Ailanthone Enhances Gastric Cancer Cell Apoptosis Through Inducing DNA Damage and Repressing Base Excision Repair&nbsp;Via Downregulating P23

doi:10.21203/rs.3.rs-130892/v1

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Ailanthone Enhances Gastric Cancer Cell Apoptosis Through Inducing DNA Damage and Repressing Base Excision Repair Via Downregulating P23

https://doi.org/10.21203/rs.3.rs-130892/v1

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Background: Chemotherapy plays an irreplaceable role in the treatment of gastric cancer (GC), but currently available chemotherapeutic drugs are not ideal. Medicinal plants are an important direction for new drug discovery.

Methods: We combined literature collection, organoid drug screening and PDX verification to search for new, effective drugs. The effect of AIL on the GC cells was investigated by CCK-8 assays, Colony Formation Assay and Cell Viability, Cell migration and invasion assay and PDX mice model. The expression of indicated RNA and proteins were measured by qRT-PCR ,IHC and western blotting. The proteins HSP90 binding to P23 or XRCC1 were identified by Co-IP.

Results: Through drug screening of GC organoids, we had determined that ailanthone (AIL) has an anticancer effect on GC cells in vitro and in vivo. We also found that AIL promoted apoptosis of GC cells through flow cytometry analysis and the expression of caspase 3 and H2AX were significantly enhanced by AIL in GC cells and tumor tissues, suggesting that AIL can induce DNA damage and apoptosis of GC cells. Further transcriptome sequencing of the PDX tissue indicated that AIL inhibited the expression of XRCC1 which plays an important role in DNA damage repair, and the results were also confirmed by western blotting. In addition, we found that AIL inhibited the expression of P23 and inhibition of P23 decreased the expression of XRCC1 through western blotting analysis, indicating that AIL can regulate XRCC1 via P23. The results of co-immunoprecipitation showed that AIL can inhibit the binding of P23 and XRCC1 to HSP90 respectively.

Conclusion: These findings indicate that AIL can induce DNA damage and apoptosis of GC cells. Meanwhile, AIL can decrease XRCC1 by downregulating P23 to inhibit DNA damage repair. The present study shed light to the new drug isolated from natural medicinal plants for GC therapy.

Cancer Biology

Ailanthone

DNA damage

BER

P23

Gastric cancer (GC) is the 5th most common cancer in the world, with a 5-year survival rate of 29%, it is 3rd in cancer-related deaths^[1].Currently, the most effective treatment is known to be surgical intervention. However, due to the fact that most GC patients were diagnosed in the late stage, they have already lost the window of opportunity for surgery. For advanced cancer, chemotherapy and perioperative chemotherapy or adjuvant chemoradiotherapy is extremely significant in improving their survival^[2]. Still, chemotherapy drugs have toxic side effects, and currently, researches are showing that not all patients are sensitive to the traditional chemotherapeutic drugs^[3]. Thus, developing a new, effective yet low toxicity drug is of utmost importance to these patients.

Medicinal plants have a long history of use in cancer treatment. Countries such as China and Japan have had thousands of years of history in cancer treatment through traditional medicine^{[4, 5]}. Today, many drugs in the clinic settings are in fact derived from such medicinal plants. For example, vincristine, paclitaxel and docetaxel are common place in modern clinical practice^{[6, 7]}. Therefore, the identification of natural compounds from medicinal plants and the development of new anticancer drugs have been gaining increasing traction over the years. The process of discovery of new drugs is inseparable from effective drug screening tools, thus we here as the largest GC diagnosis and treatment center in Southern China have established a large GC specimen banks and have successfully established multiple GC organoids and Patient-Derived Xenograft(PDX) models over the years.By using the advantages above, we combined literature collection, organoid drug screening and PDX verification to search for new, effective drugs. Fortunately,AIL turned out to be an effective compound in suppressing GC organoid growth. AIL is a medicinal plant extracted from Ailanthus altissima. It has been used in traditional Chinese medicine (TCM) to treat various diseases since ancient times. In recent years, it has been confirmed by modern scientific methods to have significant anticancer activity^[8–15].In the GC PDX model, AIL was verified to be a better suppressor of tumor growth with a lower cell toxicity. Further analysis revealed that AIL can effectively induce DNA damage in GC cells and downregulate P23 which suppresses DNA damage repair in the base excision repair (BER) pathway through regulating XRCC1.

AIL had been proven to treat castration-resistant prostate cancer by down-regulating P23.The results of our bio-information analysis showed that the high expression of P23 enhanced the DNA repair pathways, mainly the BER pathway, when compared to low expression of P23. P23 is known as an important co-chaperone of HSP90^[16]. HSP90 and its associated co-chaperones play key nucleic functions including chromatin remodeling, DNA transcription, RNA processing, DNA replication, telomere maintenance, and DNA repair^{[17, 18]}. In fact, HSP90 is involved in DNA repair and mainly depends on its co-chaperone P23, or phosphorylation to interact with the DNA metabolism proteins^[17–19].

In this study, we found that AIL can significantly inhibit tumor growth in GC cells, organoids and PDX models both in vivo and in vitro. AIL mainly inhibited DNA repair of XRCC1 by inducing DNA damage and down-regulating P23, thereby exerting anticancer effect on GC. This finding indicated that AIL could effectively kill tumors through DNA damage and inhibition of DNA repair.

