATR-Ser428 (diluted 1:1000, CST, cat. no. 2853), CDC25A (diluted 1:1000, CST, cat. no. 3652), phospho-Cdc25A-Ser124 (diluted 1:1000, Abcam, cat. no. ab156574). On the next day, the membranes were washed and then incubated for 2 hours at room temperature with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies at the dilution of 1:1000 (cat. no. A0208 and A0216; both from Beyotime, China). Labeled proteins were detected using an enhanced chemiluminescence detection system (Azure Biosystems, USA). All blots were quantified, and the data were expressed as fold changes of the GAPDH using ImageJ software.
2.4 CCK-8 Cytotoxicity Experiments
Cell Counting Kit-8 (CCK-8) Cytotoxicity experiments were used to detect the dose-response and drug combination studies. Briefly, A549 and A549/G+ cells (or cells after the transfection of siNC and siCHK1) were seeded at 3000 cells/well in 96-well plates and cultured overnight. Cells were incubated with various concentrations of gemcitabine (0, 5, 10, 50, 100, and 500 µM) for 72 h. Then, each well was added 10µl of CCK-8 reagent (Dojindo, Japan) and the plates were incubated for 1 hour at 37°C in the dark. The absorbance at 450 nm was detected by the iMark microplate reader (Bio-Rad, USA). The Probit regression model and calculate its IC50 value with SPSS 25.0 software.
2.5 Colony Formation Experiments
Cells after the transfection of siNC and siCHK1 were seeded in 6-well plates at a density of 800 cells/well, allowed to attach for 24 h, and then treated with 0 or10 µM gemcitabine for 24 h. The medium was changed in the middle term of treatment to prevent the drying and evaporation of the culture medium during long-term culture. After 14 days, stop culture and gray-white clonal spots can be seen. Cells were fixed and stained with crystal violet solution. The colonies were visualized and counted with ImageJ software.
2.6 Cell Cycle Assay
After the transfection, the cells were seeded in 6-well plates at a density of 2 × 105 cells /well, allowed to attach for 24 h, and then treated with 0 or10 µM gemcitabine for 24 h. Then, cells were harvested by trypsinization, washed with cold PBS, and fixed with 70% ethanol at 4°C overnight. Next, they were washed with PBS and incubated in the dark with Propidium Iodide (PI)/RNaseA staining solution at 37°C for 30 min. The cell cycle distribution was determined with the Flow cytometer (BD Biosciences, USA) and analyzed by Wincycle 32.
2.7 Cell Apoptosis Assay
For the apoptosis assay A549 and A549/G+ cells were use with trypsin without EDTA to harvest 48 h after drug treatment. Then, the cells were stained with the Annexin V-PE/7-AAD Apoptosis Detection Kit (KeyGEN, China) according to the manufacturer’s instructions. Stained cells were immediately analyzed using a Flow cytometer (BD Biosciences, USA). Early (Annexin V-positive, 7-AAD-negative) and late (Annexin V-positive and 7-AAD-positive) apoptotic cells were included in the determination of cell death. All the data were analyzed by FlowJo 7.6.
2.8 Immunofluorescence Assay
To obtain the morphologic images of DNA damage foci, cells were seeded at 4 × 104 per well in a 24-well plate containing a glass coverslip in each well and then treated with 0 or10 µM gemcitabine for 24 h. After treatment, the cells were fixed in 4% tissue cell fixative fluid (Solarbio, China) for 30 min at 4°C and rinsed three times with PBS. The cells were then permeabilized in 0.5% Triton X-100 (Solarbio, China) and incubated with 10% goat serum (Beyotime, China) to block nonspecific antibody binding for 2 h at room temperature. Subsequently, the cells were immunostained with antibodies against phospho-histone H2AX-Ser139 (diluted 1:400, CST, cat. no. 9718) overnight at 4°C. Followed by 1-hour incubation with Dylight 488, goat anti-rabbit secondary antibody (diluted 1:1000, Abbkine, cat. no. A23220) in the dark at room temperature. DAPI (10 ng/ml) (Solarbio, China) was incubated with cells for 15 min. And the coverslips were washed three times with PBS before mounting. Fluorescence imaging was performed using confocal laser microscopy (Leica, Germany). The cells containing more than 5 γ-H2AX foci were counted under blinded conditions.
