Anti-cancer bioactive peptide induces apoptosis in gastric cancer cells through TP53 signaling cascade

Anti-cancer bioactive peptide induces apoptosis in gastric cancer cells through TP53 signaling cascade | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-cancer bioactive peptide induces apoptosis in gastric cancer cells through TP53 signaling cascade Qimuge Suyila, Xian Li, Xiulan Su This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4286067/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Our previous studies show that anti-cancer bioactive peptide (ACBP) inhibits the initiation and progression of gastric cancer via apoptosis and cell cycle arrest, however, the mechanisms is not clear. Here, we studied the effects of ACBP in three human gastric cancer cell lines, NCI-N87 and MGC-803, and investigated effect of ACBP on the survival and morphology of the cancer cell lines, examined apoptosis and cell cycle progression, detected the expression of TP53, TP63, and TP73 in cancer cells and the expression of Bax, PUMA and Mcl-1 in xenograft mouse model. ACBP inhibited the proliferation of three cancer cells dose-dependently as did the positive control and 5-fluorouracil (5-Fu). The effect of ACBP correlated with the degree of differentiation of cancer cells, the lower the differentiation degree, the stronger the inhibitory effect. After ACBP treatment, TP53, TP63 and TP73 expression was increased in all cell lines. In xenogragft mouse model, ACBP inhibited growth of MGC-803 cell in vivo. Apoptotic related genes Bax and PUMA were up-regulated, and Mcl-1 was down-regulated. ACBP inhibited tumor cell growth by induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP63 and TP73 and its downstream apoptosis promoting genes Bax and PUMA, down-regulating antiapoptotic gene Mcl-1. chemotherapy anti-cancer bioactive peptide differentiation human gastric cancer cells tumor suppressor gene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Despite the continuous decline in the worldwide incidence of gastric cancer over the past decade, it remains one of the leading causes of cancer-related death (1,2). Currently, in China, the incidence and mortality rates of gastric cancer are higher than the global average, ranking in the top 20, suggesting that the prevention and treatment of gastric cancer in China are important for its prevention and control globally (3). To date, most studies on gastric cancer have focused on drugs that target different risk factors. 5-Fluorouracil (5-Fu) is the first antimetabolite drug synthesized from design and is the most widely used anti-gastric cancer drug in clinical practice. Clinical application has proven the prominent efficacy of 5-Fu in gastrointestinal cancer and other solid tumors, and it is often used as a positive control in studies on new anti-cancer drugs (4-6). However, 5-Fu has significant toxic side effects, which affects its clinical use. Therefore, finding drugs that target the proliferation of gastric cancer cells, promote apoptosis of gastric cancer cells, but also exert less harmful effects on human bodies has become the focus of the research. Anti-cancer bioactive peptide (ACBP) was identified from goat spleen extract after immunization with gastric cancer lysates in our laboratory. ACBP is a polypeptide with low molecular weight (ca. 8,000 Da), which was enriched and purified by centrifugation, and microcolumn high-performance liquid chromatography (MHPLC). Our previous in vivo and in vitro studies have demonstrated that ACBP can effectively inhibit the initiation and progression of gastric cancer by promoting apoptosis and inducing cell cycle arrest (7, 8). However, the relationships between the effects of ACBP and the levels of cell differentiation as well as the mechanisms of function of ACBP have yet to be investigated. In this study, three different differentiated human gastric cancer cell lines, viz., NCI-N87 (well-differentiated tubular adenocarcinoma) and MGC-803 (poorly differentiated adenocarcinoma) were used. The effects of ACBP on apoptosis and cell cycle progression of these gastric cancer cells and xenograft tumor were observed, and the possible mechanisms of ACBP-induced apoptosis, with a focus on the expression of the tumor suppressor genes TP53 signaling cascade. Materials And Methods Cell lines and cell culture The human gastric cancer cell lines NCI-N87 and MGC-803 were kindly provided by Professor Ke Yang (Medical Healthy Center, Peking University, China). The human gastric cancer cell line MKN45 was purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College (Beijing, China). Cells were cultured in RPMI 1640 medium (HyClone, Thermo Scientific, Waltham, MA, USA) containing 10% FBS (HyClone, Thermo Scientific, Waltham, MA, USA) in a 5% CO 2 incubator (Heal Force Inc., Hong Kong, China) at 37ºC. The medium was changed every other day, and cells were passaged every 3-4 days. Tumor cell survival determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells in logarithmic phase were digested with 0.25% trypsin (HyClone, Thermo Scientific, Waltham, MA, USA), and then seeded in a 96-well plate at a concentration of 4 × 103 cells/well. ACBP, at a concentration of 5.25 µg/ml, 7 µg/ml, 8.75 µg/ml, or 10.5 µg/ml, was added to the cells. 