Cell Culture

Human gastric cancer cell lines SGC7901, SNU719, AGS were provided by Nanjing Kegen Biotechnology Co., Ltd. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, placed in a humidified incubator containing 5% CO² and 95% air, subculture and all experiments were performed at 37 °C. AIL stock solution was prepared with DMSO, stored at 4 °C, and diluted with RPMI-1640 medium to the required concentration immediately before use; the final concentration of DMSO in the medium was 0.1%. Cells in the control group were treated with DMSO (0.1%) without AIL.

Human tissues and organoids

Human gastric cancer tissues were taken from patients who underwent gastric cancer surgery in the First Affiliated Hospital of Sun Yat-sen University. They agreed and signed a donation and research consent form. It was approved by the clinical scientific research and animal experiment ethics committee of the First Affiliated Hospital of Sun Yat-sen University (Lunshen [2018] No. 087). This research complied with all the human participation ethics in research. Organoid related experiments were completed in the Digestive Diseases Center of the Seventh Affiliated Hospital of Sun Yat-sen University. Organoid strains from a total of 3 patients were used.

After the tumor was excised, it was placed in 50 U/ml penicillin-streptomycin (Thermo Fisher) frozen G solution. The tissue was minced on ice and incubated in DMEM containing 1 mg/ml collagenase V (Sigma-Aldrich) for 1 hour at 37 °C. Iced PBS was added to stop the digestion, and subsequently centrifuged at 4 °C (300 rcf, 5 min). The sample was further digested with TrypLE (Thermo Fisher) at 37 °C for 5 min, and then stopped with a large amount of PBS. The suspension was filtered through 40 nylon meshes, centrifuged, and the cells were fixed in the matrix. It was then passaged with TrypLE every 2 weeks. The medium for establishing and culturing human GC organoids was as described in the literature^[20].

Colony Formation Assay and Cell Viability

AGS, SNU719, and SGC7901 cells were seeded in 6-well plates with 500 cells per well, and the experiment was repeated three times. AIL was added to the cells at different final concentrations (0, 0.05, 0.1 µM) and it was then incubated for 12 days. The cells were fixed for 30 minutes with 4% paraformaldehyde, stained with crystal violet for 30 minutes, and finally counted.

In a 96-well transparent bottom black board, 3,000 cells were seeded into each well (organoids were seeded in Matrigel). Medications were added in each well according to a 5-fold or 10-fold concentration gradient. After 72 hours, a luminometer (PerkinElmer Life and Analytical Sciences, Boston, MA) was used to determine the level of adenosine triphosphate (ATP) by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).

Cell migration and invasion assay

Transwell migration assay and matrix invasion assay were used to determine cell migration and invasion ability. In the Transwell migration assay, a small chamber (8 µm pore size; Corning) was placed in a 24-well plate, and then 5 × 10⁴ cells were suspended in 200 ul serum-free RPMI-1640 medium in the chamber; 20% pre-warmed fetal bovine serum was used in the well.The cells were incubated for a period of time (AGS/16 hours, SNU719/24 hours, SGC7901/42 hours) in 5% CO² at 37 °C, and then fixed with 4% paraformaldehyde. In the matrix invasion assay, matrix gel and serum-free 1640 medium were injected into the transwell chamber at a ratio of 1:8 for pretreatment for 2 hours. 1 × 10⁵ cells was suspended in 200 µL of serum-free RPMI-1640 medium, and pre-warmed medium containing 20% fetal bovine serum was then added to the well. The cells were incubated for a period of time (AGS/16 hours, SNU719/24 hours, SGC7901/42 hours) in 5% CO² at 37 °C, and then fixed with 4% paraformaldehyde. A cotton swab was used to gently remove cells that have not migrated or invaded the membrane. Cells were stained with 0.1% crystal violet (Sigma, St Louis, MO), and cells in 8 randomly selected field of vision were counted under a light microscope (100x magnification) to determine cell migration or invasion.

The PDX mouse model

In vivo experiments had been performed according to the Institutional Animal Care and Use Committee (IACUC). It was approved by the clinical scientific research and animal experiment ethics committee of the First Affiliated Hospital of Sun Yat-sen University (Lunshen [2018] No. 087). The experiment was conducted in the Animal Center of the First Affiliated Hospital of Sun Yat-sen University.

Balb/c nude mice (female, 8 weeks old, 19–21 g in mass) were purchased from GemPharmatech Co., Ltd (Nanjing, Jiangsu) Experimental Animal Co., Ltd. and raised in an SPF environment. PDX method was as mentioned before^[21].When the tumor volume reached about 150 mm³, they were randomly divided into three groups, and different reagents were given by intraperitoneal injection (IP). The tumor volume and body mass were measured every 3 days. The calculation formula of tumor volume is as follows: V = Πa b2/8 (where V is the tumor volume, a is the largest tumor diameter, and b is the smallest tumor diameter). Nude mice were sacrificed on day 30, and tumors were taken for measurement and weighing.