2.9 Alkaline Comet Assay
Cells were seeded at 6 × 104 per well in a 12-well plate and treated with 0 or 10 µM doses of the drugs the following day. After cells were harvested, PBS was added to re-suspend, blow and mix into single cell suspension. Add 75 µL 1% ordinary melting point agarose as the first layer of gel, then add 75 µL 0.7% low melting point agarose and 10 µL cell suspension as the second layer of gel, last add 75 µL 0.7% low melting point agarose as the third layer of gel. The cells were treated in lysate at 4°C for 3 h, followed by electrophoresis for 20 min, and stained with 0.1 mg/mL PI dye for 10 min under dark conditions. The results were observed under a fluorescence microscope. One hundred cells were counted randomly in each sample, and the tail length was measured quantitatively by CASP software to assess DNA strand breaks.
2.10 Statistical Analysis
Data were presented as the means ± SD. An unpaired two-tailed t-test or one-way ANOVA were used with Turkey, Dunnett-T3, or Dunnett post-doc test. P < 0.05 was considered significant. Data analysis was used by SPSS Statistics 25.0. Scientific graphing was performed with GraphPad Prism 8.0.
3.1 CHK1 is highly expressed in lung adenocarcinoma tissues
To clarify the correlation between CHK1 and tumor, and to understand the expression difference of CHK1 in tumor tissue and normal tissue, two databases were used for bioinformatics analysis in this study. TIMER 2.0 database showed that CHK1 was generally highly expressed in tumor tissues (Figure S1A). In the ENCORI database 526 lung adenocarcinoma tissues and 59 normal tissues were obtained. And the results showed that the expression of CHK1 in lung adenocarcinoma tissues increased by 5.17 times (P = 3.3e-40) (Figure S1B). Results from both databases showed that CHK1 overexpressed significantly in lung adenocarcinoma tissues and many other tumor tissues.
3.2 siRNA effectively interferes with CHK1 expression and successfully determined gemcitabine-resistant A549 cells
Three siRNA-CHK1 sequences were designed to verify the specificity of the sequences. The siRNA was transfected into A549 cells to interfere with the expression of CHK1. And the siNC group and siCHK1 group were set up to detect the mRNA and protein levels of CHK1 after transfection (Figure S2A, B, and C). Compared with the siNC group, the mRNA and protein levels of CHK1 in the three different siCHK1 sequences were significantly decreased after transfection, indicating that siRNA effectively interferes with the expression of CHK1 protein in cells. Combined with the results of mRNA level and protein level, the siCHK1-1 knockdown effect was the best, and siCHK1-1 was selected as the sequence fragment for all subsequent experiments.
Gemcitabine-resistant A549 cells (A549/G+) were generated by continuous administration of gemcitabine at low concentrations for 10 months. After cell culture for 24 h, sensitive cells and drug-resistant cells were cultured with a gemcitabine solutions of different concentration gradients for 72 h, and the cell survival rate under different concentrations of gemcitabine was calculated (Figure S2D). The results showed that the cell survival rate of the sensitive group was lower than that of the drug-resistant group at the same concentration except for 5 µmol/L. At the same time, the IC50 value of the sensitive group was lower than that of the resistant group (2.834 µmol/L vs 33.582 µmol/L) (Table S1). All of these indicate that the A549 cell line that induced gemcitabine tolerance before is still resistant. And it can be used for further experimental studies.
3.3 CHK1 inhibition combined with gemcitabine reduced cell proliferation ability
We initially examined whether siCHK1 enhances the cytotoxic effects of gemcitabine in A549 and A549/G+ cells. The CCK-8 assay revealed that relative cell viability was lower in the two cell lines treated with the combination of gemcitabine and siCHK1 than in those treated with each one alone (Fig. 1A and B). Similarly, combined treatment with CHK1 inhibition and gemcitabine suppressed the colony formation of two cells (Fig. 1C, D, E, and F). The results of the clone formation experiment were consistent with those of the CCK-8 assay, that is, the proliferation ability of A549 cells and A549/G+ cells could be reduced obviously by combination treatment.
We also found that A549 cells grew faster and were more sensitive to gemcitabine at the same concentration. As can be seen from the clone formation experiment, the density of A549 cells in the siNC group was higher than A549/G+ cells. And the colony cell number of A549 cells was reduced significantly in the siNC + Gem group after treatment. Meanwhile, A549/G+ cells had bigger volume and stronger adhesion ability than A549 cells, which was consistent with the characteristics of drug-resistant cells.
