EGCG synergizes the therapeutic effect of irinotecan through enhanced DNA damage in human colorectal cancer cells

Abstract Irinotecan is a kind of alkaloid with antitumour activity, but its low solubility and high toxicity limit its application. Epigallocatechin‐3‐gallate (EGCG) is one of the main bioactive components in tea. The epidemiological investigation and animal and cell experiments show that EGCG has a preventive and therapeutic effect on many kinds of tumours. Here, colorectal cancer cells RKO and HCT116 were employed, and the CCK8 proliferation test was used to screen the appropriate concentration of EGCG and irinotecan, and the effects of single and/or combined drugs on migration, invasion, DNA damage, cell cycle and autophagy of tumour cells were investigated. The results showed that EGCG combined with irinotecan (0.5 μmol L−) not only had a stronger inhibitory effect on tumour cells than EGCG or irinotecan alone but also prevented tumour cell migration and invasion. EGCG alone did not cause DNA damage in colorectal cancer cells, but its combination with irinotecan could induce S or G2 phase arrest by inhibiting topoisomerase I to cause more extensive DNA damage. EGCG also induced apoptosis by promoting autophagy with irinotecan synergistically. These results indicated that EGCG in combination with irinotecan could be a promising strategy for colorectal cancer.

form the TOP-IRI-DNA complex and stabilize it. The collision of the complex with the advancing replication fork results in a fatal double-strand break (DSB) that leads to apoptosis. 4 However, single cancer chemotherapeutic drugs tend to develop resistance or toxicity over time, 5 and the dose-limiting toxicity of irinotecan is mainly neutrophil and delayed diarrhoea, other common side effects include vomiting, myelosuppression, alopecia, dyspnoea and fever. 6 Therefore, the strategy of reducing the side effects and even increasing the antitumour activity of chemotherapeutic drugs by combination therapy has been paid more and more attention.
Some natural active compounds such as EGCG (epigallocatechin-3-gallate) and resveratrol have been revealed antitumour activity in vitro or animal experiments. 7,8 EGCG combined with chemotherapeutic drugs such as Gefitinib 9 and Bleomycin 10 can reduce both drug dose and resistance, and it also shows a good synergy effect.
Researchers are beginning to try to use these compounds as anticancer adjuvants to enhance the antitumour activity of clinical chemotherapeutic drugs. 11 Our study focused on the synergistic anti-colorectal cancer effect of EGCG combined with irinotecan, a DNA damage chemotherapeutic agent. The collaborative anticolorectal cancer effect of EGCG on irinotecan was demonstrated by cell proliferation, migration, invasion and cell cycle, and the molecular mechanism of EGCG was discussed in terms of DNA damage and autophagy pathway. from Proteintech Group. Goat anti-Rabbit IgG-FITC antibody was purchased from Hua'an. Annexin V-FITC Apoptosis assay kit was purchased from Vazyme. A cell cycle and apoptosis assay kit was purchased from Beyotime Biotechnology.

| Cell culture
RKO cells were resuscitated from our laboratory, and HCT116 cells were obtained from NuoHe Bio-Tech; all the cells were grown in RPMI-1640 medium (Thermo Fisher Scientific) containing 10% (vol/vol) foetal bovine serum (Zhong Qiao Xin Zhou Biotechnology) and added 1% (vol/vol) Penicillin-Streptomycin (HyClone). Cells were cultured in a Forma Series II Water Jacket CO 2 Incubator (Thermo Fisher Scientific Instruments) at 37°C in a 5% CO 2 atmosphere.

| Cell proliferation assay
EGCG was dissolved in PBS to form a 21.8 mmol L − reserve liquor and irinotecan in DMSO to 50 mmol L − and then diluted to the corresponding working concentration by RPMI-1640 serum-free medium. RKO and HCT116 cells were inoculated in a 96-well plate and then incubated at 37°C for 60 minutes more with 10% (vol/vol) of CCK8 dissolved in serum-free medium RPMI-1640; after 24 hours of irinotecan and/or EGCG treatment, the absorbance at 450nm was measured by iMark (Bio-Rad Laboratories).