Biochemistry, histology and immunohistochemical staining

In order to evaluate the toxicity of Ailanthone, mice with subcutaneously transplanted tumor were sacrificed at the end of the experiment, and blood serum, liver and kidney were harvested. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Tumor or mouse tissue samples were immediately fixed in 10% neutral buffered formalin for 24 hours, gradually dehydrated in a solution with increasing ethanol content (75, 85, 95 and 100%, v/v), and finally embedded in paraffin blocks. Liver and kidney were fixed by paraffin-embedded formalin and cut into 3 mm sections. H&E staining was performed by the experimental histopathology laboratory in accordance with standard procedures. Observations were made under light microscope (100X magnification). Immunohistochemical (IHC) staining: Collect tissues after fixation, dehydration, embedding and sectioning. After heat-mediated antigen repair in 10 mM sodium citrate buffer (pH 6.2) or Tri-EDTA, 1.6% hydrogen peroxide was used to block endogenous peroxidase. Ki67(1: 1000, Abcam, ab15580), H2AX (1: 100, Proteitech, 10856-1-AP), Caspase3 (1:200, Proteitech, 19677-1-AP), XRCC1 (1: 100, Abcam, ab44830), P23 (1: 200, Proteitech, 10824-1-AP), HSP90 (1: 400, Proteitech, 60318-1-Ig, Mouse), and AKT (1: 500, Proteitech, 10176-2-AP) were used as a primary antibody for immunohistochemistry tests according to the protocol. The samples were stained with hematoxylin (HE) and eosin to indicate the nucleus and cytoplasm, respectively. DAB staining was performed and positive cells were counted in 5 random field of views per slide.

Quantitative real-time PCR

AGS, SNU719, and SGC7901 cells were sown in 6-well plates and incubated with different concentrations of AIL for 12 hours. AG RNAex Pro reagent (Accurate Biology, CAT#AG21102) was used to extract total RNA from tissue samples or cell lines. Evo M-MLV reverse transcriptase premix (Accurate Biology CAT#AG11706) was used to synthesize cDNA from 2 µg RNA of each sample. SYBR Green Premix Pro Taq HS qPCR kit (Accurate Biology, CAT# AG11701) was used for qRT-PCR. Each sample was tested 3 times. The data was analyzed using the 2-ΔΔCT calculation method. The primers were as follows:

GAPDH forward 5’-GACCCCTTCATTGACCTCAA-3’;

GAPDH reverse 5’-TGCTTCACCACCTTCTTGAT-3’;

HSP90 forward: 5’-CATAACGATGATGAGCAGTACGC-3’;

HSP90 reverse: 5’- GACCCATAGGTTCACCTGTGT-3’;

P23forward:5’- GAAAGCACAGTAATCACTGGTGT-3’;

P23 reverse:5’- ACGGTAGTCCAATAGAGCAACC-3’;

XRCC1 forward: 5’- TCAAGGCAGACACTTACCGAA-3’;

XRCC1 reverse:5’-TCCAACTGTAGGACCACAGAG-3’.

Western blotting

The cells were processed differently according to their corresponding requirements, and the protein concentration is determined by BCA Protein Assay Kit (KGI Biosciences), using SDS-PAGE Sample Loading Buffer, 5X (Beyotime) at 95° for 8 minutes. The lysate is separated on a polyacrylamide gel and transferred to nitrocellulose. The blot was detected with a specific antibody, and then the membrane was detected with the LI-COR Odyssey CLx Infrared Imaging System (LI-COR Biotechnology, Lincoln NE). Antibodies were H2AX (1: 1000, Proteintech, 10856-1-AP), XRCC1 (1: 1000, Abcam, ab44830), Caspase3 (1: 400, Proteintech, 19677-1-AP), P23 (1: 500, Proteintech, 10824-1-AP), AKT (1: 2000, Proteintech, 10176-2-AP), HSP90 (1: 1000, Proteintech, 60318-1-Ig), Tubulin (1: 1000, Proteintech, 10068-1- AP), GAPDH (1: 20000, Proteintech, 60004-1-Ig).

Immunoprecipitation

The protein expression level was determined by the western blotting results of cytoplasm or nuclear lysate. Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, CAT#P0028) was used to prepare cytoplasmic lysates and nuclear lysates, and protein concentration was measured. 2,000 µg of protein lysate (prepared 500 µL) was mixed with primary antibody and incubated overnight in a 4° shaker. 50 µL of protein A/G agarose (Thermo Fisher Scientific, CAT#20424) beads was washed 3 times with lysate, mixed in the above-mentioned protein mixture with the primary antibody and incubated overnight in a 4° shaker. The mixture was washed 6 times with lysis buffer (Thermo Fisher Scientific, CAT#87787) before SDS-PAGE Sample Loading Buffer was added, and then heated to 95° for 10 min at 5X (Beyotime), centrifuged at 3,000 rpm for 3 min, the supernatant was carefully aspirated and the protein level was finally detected via western blotting. Antibodies used were: XRCC1 (1: 100, Abcam, ab44830), P23 (1: 200, Proteintech, 10824-1-AP), HSP90 (1: 400, Proteintech, 60318-1-Ig), AKT (1: 400, Proteintech, 10176-2-AP).