3.4 CHK1 inhibition combined with gemcitabine induce cell apoptosis
To evaluate the potential induction of cell apoptosis, we treated the two cell lines with the reagents alone or in combination with the CHK1 inhibition. And after 48 h of treatment, exposed to an Annexin V/7-AAD solution to allow cell apoptosis determination by flow cytometry. In the bivariate flow cytometry scatter plot, the upper right quadrant stands for the late apoptotic cells (AV+/7-AAD+ cell population); the lower right quadrant signifies early apoptotic cells (AV+/7-AAD− cell population); the lower left quadrant stands for living cells (AV−/7-AAD− cell population); the upper left quadrant stands for the mechanical injury cells (AV−/7-AAD+ cell population). The results showed that the total apoptosis rate (early apoptosis rate + late apoptosis rate) increased significantly in both groups cells after combined treatment (Table 1). In addition, after CHK1 inhibition and gemcitabine treatment, both early and late apoptotic cell populations of A549 cells were significantly increased, and the number of viable cells decreased significantly. However, in A549/G+ cells, the number of early apoptotic cells increased, and that of late apoptotic cells did not change (Fig. 2). We believed that under the same treatment conditions, A549/G+ cells were partially resistant to apoptosis after combined treatment, and inhibition of CHK1 alone also significantly changed the apoptosis rate of the two cell lines.
Subsequently, the expression levels of apoptosis-related proteins Cleaved PARP, Bcl-2, and Bax were detected by Western blot. Cleaved poly(ADP-ribose)-polymerase (Cleaved-PARP) is an indicator of apoptosis. Results showed that compared with the siNC group, Cleaved PARP in the siCHK1 group and siCHK1 + Gem group were increased, indicating an increased cell apoptosis rate. Bcl-2 was an anti-apoptosis protein, and both cells showed low expression of Bcl-2 after combined treatment. Meanwhile, the expression of pro-apoptotic indicator Bax did not change. Using the Bcl-2/Bax ratio to indicate the overall apoptosis level, the apoptosis degree of the siCHK1 + Gem group of both cells increased significantly, indicating an increased apoptosis rate, which was consistent with the results of the flow cytometry apoptosis experiment (Fig. 3).
3.5 CHK1 inhibition combined with gemcitabine induced DNA damage response and affected homologous recombination repair ability
We have observed the degree of DNA double strand breaks caused by combined treatment in the same group of A549 and A549/G+ by immunofluorescence test and neutral comet assay. γ-H2AX is a marker of DNA double-strand fracture damage. Immunofluorescence results showed that the siCHK1 + Gem group produced a large proportion of γ-H2AX green fluorescence. And most of the green fluorescence is around the periphery of the nucleus. It was not the focalization of γ-H2AX but the panucleation of γ-H2AX, and it was the result of more DNA damage and DNA replication stress caused by the combined treatment. In the A549 cell line, compared with the siNC group, the siCHK1 and siCHK1 + Gem groups showed different degrees of cell morphology changes, such as nuclear vacuolation, and apoptotic bodies. But A549/G+ cells did not obviously show such morphological change. All of these indicated that the combined treatment had a more intense damage effect on sensitive cell strains (Fig. 4A and C).
The neutral comet experiment can effectively detect and quantitatively analyze the degree of DNA damage in cells. The more serious the damage is the more DNA chain breaks. Under the same electrophoresis conditions, the longer the distance of cell migration and the longer the tail length are. The results showed that both siCHK1 + Gem groups produced a long comet trailing phenomenon (Figs. 4B and D).
Subsequently, we detect the expression levels of γ-H2AX and Rad51 with a Western blot assay. The results showed that compared with the siNC group, the expression level of γ-H2AX in the siCHK1 group and the siCHK1 + Gem group was significantly increased, indicating that the combined treatment increased the accumulation of γ-H2AX produced by DNA damage. A549 cells damaged by gemcitabine treatment alone, and the expression of γ-H2AX increased, but it did not affect A549/G+. The pCHK1 was activated and its expression increased under the action of drugs. Rad51 is a key protein in the HR repair pathway. The stronger the HR repair ability, the higher the expression level of Rad51. The result showed that the expression of Rad51 in A549 cells was decreased after CHK1 inhibition, indicating that medication resulted in a relatively reduced repair ability. While the expression level of Rad51 was enhanced in A549/G+ cells after gemcitabine treatment, indicating that drug-resistant cells had better repair ability than sensitive cells. The combined treatment reduced the expression of Rad51 in A549 cells. For A549/G+ cells, the expression level of Rad51 in the siCHK1 + Gem group was lower than that in the siNC + Gem group, indicating that the combined treatment reduced the activity of homologous recombination and the repair ability was relatively reduced. At this time, the repair ability could not completely repair the cell damage, resulting in cell death (Fig. 5).