| Cell migration assay
Cells were inoculated in a 12-well plate one day ahead of schedule.
When the cells adhered to the wall and converged to 80%-90%, a 200-μL pipet tip was used to split the cells. After washed off the cells with PBS, the serum-free medium was reloaded, and the appropriate concentration of irinotecan and/or EGCG was added. A DM2500 fluorescence microscope (Leica Microsystem) was applied to observe and photograph.

| Cell invasion test
The cells in the logarithmic growth phase were replaced by serum-free medium and then starved for 24 hours. 2 mL Matrigel was thawed in 4°C ahead of time and mixed with 98 mL serum-free medium, and the concoction was placed in the centre of the upper chamber for 3 hours at 37°C to polymerize. A 600 μL medium containing 20% serum was added to the lower chamber. The upper chamber of Matrigel was carefully placed into the 24-well. The cells digested by trypsin were cultured for 24 hours with a density of 1.5 × 10 5 cells/mL, and 300 μL serum-free cell suspension was laid in the upper chamber.
After incubation, the culture medium was discarded from the upper chamber, then washed with PBS and gently wiped off the remaining cells and Matrigel with a cotton swab. The cells were immersed in methanol solution for 15 minutes, and then, the cells were stained in 0.1% methanol-soluble crystal violet dye for 15 minutes, and the purple colour was washed off by pure water. The upper chamber was placed in a 37°C incubator to dry, and cells were photographed with a DM2500 fluorescence microscope.

| Western blot
Protein was extracted from the treated RKO and HCT116 cells in a 6-well plate with RIPA lysate (1% protease), separated by 10% SDS-PAGE and transferred to the NC membrane (Pall Corporation, New York, USA). 0.05% TBST solution with 5% skimmed milk powder was applied to block at room temperature for 1 hour. The membrane was incubated with the corresponding antibody in 0.05% TBST solvent at 4°C overnight, and the HRP-linked secondary antibody (Signal Antibody) was added at room temperature for 1 hour. ECL substrate (JuHeMei Biotechnology) was utilized and photograph was obtained by Universal Hood II Gel Imaging System (Bio-Rad Instrument).

| Immunofluorescence
According to the CST product instructions, in short, the cells were seeded on a 12-well plate, fixed with 4% paraformaldehyde for 15 minutes and washed three times with PBS for 5 minutes each.
After blocking (1 × PBS/5% normal serum/0.3% Triton TM ×-100) for 60 minutes, the antiγ-H2AX antibody in dilution buffer (1 × PBS/1% Triton TM ×-100) was added for 24 hours in 4°C. The cells were rinsed three times and hatched by Goat anti-Rabbit IgG-FITC antibody for 1 hour at room temperature. Then, the plate was stained with DAPI for 5 minutes and took photographs with a fluorescence microscope.

| Cell cycle distribution measured by flow cytometry
Cells treated in a 12-well plate are operated according to the manufacturer's instructions. In short, the cells were digested with trypsin and centrifuged at 1500 g for 5 minutes to collect in a tube. Add 1 mL pre- was employed for DNA content analysis using the PI channel.

| Cell apoptosis detected by flow cytometry
Cells treated in a 12-well plate are managed according to the manufacturer's instructions. Briefly, the cells were digested with trypsin without EDTA and centrifuged at 2000 g for 5 minutes. After rinsing with PBS and centrifuging for 5 minutes at 1000 g, Annexin V-FITC and PI staining were applied for 15 minutes and detected on the flow cytometry through FITC and PI channels.