Immunofluorescent(IF) staining

The collected organoids were fixed for 16–20 hours, then embedded, sliced, baked, and deparaffinized. After heat-mediated antigen retrieve in 10 mM sodium citrate buffer (pH 6.2) or Tri-EDTA, 1.6% hydrogen peroxide was used to quench endogenous peroxidase, then blocked with PBS containing 5% BSA and 0.2% Triton- X. The primary antibody was incubated in blocking buffer at 4 °C for 16–20 hours. The fluorescent secondary antibody was incubated for 1 hour at 20 °C, and then incubated in DAPI for 10 minutes. Fluorescence staining was imaged on a Zeiss LSM 780 confocal microscope. Primary antibodies used were: Ki67 (1: 1000, Abcam, ab15580), H2AX (1: 400, Proteintech, 10856-1-AP), Caspase3 (1: 400, Proteintech, 19677-1-AP ), XRCC1 (1: 100, Abcam, ab44830), P23 (1: 200, Proteintech, 10824-1-AP), HSP90 (1: 400, Proteintech, 60318-1-Ig), AKT (1: 400, Proteintech, 10176-2-AP). Secondary antibodies used were: Donkey anti-Rabbit /Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, A-21206), Donkey anti-Rabbit /Alexa Fluor 546 (1:500, Thermo Fisher Scientific, A11040), Donkey anti-Mouse /Alexa Fluor 488 (1:000, Thermo Fisher Scientific, A-21202), Donkey anti-Mouse/Alexa Fluor 546 (1:500, Thermo Fisher Scientific, A10036).

RNA isolation and microarray

Total RNA was extracted from tissue samples, the concentration and purity of the extracted RNA were detected by Nanodrop 2000, RNA integrity was detected by agarose gel electrophoresis, and RIN value was determined by Agilent 2100. A single library construction requires that the total amount of RNA is not less than 5 µg, concentration ≥ 200 ng/µL, and the OD260/280 is between 1.8 and 2.2. mRNA capture and library preparation were completed by the advanced sequencing equipment of Shanghai Origingene Bio-pharm Technology Co., Ltd. using KAPA mRNA HyperPrep kit (Roche). Three biological library was sequenced on the Illumina Truseq TM RNA sample prep Kit platform of the facility, and each sample produced an average of 25 million single-ended reads of 75 bp. The high-quality DNA sequencing obtained after quality control was compared with the designated reference genome. For the PDX sample, it was first compared to the mouse reference genome. After eliminating the mouse data, the remainder was then compared to the human reference genome retrieved from Ensembl database (genome version GRCh38, gene annotation information Ensemble 92). Before alignment, cutadapt (version 1.9.1) was used for quality trimming and adaptor removal of the original reading. Using the annotation release 86 as a reference, read was sequenced on human genome GRCh38 using RSEM 1.3.0 and STAR 2.5.2, and the subsequent gene levels were counted. In R program (version 3.6.1), the DESeq2 package (version 1.24.0) was used for normalization and differential expression analysis of raw count data. Regularized logarithmic transformation was performed on the rlog function.

Gene Set Enrichment Analysis (GSEA)

GSEA was performed with the software (GSEA V4.0.3) developed by the Broad Institute of Massachusetts Institute of Technology and Harvard University (https://www.gsea msigdb.org/gsea/index.jsp). For the RNA-seq datasets of low and high expressionof P23, OXA group and AIL group, the normalized RNA read count was used for analysis, and the following settings were applied: number of permutations = 1000, type of permutation = gene set, enrichment statistics = weighting, measurement of gene ranking = signal noise. For the TCGA gastric cancer data set, the samples were grouped according to their expression above or below the median value. The normalized RSEM read count was used for analysis, and the following settings were applied: number of permutations = 1000, permutation type = phenotype, enrichment statistics = weighting, measurement of gene ranking = signal 2 noise. Recognized marker gene set 40, KEGG pathway or gene ontology (GO) terms and false discovery rate (FDR q) < 0.05 were set as significant enrichment.

Screening of differentially expressed genes (DEGs)

The expectation-maximization algorithm of RNA-Seq was used to normalize the 3-level transcriptome data of the data set, and the logarithmic transformation of all gene expression values was performed. Approximate data were normalized by quantiles and were normally distributed^[22]. In this study, the R program package limma v3.28.14 was used to analyze the differential genes in the gene expression data, and its mRNA satisfied P < 0.01, false discovery rate (FDR) was < 0.01 and |log2 fold change (FC)| was > 1.5, where P < 0.05 indicated that the hypothesis test was statistically significant. FDR is a control indicator for the error rate of the hypothesis test. As an evaluation index of the selected differential genes, the number of false rejections was proportional to the number of null hypotheses rejected. FC is usually used to describe the degree of change from the initial value to the final value. In this study, the ratio of tumor tissue gene expression value to normal tissue gene expression value was used, also known as the fold change. The heatmap and volcano map of the differential genes were constructed in R language for visual comparison.

Flow cytometry and FACS

Flow cytometric analysis: Single cells prepared from organoids was suspended in PBS containing 10% FBS. The single cell suspension was stained with a fluorescently-labeled isotype control antibody or experimental antibody in the dark at 4 °C for 30 minutes, and washed three times in FACS buffer. The cells were filtered through a 40-HCLM nylon mesh and incubated in 3 HCLM DAPI in FACS buffer. Single-color stains and Fluorescence Minus One control were used when necessary. Annexin V-PI apoptosis assay and cell-cycle analysis were performed using Annexin V-FITC apoptosis detection kit (KeyGEN BioTECH, KGA105) and apoptosis detection kit (KeyGEN BioTECH, KGA512) according to the manufacturer's protocol. Cells were sorted and analyzed by CytoFlex LX (Beckman). FlowJo 10 software was used to analyze the data.Flow sorting: After the prepared organoid suspension single cell was fixed (Cytofix/Cytoperm Soln Kit, CAS: 554714), P23 was added (1:100, Thermo Fisher Scientific, MA3-414) and incubated at 4° for 30 min, then washed three times in FACS buffer, incubated with the fluorescent secondary antibody Donkey anti-Rabbit /Alexa Fluor 488 (1:200, Thermo Fisher Scientific, A-21206) for 15 minutes, and lastly dead cells were excluded after staining with DAPI. The cells were sorted on a SH800S (SONY) flow cytometer, and P23(+) and P23(-) cell populations of the GC organoid strains were separated by flow cytometry. The cells were collected in cold PBS supplemented with 10% fetal bovine serum, and the final sorted cell population was subjected to other experiments.