3.6 CHK1 inhibition combined with gemcitabine resulted in S phase arrest and RRM2 down regulation
Cell cycle experiments were performed on A549 and A549/G+ cells in the same group to observe the cell cycle changes caused by CHK1 inhibition combined with gemcitabine. The results showed that gemcitabine increased the number of cells in the G1 phase and decreased the number of cells in the S phase in the siNC + Gem group in A549 cells, indicating that gemcitabine can block A549 cells in the G1 phase. As for A549/G+ cells, the number of cells in the S phase increased in the siNC + Gem group under gemcitabine treatment. These indicated that the drug could block A549/G+ cells at the S phase, which also manifested that A549/G+ could partially tolerate gemcitabine treatment, making the cell cycle passed the G1 phase.
Compared with the siNC group, the siCHK1 group had more S phase cells in A549. The S phase cell number of the siNC + Gem group decreased after drug treatment. But the siCHK1 + Gem group could partially recover the reduced S phase, increasing the S phase cell number, which demonstrated that more A549 cells could pass the G1 phase and reach the S phase after combined treatment (Fig. 6A and B). The same phenomenon also appeared in A549/G+ cells, siCHK1 group increased the number of S phase cells, siCHK1 + Gem group further significantly increased the proportion of S phase cells. All of this indicated that after combined treatment, drug-resistant cells might be more likely than sensitive cells to pass the G1 phase and finally be blocked in the S phase (Fig. 6C and D). Damaged cells are not repaired and enter the abnormal cell cycle, resulting in mitosis disaster.
Then, we used Western blot to detect the expression levels of cell cycle-related protein Cyclin A2, Cyclin E1, RRM2, CDK2, and pCDK2. It is a necessary condition for initiating and progressing G2 phase events that CDK2 binds Cyclin A2. Meantime, CDK2 needs to bind Cyclin E1 to trigger the S phase. When CDK2 dephosphorylation is activated, the level of its phosphorylated form pCDK2 (Y15) reduces. The results showed that the expression levels of free CDK2, Cyclin A2, and Cyclin E1 decreased after CHK1 knockdown treatment in both cell lines whether to add gemcitabine or not (Fig. 7). These indicated the binding between CDK2/Cyclin A2 and CDK2/Cyclin E1 was increasing. These complexes can induce the cells to enter the subsequent S and G2 phases. The expression of pCDK2 (Y15) decreased after activation, showing that CDK2 further completed the binding with cyclin A2 and E1. As Fig. 7, under the action of gemcitabine, RRM2 was activated, and then its expression increased, while siCHK1 + Gem group treatment reduced RRM2. The results indicated that the combined treatment made cells sensitive to gemcitabine and promoted cell death. In short, it helped cells overcome resistance to gemcitabine.
3.7 CHK1 inhibition combined with gemcitabine affects the ATR-CHK1-CDC25A pathway to overcome gemcitabine resistance
By reviewing the literature on cellular replication stress and upstream and downstream pathways related to CHK1 protein, Western blot was used to detect the expression of ATR-CHK1-CDC25A pathway-related proteins ATR, pATR (Ser428), CHK1, pCHK1 (Ser345), CDC25A and pCDC25A (Ser124) in each group of cells. The results showed that there was no significant change in protein levels of ATR and pATR in each group of A549 cells, but ATR and pATR were all activated by drugs in A549/G+ cells. The expression levels of CHK1 and pCHK1 decreased significantly after knockout treatment. Gemcitabine activated pCHK1 and made its expression level increased. According to relevant literature, pCHK1 inhibits CDC25A through phosphorylation. CDC25A, as a dephosphorylated protein, decreases its phosphorylation state when activated. pCDC25A expressed significantly reduced after CHK1 knockdown (Fig. 8).
When drugs damage cells, the ATR-CHK1-CDC25A pathway will be activated. Under normal regulation, ATR-CHK1 regulates CDC25A negatively, so CDC25A will not be activated, and neither will its downstream CDK2. That is, CDK2 cannot bind to Cyclin A2 and Cyclin E1. The CDK2/Cyclin A2 and CDK2/Cyclin E1 complexes could not be formed between the two cells, leading to the failure of cell cycle progression, and the two cell lines were blocked, with A549 cells blocked in the G1 phase and A549/G+ cell blocked in S phase. After siRNA inhibited CHK1's expression, CDC25A was activated to release the phosphorylated group. And CDK2 was activated and combined with Cyclin A2 and Cyclin E1, resulting in the damaged cells crossing the monitoring point of cell cycle detection and entering the subsequent cell cycle. These could cause mitotic disaster finally. Thus, resistant cells A549/G+ can achieve the effect of overcoming gemcitabine resistance (Fig. 9).