| Autophagy observed by MDC staining
According to the method in literature, 12

| Data processing and statistical analysis
All the experiments were repeated 3 times, and the data format was mean ± standard deviation. GraphPad Prism 7 (Graph Pad Software) was used for data statistics and mapping. The significance test was performed by one-way ANOVA and multiple comparison with Bonferroni correction. The data from flow cytometry were processed by FlowJo (TreeStar) or ModFit (Verity Software House).

| EGCG and irinotecan synergistically inhibited the proliferation of colorectal cancer cells
To investigate the synergistic effect of EGCG on the antitumour cell proliferation of irinotecan, Cell Counting Kit-8 was used to determine the 24-hour inhibition rate of EGCG and/or irinotecan. As shown in

| EGCG and irinotecan synergistically inhibited the migration and invasion of colorectal cancer cells
The basic principle of the wound healing experiment is to create a blank, cell-free area in the monolayer of cells on the board. After a period of cultivation, the cells will come into contact with each other again. It is often used to detect the migration ability of tumour cells. As shown in Figure 2 (A, B) and Figure S1, either irinotecan alone, EGCG alone or combination of the two drugs inhibited the migration of both colorectal cancer cells, and the co-treatment with EGCG and irinotecan led to significantly additive inhibition than either drug alone.

| EGCG augmented irinotecan-induced DNA damage
Irinotecan and its derivatives, which inhibit topoisomerase I and cause DNA damage, are often used as chemotherapy drugs to treat various cancers. 13,14 EGCG also induces DNA damage in human normal and cancer cells. 15,16 Irinotecan increased the DNA damage marker γ-H2AX in nuclear compared with the control group.
EGCG alone did not cause significant DNA damage but combined with irinotecan induced more severe DNA damage. Besides, immunofluorescence results indicated that HCT116 cells with or without irinotecan accumulated in chromatin and formed nuclear fragments, and the nuclear volume increased significantly ( Figure S3). WB results showed that the concomitant effect of EGCG on DNA damage may be related to the inhibition of topoisomerase I and increased with time (Figure 3 A, B).
EGCG can increase the cleavage of ataxia telangiectasia-mutated (ATM) and p-ATM proteins by co-treatment with irinotecan. In RKO and HCT116 cells, when the concentration of EGCG was 50 μmol L − and 20 μmol L − , respectively, the combination induced more obvious ATM fragmentation than any single drug. During apoptosis, the cleavage of ATM produces a kinase-free protein that, through its DNA binding ability, inhibits DNA repair and DNA damage signals in a trans-dominant-negative manner. Therefore, the expression of the S-139 phosphorylated product of γ-H2AX was positively correlated with the disintegration of ATM ( Figure 3A). 17

| EGCG combined with irinotecan induced cell cycle arrest
The eukaryotic cell cycle is usually divided into G1, S, G2 and M phases. Cell cycle checkpoints composed of receptors, mediators, transducers and effectors are especially important for cell growth and cell fate. The G1, S and G2 stages are closely related to DNA F I G U R E 1 CCK8 assay to detect the proliferation of RKO and HCT116 cells treated with irinotecan and/or EGCG at different concentrations after 24 h. A-C, are irinotecan, EGCG, 0.5μM irinotecan + EGCG in RKO cells, and D-F, in HCT116 cells. *P < .05, **P < .01, ***P < .005, ****P < .001 (compared with the control group); # P < .05, ## P < .01, ### P < .005, #### P < .001 (compared with IRI 0 µmol L − group)   F I G U R E 5 Effects of EGCG on autophagy in RKO and HCT116 cells pretreated with irinotecan, WB was applied to detect the expression of autophagyrelated proteins of RKO A, B and HCT116 C, D cells of autophagy, EGCG combined with irinotecan reduced autophagy and decreased apoptosis. In contrast, autophagy was further promoted and apoptosis increased when treated with autophagy inducer rapamycin (RA) (Figure 6 and Figure S7, Figure S5).