Clinical gastric cancer samples

The sample data were procured from the prospectively collected GC database of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. We reviewed the treatment records of patients undergoing gastric cancer resection from January 2010 to May 2012. The inclusion criteria were pathological TNM stages I–III gastric adenocarcinoma patients whom underwent radical surgery patients. Exclusion criteria: other history of malignant tumors, neoadjuvant therapy, loss to follow-up, no tissue samples. A total of 93 cases of tissue samples were collected from surgical specimens, and informed consent was obtained from the patients. This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. The detailed clinical characteristics of the patients were shown in Table 1. The endpoint of the study was recurrence. The specimen tissues were tested by IHC, and the results were interpreted by two independent pathologists. They were blinded to the specific diagnosis and prognosis of each case, and used a semi-quantitative method for scoring. Among the samples, more than 10% of tumor cells staining were positive. The staining intensity was divided into negative, weak, moderate and strong. Low P23 expression was detected in the negative and low intensity samples, and medium and strong intensity returned with high P23 expression. Subsequently, the disease-free survival of patients in the high P23 expression group (n = 42) and low P23 expression group (n = 51) were evaluated. Progression free survival is defined as the duration from tumor resection to progressive disease. Follow up was completed in intervals of 3 months (0–2 years), 6 months (2–4 years), and once a year until recurrence or June 2020. The follow-up study included abdominal computed tomography and postoperative physical examination.

Table 1

Research status of 8 medicinal plants
Medicinal plants	Tumor type	References
Chrysin	Prostate Cancer,Ovarian Cancer	365
Hesperidin	Cholangiocyte,Oral Carcinogenesis	282
Ailanthone	Prostate Cancer,Lung Cancer	25
Indirubin	Prostate Cancer,Promyelocytic Leukemia༌Glioma	157
Oridonin	Fibrosarcoma, Diffuse Large B Cell Lymphoma༌ Leukemia	261
Celastrol	Melanoma,Breast Cancer༌Prostate Cancer	321
Ursolic acid	Colorectal Cancer,Breast Cancer༌Leukemia	563
Pseudolaric Acid B	Glioma,Lung Cancer	59
Wedelolactone	Prostate Cancer,pancreatic cancer	45

Table 2

Characteristics of 93 recurrence gastric cancer patients
Characteristics			Low(51)	High(42)	p
Sex	Female	26	16	10	0.419
	Male	67	35	32	0.419
Diferentiation	Well	62	41	21	0.003
	Moderate	26	7	19
	Poor	5	3	2
pT stage	T1	22	13	9	0.870
	T2a	13	6	7
	T2b	28	14	14
	T3	21	13	8
	T4	9	5	4
pN stage	N1	58	32	26	0.946
	N2	25	14	11
	N3	10	5	5
pM stage	M0	84	46	38	0.851
	M1	9	5	4	0.851

Statistical analysis

SPSS 22.0 software was used for statistical analysis. Data were expressed as mean ± standard deviation (s.d.), and P < 0.05 was considered statistically significant. Tumor volume was measured repeatedly using a general linear model. The difference between categorical variables was tested using χ2 test, and the difference between two groups was tested using student’s t test. Kaplan-Meier curve and log-rank test were used to analyze the clinical data of gastric cancer patients.

AIL has a good inhibitory effect on GC

TCM has played an important role throughout human history before development of modern medicine took over. So far, many drugs are monomers that were first inspired by it and subsequently extracted from plants, such as vincristine, which has been widely used in clinical treatment of cancer^[7], and artemisinin which saved countless lives from malaria^[23].From the above, it is fair to say seeking inspiration from ancient methodologies like TCM is still an important way to discover new medicines. From the 166 kinds of medicinal plants with anticancer effects recorded in the Pharmacopoeia of the People’s Republic of China (2000 edition)^[24],we have selected 9 kinds of medicinal plants(Table 1)that had been proven through modern science in recent years to have relatively good anticancer effects (Figure S1-D). We then conducted drug susceptibility tests on three strains of GC organoids (Figure S1-ABC) we had established. Fortunately, AIL excelled above the rest (Fig. 1-A),. AIL is an herbal monomer extracted from the bark of Ailanthus altissima^[25], which in recent years has been proven to have obvious anticancer effect on multiple cancer types. In order to verify whether AIL has the potential to inhibit the growth of GC cells, we evaluated its effect on GC cell growth and colony formation. The results showed that AIL significantly inhibited the growth of AGS, SNU719 and SGC 7901 cell lines (Figure S2-AB). We also evaluated its effect on the spheroidizing ability of GC organoids, and the results showed that AIL inhibited the spheroidizing ability of GC organoids significantly (Fig. 1-BCD). We further verified its effect on GC PDX models and found that it also had a good inhibitory effect in vivo (Fig. 1-EFG,Figure S6-BC). There was no significant weight loss in mice during treatment as well (Figure S3-B), with no significant difference in serum ALT and AST levels post treatment (Figure S3-A). There was no damage found on liver and kidney function (Figure S3-C). Additionally, AIL exerted a significant inhibitory effect on GC cell migration and invasion in transwell cell migration and invasion assays (Figure S4-ABCD). In summary, these results indicated that AIL could significantly inhibit GC cell in vitro and in vivo, while maintaining a low cell toxicity edge. However, we do not know the mechanism by which AIL inhibits the proliferation of GC cells. To achieve this end, we had further explored its mechanisms.