Lung cancer is a malignant tumor with high incidence and mortality in China and in the world, while non-small cell lung cancer accounts for about 85% of all lung cancers [21]. Previous studies have demonstrated synergies between gemcitabine and other anticancer agents in experimental models[22, 23]. A combination of gemcitabine with other anticancer drugs is a first-line treatment option. However, acquired drug resistance often leads to clinical lung cancer treatment failure. In this study, we demonstrated that inhibition of CHK1 enhanced the activity of gemcitabine. In addition, the combination therapy proposed in this study showed synergistic effects even in chemotherapy-resistant lung cancer A549/G+, suggesting the possibility of overcoming drug resistance.
CHK1 is an all-important protein kinase in cell cycle detection sites, which regulates the activity and expression of downstream target proteins through signal transduction and amplification to block cell cycle. When DNA damage occurs, CHK1 can provide time for DNA damage repair by inducing cell cycle delay or arrest [24]. When CHK1 is inhibited, DNA damage cannot be repaired without cell cycle arrest, and cells carry the damaged DNA into mitosis, resulting in cell death. Some studies have reported that CHK1 inhibitors increase the activity of various DNA damage or DNA synthesis inhibition therapies in different cancer cells [25], such as etoposide [26], cisplatin [27–29], gemcitabine [30–33] and PARP inhibitors [34]. Our results are consistent with those of these reports.
Continuous exposure of cells to genotoxic substances can damage DNA. In response to DNA damage, cells have evolved a complex mechanism called the DNA damage response that detects damage and promotes DNA repair. DNA single-strand breaks are discontinuations of one of the DNA double strands monitored by ATR that subsequently activates the downstream protein CHK1. Unrepaired single strand breaks lead to DNA replication stress, which is transformed into DSB during the S phase, leading to genomic instability[35]. Immunofluorescence results showed that the coverage of green fluorescence was not the same as common foci, which mostly surrounded the periphery of the nucleus. Instead of γ-H2AX focusing, it was a kind of γ-H2AX panucleation, which is the outcome of broader DNA damage and DNA replication stress caused by the combined treatment. This extensive and uniform phosphorylation of nuclear γ-H2AX is proportional to the intensity of induced replication stress. Moeglin et al. [36]found that increasing combinations of ATR and CHK1-inhibiting drugs were used to treat cancer cells, and the cell death was positively correlated with the appearance of pan-nuclear γ-H2AX pattern. Ewald et al. [37] proposed that the dense pan-nuclear location of γ-H2AX after the deletion of the detection point may mark the change of the entire nuclear DNA structure. In clinical application, the panuclear γ-H2AX phosphorylation pattern can be used as an indicator of drug efficacy in replicating stress-dependent cell death.
Much literature found that CHK1 protects cells from DNA damage-induced replication stress by regulating the HR pathway and that chemical sensitization of CHK1 inhibition is due to blocking the HR pathway [26, 38]. In addition, Rad51 is not only a vital protein in the HR pathway, and its gene family plays different roles in the repair of double-strand break, replicative stress, and meiosis, but also a critical regulator of DNA fidelity. And Rad51 has the function of discovering and invading homologous DNA sequences and can repair DNA accurately and timely [39]. Yang et al. thought that DNA repair ability plays a vital role in drug resistance of tumor cells [40]. On the other hand, Li et al. [41] demonstrated that inhibition of DNA repair ability could enhance cell sensitivity to cisplatin in cisplatin-resistant A549 cells. In addition, DSB repair induced by chemotherapy drugs may start tumor cells to develop drug resistance [42]. Our results showed that the combined treatment reduced the expression of Rad51 in both A549 cells and A549/G+, suggesting that the combined treatment could inhibit the HR repair pathway. Ultimately, catastrophic DNA damage accelerates the process of apoptosis, which provides a new option for improving gemcitabine sensitivity in A549.
Cell cycle results showed that under the treatment of gemcitabine, A549 cells were blocked in the G1 phase, while A549/G+ cells were blocked in the S phase, indicating that A549/G+ could partially tolerate gemcitabine treatment. As everyone knows that the G1 phase is the main regulatory point for cell cycle initiation and cell proliferation. The successful transformation from the G1 phase to the S phase indicates that the cell can complete DNA replication, and then complete cell division and synthesis of other related biological macromolecules [43, 44]. Therefore, the failure of cell cycle arrest in the G1 phase may be the main reason for drug resistance of A549/G+ cells. However, after CHK1 inhibition, both cells were more blocked in the S phase, and the proportion of A549/G+ blocked in the S phase was significantly increased. These suggested that drug-resistant cells may be more likely than sensitive cells to pass the G1 phase and finally be arrested in the S phase.