| D ISCUSS I ON
Green tea is one of the most popular and widely consumed beverages in the world, and green tea polyphenols are promising phytochemicals with chemoprophylaxis and chemical protection.
EGCG is one of the most abundant catechins in green tea and one of the most studied tea components. Many epidemiological studies have shown that green tea consumption can be a good prevention of various types of cancer and reduce cancer metastasis. [19][20][21] The anticancer effect of EGCG has been demonstrated in many cell and animal experiments. For example, the combination of EGCG at 0.1 μg/ mL and 5-FU can significantly inhibit the proliferation of head and neck squamous cell carcinoma (HNSCC) cells. 22 In the concomitant treatment of EGCG and taxol, docetaxel, the synergistic effect even F I G U R E 6 Effects of autophagy on apoptosis of RKO and HCT116 cells induced by EGCG combined with irinotecan. After MDC staining, the representative graph of autophagy changes in RKO A, and HCT116 B, cells was detected by flow cytometry, and the expression of autophagy-related proteins in RKO C, and HCT116 D, cells were measured by WB. Representative graph of apoptosis rate in RKO E, and HCT116 F, cells, and the data were fitted with FlowJo software. *P < .05,**P < .01,***P < .005,****P < .001 eliminated xenografts of human prostate cancer cell line PC-3ML in mice. 23 Our results also suggested that EGCG in combination with irinotecan has a synergistic inhibitory effect on RKO and HCT116

cells. Through cell proliferation, migration and invasion experiments,
we demonstrated that EGCG and irinotecan have a synergistic antitumour effect (Figures 1, 2) (Tables 1 and 2).
Similar to irinotecan, EGCG has also been reported to inhibit topoisomerase I in colon cancer cells. 24 Our experiments demonstrated for the first time that EGCG and irinotecan can synergistically inhibit the activity of topoisomerase I, leading to more extensive DNA damage ( Figure 3). ATM is the major inducer of DNA double-strand breaks (DSBs) and the main conduction factor, which activates downstream proteins and induces DNA damage response. 25 ATM cleavage appears to occur in parallel with apoptosis, which has also been reported in cisplatin-induced renal cell injury, 26 and it does not eliminate the binding activity with the end DNA strand. 17 Consistent with the reported results, our findings also suggested that EGCG promotes ATM fragmentation and cell apoptosis. (Figure 3 and Activation of CDK4 and CDK6 affects the early stages of G1; these CDKs bind to Cyclin D and phosphorylate RB, preventing them from binding and inhibiting E2F transcription factors, which are necessary for the transcription of G1/S conversion and promotion of the next phase of the cell cycle. 27 Our results suggested that EGCG induced S phase or G2 phase arrest by synergizing with irinotecan in promoting DNA damage (Figure 4).
DNA damage can induce autophagy. 28 DNA damage chemotherapeutic drugs such as camptothecin, 29 etoposide 30 and ionizing radiation 31 have been shown to induce autophagy as well as initiate cell cycle arrest. ATM may be involved in autophagy. When cells are exposed to genotoxic or oxidative compounds, ATM is activated, and the mTORC1 signalling pathway is inhibited to induce autophagy. 32 The effect of EGCG on autophagy has been studied pre-clinically, in regulating autophagy and cell death. 35 When human colorectal cancer cells were treated with sublethal chemotherapy drugs such as melphalan and autophagy inducers such as rapamycin, autophagy was converted to apoptosis by cleavage of the key molecule Beclin-1 induced by Caspase-8. 36 Our results showed that EGCG induces autophagy in a dose-and time-dependent manner.
Irinotecan alone did not appear to induce autophagy, but it stimulated Beclin-1 cleavage, and EGCG enhanced autophagy ( Figure 5).
Concerning the results of apoptosis, together with the impact of autophagy induction and inhibition on apoptosis, we hypothesized that EGCG could turn autophagy into apoptosis by promoting autophagy and Beclin-1 cleavage, which increased the toxicity of irinotecan in colorectal cancer cells.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Foundation of China, Grant No. 81803967.

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.