AIL promotes apoptosis through inducing DNA damage

In order to clarify the question of how AIL inhibits the proliferation of GC cells, we conducted a series of exploratory experiments. Cell cycle analysis and apoptosis analysis were performed via flow cytometry on the GC organoids after AIL treatment. The results showed that AIL did not induce cycle arrest on the cells (Figure S5-AB), but it did significantly induced apoptosis (Fig. 2- AB). We also explored whether AIL increases DNA damage in GC cells. Western blotting and IF analysis found that AIL did induce DNA damage in GC cells (Fig. 2-CDEFGH), and it was concentration (Fig. 2-EFG) and time dependent (Fig. 2-H). It was revealed that AIL has a stronger ability to induce DNA damage(Fig. 2-CDEFGHIJ). In this regard, we speculate that AIL may be able to inhibit DNA repair, thereby causing continuous DNA damage.

AIL inhibits DNA repair in GC cells

To prove the above hypothesis, we sequenced the transcriptomes of the PDX tissues. The pathway enrichment analysis revealed that the BER pathway associated with DNA repair was mainly regulated by AIL (Fig. 3-A); the differential gene expression analysis suggested that the AIL group had a significant inhibitory effect on the key gene XRCC1 of the BER pathway (Fig. 3-B)^[26–28].Subsequent experiments also confirmed that AIL has a good inhibitory effect on XRCC1 of GC cells (Fig. 3-CDFG). In previous study, AIL had been proven to treat castration-resistant prostate cancer by down-regulating P23^{[8, 29]}. Bioinformatics analysis showed that P23 is closely related to DNA repair-related pathways, that is to say that high expression of P23 leads to higher enrichment of the BER, HR, NHEJ, and MMR pathways when compared with low expression of P23, indicating that P23 promoted DNA repair (Figure S6-A). Western blotting also confirmed that AIL can significantly inhibit the expression level of P23 in GC cells (Fig. 3-CD). As an important co-chaperone of HSP90, P23 plays a key role in HSP90's participation in chromatin remodeling, DNA transcription, RNA processing, DNA replication, telomere maintenance and DNA repair. In fact, involvement of HSP90 in DNA repair requires its co-chaperone P23 or phosphorylation to interact with DNA metabolism proteins. However, our results showed that AIL did not increase the expression of HSP90 (Fig. 3-D).Instead, we noted that AIL is not an ATP-competitive inhibitor of HSP90 like 17-AAG, which increases the expression of HSP90 protein^[30]. Interestingly, AIL could sufficiently inhibit the AKT protein expression of GC (Fig. 3-D). We further compared the inhibitory effects of 17-AAG, CEL and AIL on XRCC1, and found that AIL has significant advantages as well (Fig. 3-E).The IHC of PDX tissue also confirmed that AIL inhibits the expression of P23, HSP90 and AKT (Fig. 3-FG,Figure S6-BC). These results indicated that the DNA damage of GC cells induced by AIL was mainly related to the inhibition of the BER pathway.

AIL regulates XRCC1 through P23 to inhibit DNA repair

Although we had confirmed the effects of AIL on P23, HSP90, and XRCC1, the regulatory relationship between them was still unclear. In the past, the HSP90 chaperone system was thought to occur in the cytoplasm^[31].It is becoming increasingly clear that HSP90 and its associated chaperone proteins have important functions in the nucleus, including chromatin remodeling, DNA transcription, RNA processing, DNA replication, telomere maintenance and DNA repair ^{[32, 33]}.The involvement of HSP90 in DNA repair depended upon its co-chaperone P23, or phosphorylation to interact with DNA metabolism proteins^[32–34].In cancer cells, HSP90 and P23 bind stably to form a super-chaperone complex, which is the activated state of the protein^[35].The existence of P23 is not solely limited to HSP90, because P23 also fulfills a variety of functions unrelated to HSP90 in cells^[17].In a prostate cancer study, it was found that by inhibiting P23, the client protein, androgen receptor of HSP90 can be significantly inhibited. As an important client protein of HSP90, the important scaffold protein XRCC1 efficiently promotes the repair of DNA single-strand breaks (SSBs)^{[36, 37]}.If the SSBs are not properly repaired, they may convert into double-strand breaks during DNA replication, eventually leading to genetic instability and apoptosis^[38].Therefore, we theorize that AIL inhibits the interaction between P23 and HSP90 by down-regulating P23, thereby affecting the interaction between HSP90 and its client protein XRCC1.In order to verify the regulatory axis of this molecular mechanism, we first proved the regulatory effect of P23 inhibitor (CEL) on HSP90 and XRCC1 (Fig. 4-A). To verify that AIL functions through P23,we separated P23(+) GC organoid and P23(-) GC organoid strains through flow sorting (Fig. 4-B). qPCR analysis found that P23(+) GC organoids upregulated XRCC1, while P23(-) GC organoids inhibited XRCC1 (Fig. 4-CD). Subsequently, we verified the inhibitory effect of HSP90 inhibitors on XRCC1 in GC cells (Fig. 4-E). At this point, we had basically confirmed that AIL inhibited XRCC1 by downregulating P23. Further bioinformatics analysis found that HSP90 is positively related to DNA repair, that is, as HSP90 is upregulated, its DNA repair ability gets stronger (Figure S7-A). In summary, AIL has a obvious tumor suppressing effect because it can induce DNA damage while inhibiting its repair in GC cells. For this reason, we further compared the spheroidization ability of GC organoid cells treated with 17-AAG, CEL and AIL respectively and the results showed that the anticancer effect of AIL was excellent. This showed that P23 was not only upstream of XRCC1, but it can also regulate XRCC1.