RR is a tetramer consisting of two homodimeric subunits. Every subunit contains ribonucleotide reductase regulatory subunit M1 (RRM1) and RRM2 [45, 46]. Jin et al. [47] found differential enrichment in cell cycle, P53 signaling pathway, DNA replication, apoptosis, and cancer pathways in patients with high expression of RRM2. Overexpression of RRM2 is common in chemotherapy-resistant cancer cells and patients. Therefore, RRM2 is usually considered a tumor promoter and tumor therapeutic target. Because RRM2 mediates resistance to many chemotherapy drugs, it can become a predictor of chemotherapy sensitivity and prognosis[48]. In addition, the upregulation of RRM2 is related to gemcitabine resistance of different clinical tumors [49]. Our study found that the expression of RRM2 decreased after CHK1 inhibition, indicating that the two cell lines became sensitive to drug action after CHK1 inhibition, which helped A549/G+ cells overcome their drug resistance.
When DNA fracture occurs, CHK1 is activated by ATR phosphorylation, forming a classical signal transduction pathway, namely, the ATR-CHK1-CDC25A signaling pathway, which can co-regulate cell cycle progression with CDK2. CDKs must bind to corresponding Cyclins to function in the cell cycle. Cyclin E1 can bind to CDK2 or CDK3, promote the transformation of cells from the G1 phase to the S phase, and shorten the time for cells to pass the detection point of the phase G1/S, thus leading to genomic instability. Therefore, abnormal expression of Cyclin E1 can mediate chemotherapy resistance [50, 51]. Cyclin A2 is usually associated with cell proliferation and is mainly expressed in the S phase, mediating the initiation and transport of the S phase, and is often highly expressed in human cancers [52]. Cyclin A2 has been shown to be critical for DNA synthesis by initiating and regulating DNA replication and promoting G2/M transition by binding CDK2[53]. CDK2 is also responsible for the S phase, and its binding with Cyclin E1 is a necessary condition for initiating the S phase. Our study confirmed that when we inhibited the expression of CHK1, the expression of free Cyclin A2, Cyclin E1, and CDK2 decreased, indicating that more CDK2 completed the binding with Cyclin A2 and Cyclin E1. The combination of the two complex causes the damaged cells to enter the later S and G2 phases, facilitating the transition of the cell cycle and causing the mitotic catastrophe.
Although our paper has some vital results, our study has limitations. We investigated the effects of CHK1 inhibition on lung cancer cell line A549 and its gemcitabine resistance cells A549/G+. However, our findings cannot be simply generalized to all lung adenocarcinoma cells. It is unclear whether there may be other repair pathways after CHK1 affects DNA damage.
Our results found that CHK1 inhibition combined with gemcitabine had a better anti-tumor effect on A549 cells. Furthermore, our study demonstrates the promise of drugs that target the detection of checkpoint kinase and DNA replication stress responses as therapeutic strategies for lung cancer. Therefore, we support the development of this approach for clinical studies of CHK1 inhibitors in combination with gemcitabine in patients with advanced lung cancer.
Conflict of Interest:
None to report
- Nawaz K, Webster RM. The non-small-cell lung cancer drug market. Nat Rev Drug Discov, 2016, 15(4):229–230.
- Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin, 2021, 71(3):209–249.
- Cao M, Chen W. Epidemiology of lung cancer in China. Thorac Cancer, 2019, 10(1):3–7.
- Gridelli C, Rossi A, Carbone DP, et al. Non-small-cell lung cancer. Nat Rev Dis Primers, 2015, 115009.
- Politi K, Ayeni D, Lynch T. The Next Wave of EGFR Tyrosine Kinase Inhibitors Enter the Clinic. Cancer Cell, 2015, 27(6):751–753.
- Mini E, Nobili S, Caciagli B, et al. Cellular pharmacology of gemcitabine. Ann Oncol, 2006, 17 Suppl 5v7-12.
- Baker CH, Banzon J, Bollinger JM, et al. 2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase. J Med Chem, 1991, 34(6):1879–1884.
- Li Y, Wang LR, Chen J, et al. First-line gemcitabine plus cisplatin in nonsmall cell lung cancer patients. Dis Markers, 2014, 2014960458.
- Jones RM, Kotsantis P, Stewart GS, et al. BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine. Mol Cancer Ther, 2014, 13(10):2412–2421.
- Patil M, Pabla N, Dong Z. Checkpoint kinase 1 in DNA damage response and cell cycle regulation. Cell Mol Life Sci, 2013, 70(21):4009–4021.
- Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature, 2004, 432(7015):316–323.
- Carrassa L, Damia G. DNA damage response inhibitors: Mechanisms and potential applications in cancer therapy. Cancer Treat Rev, 2017, 60139–151.
- Sanchez Y, Wong C, Thoma RS, et al. Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science, 1997, 277(5331):1497–1501.