AIL inhibits the binding of P23 to HSP90 and HSP90 to XRCC1

As an important co-chaperone of HSP90, P23 binds stably with HSP90 in cancer cells to form a super chaperone complex^[39]it could also bind with HSP90 without the presence of any client protein^[40];unlike XRCC1, which requires to bind with HSP90 as a chaperone molecule in order to participate in DNA repair^[41].Since AIL can inhibit the expression of P23 and XRCC1, does it also inhibits the interaction between P23 or XRCC1 and HSP90 at the same time? Through immunocolocalization and co-immunoprecipitation, we proved that both of P23 and XRCC1 can bind with HSP90 in GC cells, and AIL can inhibit their combination (Fig. 5-ABC). The above results revealed that AIL not only inhibited the expression of P23 and XRCC1, but it also inhibited the binding of P23 to HSP90, and HSP90 to XRCC1.

High expression of P23 is related to GC recurrence

P23 is proven to promote breast cancer metastasis and affect its prognosis ^[42]; it also promotes the invasion and metastasis of prostate cancer^{[42, 43]}, and targeting P23 can treat castration-resistant prostate cancer^[8]. In this study, we had proven that AIL can target P23 in GC cells. In order to explore the clinical impact and status of P23 on gastric cancer, we had enrolled patients with GC recurrence from Sun Yat-sen University’s Gastric Cancer Research Center. We used IHC of tumor tissues to detect the expression level of P23 and found that P23 was highly expressed in the specimens of patients who relapsed (Fig. 6-A). The recurrence time of patients with high expression of P23 was significantly shorter than that of patients with low expression (Fig. 6-B). It showed that P23 promoted GC recurrence, and P23 could be used as an important index for clinical prediction of postoperative recurrence in patients.

Chemotherapy plays a pivotal role in GC treatment^[2], however, even with a variety of chemotherapeutics and targeted therapy currently available in clinic, its therapeutic effect is still far from satisfactory. As the fifth most common cancer in the world, GC is still the third leading cause of cancer-related death with only a 5-year survival rate of merely 29%^[1]. Therefore, it is crucial to find new drugs for its treatment. In this study, we selected medicinal plants with anticancer effects from the "Pharmacopoeia of the People's Republic of China" (2000 edition), combining with literature meta-screening, and applications of organoids established by our center as a powerful drug screening tool^[44–46],we had successfully screened out the monomer AIL which can significantly inhibit the proliferation of GC cells, induce apoptosis, and has a good anticancer effect in vivo.

Organoids, as multi-cell clusters constructed by three-dimensional culture in vitro, have the ability to self-renew and self-organize, thus maintaining the physiological structure and function of their source tissues^[47]. With this advantage, organoids quickly became powerful tools for tumor and stem cell biology research^[44].In this study, we used organoids as research tools and confirmed that AIL can effectively inhibit the proliferation of GC cells in vitro and in vivo. Although AIL has no cycle arrest effect on GC cells, it can significantly induce GC cell apoptosis. Through transcriptome sequencing of the PDX tissues, we can observe that the DMSO group was enriched in the BER and HR DNA repair pathways, thus proving that AIL could suppress DNA repair pathways; differential gene expression analysis suggested that the AIL group had a significantly strong inhibitory effect on XRCC1, the key gene in the BER pathway. Subsequent experiments also confirmed that AIL has a good inhibitory effect on XRCC1 in GC cells. Therefore, we speculated that AIL could inhibit DNA damage repair while inducing DNA damage in GC cells.

XRCC1 as an important gene for DNA repair in the BER pathway^{[26, 35]}can interact with PARP1, DNA ligase III and Pol proteolysis to promote effective repair of DNA SSBs^{[36, 37]}. If SSBs are not repaired promptly and properly, they may convert into DSBs during DNA replication, leading to apoptosis^[38]. XRCC1 function depends on the binding and stability of HSP90^[41].By binding and stabilizing XRCC1, the phosphorylated form of HSP90 promotes the formation of additional XRCC1 complexes because in the absence of HSP90 or HSP90 binding, free XRCC1 will be removed by ubiquitin-mediated degradation^[41].In recent years, more and more evidence has shown that HSP90 played an important role in cell homeostasis, transcription regulation, chromatin remodeling and DNA repair^{[17, 18, 35]}.In fact, P23 not only showed overlapping interactions with HSP90, but it also has more interactions independent of HSP90^{[17, 50]}. Our study also found that P23 can promote DNA repair. It has been reported that AIL can treat castration-resistant prostate cancer by inhibiting P23^[8]. Surprisingly, our study also verified that AIL can significantly inhibit P23 in GC cells. At the same time, AIL can also inhibit the protein binding of P23 to HSP90, and HSP90 to XRCC1. Additionally, AIL can also inhibit AKT and binding of AKT to HSP90. AKT can mediate DNA damage repair ^[51] ,and act as a client protein of HSP90 while interacting with HSP90^[52].It is possible that the DNA damage induced by AIL itself and its other effect of DNA repair inhibition by blocking multiple signal pathways of GC cells led to synthetic lethality^[53].

It is known that P23 plays a key role in DNA repair, and through this study, we confirmed that AIL can significantly inhibit P23. We further proved that downregulation of P23 can inhibit HSP90 and XRCC1, and interestingly, while dividing GC organoids into P23(+) and P23(-) groups through flow sorting, it showed that the P23(+)group had upregulated XRCC1, while the P23(-)group had downregulated XRCC1. All these indicated that P23 plays a central role in the P23/HSP90/XRCC1 axis. Other researches have revealed that the expression of P23 is increased in some cancers. For example, highly expressed P23 promotes breast cancer progression and poor prognosis by increasing lymph node metastasis and drug resistance^[54]; while cells with high expression of P23 in prostate cancer acquires a more aggressive phenotype, which promotes its invasion and metastasis, thereby leading to disease progression and a worse prognosis ^[55] .However, research on clinical prognosis in GC has not been carried out yet. Our comparative study on the recurrence time of GC patients with low through high expression of P23 through immunohistochemical methods found that patients with high expression of P23 had a shorter relapse time, indicating that P23 is an important factor affecting the recurrence and development of GC. Therefore, it provides an important foundation for the targeted therapy of P23.

In summary, we clarified the anticancer effect of AIL in gastric cancer through GC cell lines, organoids and PDX. Combined with the results of transcriptome sequencing, it is clear that not only can AIL induce DNA damage, it also inhibits DNA repair through downregulating P23 which in turn inhibits the DNA repair capability of XRCC1, thereby exerting a tumor suppressing effect on GC. This finding indicates that AIL can effectively kill tumors through DNA damage and inhibition of DNA repair.

AIL: ailanthone; OXA: oxaliplatin; GC: gastric cancer; TCM: traditional Chinese medicine; PDX: patient-derived xenograft; XRCC1:X-ray repair cross-complementing protein 1; BER: base excision repair; BSA: bovine serum albumin; FBS: fetal bovine serum; CEL: celastrol; ORI: oridonin; CHR: Hesperidin; CHR: Chrysin; IND: Indirubin; UA: Ursolic acid; WED: Wedelolactone; ALT: alanine aminotransferase; PAB: Pseudolaric Acid B; IHC: immunohistochemistry; FITC: fluorescein isothiocyanate; AST: aspartate aminotransferase; IF: immunofluorescence；IOD:Integrated option density

Ethics approval and consent to participate

The current study was performed with approval from the Ethics Committee of The First Affiliated Hospital, Sun Yat-sen University. All patients provided written informed consent. The animal experiments was performed under the approval of committee on the Ethics of Animal Experiments of The First Affiliated Hospital, Sun Yat-sen University.

Consent for publication

All authors agree to submit the article for publication.

Availability of data and materials

The datasets from the current study are available from the corresponding author on reasonable request. Publicly available data was obtained from the GEO database (https://www.ncbi.nlm.nih.gov/gds) and TCGA database (https://portal.gdc.cancer.gov/).

Competing interests

The authors declare no conflict of interest.

Funding

National Natural Science Foundation of China (82073148), Sanming Project of Medicine in Shenzhen (SZSM201911010), Shenzhen Key Medical discipline Construction Fund (SZXK016) and the Science & Technology Planning Project of Guangdong Province (2018B030317001、2019A030317005).

Authors' contributions

Conception and design, CZ, YH, and HL; development of methodology or material support, MH, WL, XW, WC, XX, TH, YZ, HZ, WW, GZ and ZH; acquisition of data, CW, HL and XW; drafting of the manuscript, CW; revision of the manuscript, CZ, YH and MH; obtaining funding and study supervision, CZ, YH and QS. All authors provided final approval of the submitted and published versions of this manuscript.

Acknowledgements

Not applicable.

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Ailanthone Enhances Gastric Cancer Cell Apoptosis Through Inducing DNA Damage and Repressing Base Excision Repair Via Downregulating P23

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Abstract

Figures

Background

Methods And Materials

Cell Culture

Human tissues and organoids

Colony Formation Assay and Cell Viability

Cell migration and invasion assay

The PDX mouse model

Biochemistry, histology and immunohistochemical staining

Quantitative real-time PCR

Western blotting

Immunoprecipitation

Immunofluorescent(IF) staining

RNA isolation and microarray

Gene Set Enrichment Analysis (GSEA)

Screening of differentially expressed genes (DEGs)

Flow cytometry and FACS

Clinical gastric cancer samples

Statistical analysis

Results

AIL has a good inhibitory effect on GC

AIL promotes apoptosis through inducing DNA damage

AIL inhibits DNA repair in GC cells

AIL regulates XRCC1 through P23 to inhibit DNA repair

AIL inhibits the binding of P23 to HSP90 and HSP90 to XRCC1

High expression of P23 is related to GC recurrence

Discussion

Conclusion

Abbreviations

Declarations

References

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