- Rundle S, Bradbury A, Drew Y, et al. Targeting the ATR-CHK1 Axis in Cancer Therapy. Cancers (Basel), 2017, 9(5).
- Niida H, Nakanishi M. DNA damage checkpoints in mammals. Mutagenesis, 2006, 21(1):3–9.
- Montano R, Thompson R, Chung I, et al. Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo. BMC Cancer, 2013, 13604.
- Smits VAJ, Gillespie DA. DNA damage control: regulation and functions of checkpoint kinase 1. FEBS J, 2015, 282(19):3681–3692.
- Goto H, Natsume T, Kanemaki MT, et al. Chk1-mediated Cdc25A degradation as a critical mechanism for normal cell cycle progression. J Cell Sci, 2019, 132(2).
- van Harten AM, Buijze M, van der Mast R, et al. Targeting the cell cycle in head and neck cancer by Chk1 inhibition: a novel concept of bimodal cell death. Oncogenesis, 2019, 8(7):38.
- Liang A-L, Du S-L, Zhang B, et al. Screening miRNAs associated with resistance gemcitabine from exosomes in A549 lung cancer cells. Cancer Manag Res, 2019, 116311–6321.
- McGuire S. World Cancer Report 2014. Geneva, Switzerland: World Health Organization, International Agency for Research on Cancer, WHO Press, 2015. Adv Nutr, 2016, 7(2):418–419.
- Teng JP, Yang ZY, Zhu YM, et al. Gemcitabine and cisplatin for treatment of lung cancer in vitro and vivo. European review for medical and pharmacological sciences, 2018, 22(12):3819–3825.
- Peters GJ, Bergman AM, Ruiz van Haperen VW, et al. Interaction between cisplatin and gemcitabine in vitro and in vivo. Semin Oncol, 1995, 22(4 Suppl 11):72–79.
- Brooks T, Wayne J, Massey AJ. Chk1 inhibition induces a DNA damage bystander effect in cocultured tumour cells. DNA Repair (Amst), 2021, 101103099.
- Calvo E, Chen VJ, Marshall M, et al. Preclinical analyses and phase I evaluation of LY2603618 administered in combination with pemetrexed and cisplatin in patients with advanced cancer. Invest New Drugs, 2014, 32(5):955–968.
- Fan Z, Luo H, Zhou J, et al. Checkpoint kinase–1 inhibition and etoposide exhibit a strong synergistic anticancer effect on chronic myeloid leukemia cell line K562 by impairing homologous recombination DNA damage repair. Oncol Rep, 2020, 44(5):2152–2164.
- Endersby R, Whitehouse J, Pribnow A, et al. Small-molecule screen reveals synergy of cell cycle checkpoint kinase inhibitors with DNA-damaging chemotherapies in medulloblastoma. Sci Transl Med, 2021, 13(577).
- Hsu W-H, Zhao X, Zhu J, et al. Checkpoint Kinase 1 Inhibition Enhances Cisplatin Cytotoxicity and Overcomes Cisplatin Resistance in SCLC by Promoting Mitotic Cell Death. J Thorac Oncol, 2019, 14(6):1032–1045.
- Sen T, Tong P, Stewart CA, et al. CHK1 Inhibition in Small-Cell Lung Cancer Produces Single-Agent Activity in Biomarker-Defined Disease Subsets and Combination Activity with Cisplatin or Olaparib. Cancer Res, 2017, 77(14):3870–3884.
- Isono M, Okubo K, Asano T, et al. Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells. Sci Rep, 2021, 11(1):10181.
- Morimoto Y, Takada K, Takeuchi O, et al. Prexasertib increases the sensitivity of pancreatic cancer cells to gemcitabine and S–1. Oncol Rep, 2020, 43(2):689–699.
- Drápela S, Khirsariya P, van Weerden WM, et al. The CHK1 inhibitor MU380 significantly increases the sensitivity of human docetaxel-resistant prostate cancer cells to gemcitabine through the induction of mitotic catastrophe. Mol Oncol, 2020, 14(10):2487–2503.
- Liang M, Zhao T, Ma L, et al. CHK1 inhibition sensitizes pancreatic cancer cells to gemcitabine via promoting CDK-dependent DNA damage and ribonucleotide reductase downregulation. Oncol Rep, 2018, 39(3):1322–1330.
- Parmar K, Kochupurakkal BS, Lazaro J-B, et al. The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition. Clin Cancer Res, 2019, 25(20):6127–6140.
- Caldecott KW. Single-strand break repair and genetic disease. Nat Rev Genet, 2008, 9(8):619–631.
- Moeglin E, Desplancq D, Conic S, et al. Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress. Cancers (Basel), 2019, 11(3).
- Ewald B, Sampath D, Plunkett W. H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation. Mol Cancer Ther, 2007, 6(4):1239–1248.
- Meyer F, Becker S, Classen S, et al. Prevention of DNA Replication Stress by CHK1 Leads to Chemoresistance Despite a DNA Repair Defect in Homologous Recombination in Breast Cancer. Cells, 2020, 9(1).
- Scully R, Panday A, Elango R, et al. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol, 2019, 20(11):698–714.
- Yang S-Y, Li Y, An G-S, et al. DNA Damage-Response Pathway Heterogeneity of Human Lung Cancer A549 and H1299 Cells Determines Sensitivity to 8-Chloro-Adenosine. Int J Mol Sci, 2018, 19(6).
- Li X-Q, Ren J, Chen P, et al. Co-inhibition of Pol η and ATR sensitizes cisplatin-resistant non-small cell lung cancer cells to cisplatin by impeding DNA damage repair. Acta Pharmacol Sin, 2018, 39(8):1359–1372.
- Gil Del Alcazar CR, Todorova PK, Habib AA, et al. Augmented HR Repair Mediates Acquired Temozolomide Resistance in Glioblastoma. Mol Cancer Res, 2016, 14(10):928–940.
- Kar S. Unraveling Cell-Cycle Dynamics in Cancer. Cell systems, 2016, 2(1).
- Dalton S. Linking the Cell Cycle to Cell Fate Decisions. Trends in cell biology, 2015, 25(10):592–600.
- Bothou C, Sharma A, Oo A, et al. Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment. Cancers (Basel), 2021, 13(16).
- Thelander L, Eriksson S, Akerman M. Ribonucleotide reductase from calf thymus. Separation of the enzyme into two nonidentical subunits, proteins M1 and M2. J Biol Chem, 1980, 255(15):7426–7432.
- Jin C-Y, Du L, Nuerlan AH, et al. High expression of RRM2 as an independent predictive factor of poor prognosis in patients with lung adenocarcinoma. Aging (Albany NY), 2020, 13(3):3518–3535.
- Zhan Y, Jiang L, Jin X, et al. Inhibiting RRM2 to enhance the anticancer activity of chemotherapy. Biomed Pharmacother, 2021, 133110996.
- Aye Y, Li M, Long MJC, et al. Ribonucleotide reductase and cancer: biological mechanisms and targeted therapies. Oncogene, 2015, 34(16):2011–2021.
- Gorski JW, Ueland FR, Kolesar JM. CCNE1 Amplification as a Predictive Biomarker of Chemotherapy Resistance in Epithelial Ovarian Cancer. Diagnostics (Basel), 2020, 10(5).
- Zhao Z-M, Yost SE, Hutchinson KE, et al. CCNE1 amplification is associated with poor prognosis in patients with triple negative breast cancer. BMC Cancer, 2019, 19(1):96.
- Yasmeen A, Berdel WE, Serve H, et al. E- and A-type cyclins as markers for cancer diagnosis and prognosis. Expert Rev Mol Diagn, 2003, 3(5):617–633.
- Blanchard JM. Cyclin A2 transcriptional regulation: modulation of cell cycle control at the G1/S transition by peripheral cues. Biochem Pharmacol, 2000, 60(8):1179–1184.
Table 1. Total apoptosis rate of CHK1 inhibition combined with gemcitabine
|
Group |
A549（ |
A549/G+（ |
|
siNC |
18.47 ± 3.31 |
14.02 ± 1.36 |
|
siCHK1 |
29.97 ± 1.83 |
23.97 ± 1.86 |
|
siNC+Gem |
23.40 ± 2.84 |
16.06 ± 0.34 |
|
siCHK1+Gem |
45.60 ± 3.90 |
42.90 ± 0.83 |
）
- FigureS1.jpg
Figure S1. Expression of CHK1 in tumors and normal tissuesA: Expression level of CHK1 in tumors and normal tissues; B: Expression level of CHK1 in lung adenocarcinoma and normal tissues. ***: P<0.001, **: P<0.01, *: P<0.05.
- FigureS2.jpg
Figure S2. Validation of siRNA fragments and A549 gemcitabine resistant cellsA: Difference of CHK1 mRNA levels between siNC group and siCHK1 group after siRNA transfection; B, C: Differences of CHK1 protein levels between siNC group and siCHK1 group after siRNA transfection; D: Cell survival rate of A549 sensitive and resistant strains. ***: P<0.001, **: P<0.01.
- TableS1.docx
Table S1. Semi-inhibitory concentrations of gemcitabine sensitive and resistant cells