5-Fu at a concentration of 0.01 µmol/l, 0.1 µmol/l, 1 µmol/l, or 10 µmol/l was used as the positive control and the same volume of saline was used as the negative control. After 24, 48, or 72-h incubation, 20 µl of MTT (Sigma-Aldrich, St Louis, MO, USA), at a concentration of 5 mg/ml, was added to each well and the cells were cultured for another 4 h. Then, the supernatant was discarded, 150 µl dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA) was added, and the plate was shaken for 10 min to fully dissolve the formazan before using a microplate reader to measure the absorbance at 570 nm. Samples exposed to each drug concentration were processed in triplicate and the average was calculated. Samples of each drug concentration were run in triplicate and the average cell inhibition rate (IR) was calculated using the following formula: IR = (absorbance of control group - absorbance of the experimental group)/absorbance of control group × 100%, and an average value was calculated for each well. Hematoxylin and eosin (HE) staining Three cell lines were seeded on coverslips. Cells were treated with saline (Ctrl), active peptide (ACBP), or positive control group (5-Fu) were stained with hematoxylin and eosin. Cellular morphology was observed. Apoptosis and cell cycle analysis Cells were routinely digested with 0.25% trypsin and washed thoroughly with phosphate-buffered saline (PBS) The cells for apoptosis assay were used the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) and proceeded according to manufacturer’s instruction. Briefly, 5 × 105 cells were collected and resuspended in 500 ml Binding Buffer. Then, 5 µl Annexin V-FITC and 20 µl propidium iodide (PI) was added and the mixture was mixed well. The reaction proceeded in the dark at room temperature for 15 min and cells were analyzed by flow cytometry (Becton Dickinson Co., Franklin Lakes, CA, USA) within 1 h to detect cell apoptosis. The cells for cell cycle analysis were used Cycletest Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA) and proceeded according to manufacturer’s instruction. Briefly, cells were collected and washed three times in cold PBS, and then incubated with 15 µl RNAse (100 µg/ml) at 4ºC in the dark for 15 min. Subsequently, after addition of 500 µl PBS containing 50 µg/ml PI, cells were incubated in the dark at 4ºC in the dark for 30 min and then analyzed by flow cytometry. A part of xenograft tumor was minced into small pieces by scissors and digested in collagenase IV solution for 1-2 hr at 37 °C to get single cell suspension. Apoptosis and cell cycle analysis was performed as described above. Semi-quantitative RT-PCR analysis Total RNA was extracted from control cells or cells treated with ACBP or 5-Fu by using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The amount of total RNA was measured by a UV spectrophotometer. At 37ºC, 2 µg of total RNA was digested with DNase, and cDNA was synthesized using PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan). RT-PCR was conducted in a 20-μL reaction volume containing 4 μL PrimeScript buffer (5 ×), 20 units RNase Inhibitor, 200 units PrimeScript RTase, 1 μL Oligo dT primer (50 μM), 1 μL dNTP (10 mM), and 2 μg RNA. The RT-PCR mix was incubated at 30°C for 10 min, followed by incubation at 42°C for 60 min, and 95°C for 5 min. The primer sequence for TP53 was F: 5′-ATGAAGCTCCCAGAATGCCA-3′, R: 5′-AATCAACCCACA GCTGCACA-3′, and the amplified product was 232 bp. The primer sequence of TP63 was F: 5-′CTGCCCTGACCCTTACATC-3′, tR: 5′-CAGTCCATGCTAACTCAATC-3′, and the amplified product was 267 bp. The primer sequence of TP73 was F: 5′-GGAACCAGACAGCACCTAC-3′, R: 5′-GTTGTGCGTAGGG CGAGTG-3′, and the amplified product was 167 bp. The primer sequences of GAPDH were F: 5′-AGAAGGCTGGGGCTCATTTG-3′, R: 5′-AGGGGCCATCCACAGTCTTC-3′, and the amplified fragment was 258 bp. PCR was conducted in a 50-μL reaction volume containing 5 μL Ex Taq buffer (10 ×; TaKaRa, Otsu, Japan), 4 μL dNTP (2.5 mM; TaKaRa, Otsu, Japan), 0.2 μM of each forward and reverse primer, 0.25 μL (5 U/μL) TaKaRa Ex Taq (TaKaRa, Otsu, Japan), and 1 μL cDNA. PCR reaction conditions were as follows: initial denaturation at 94ºC for 4 min; 28-32 cycles of denaturation for 30 s at 94ºC, annealing for 30 s at 60-62ºC (temperature according to the gene of interest), and extension for 30 s at 72ºC; and final extension at 10 min at 72ºC. PCR products were separated by 2% agarose gel electrophoresis (containing 0.15 g/l ethidium bromide), and the images obtained were analyzed with a gel imaging and analysis system (Heal Force Inc., Hong Kong, China). ELISA assay for protein levels Cell suspension samples were collected from the ACBP, 5-Fu, and control groups. The samples or standards were added to the 96-well plates pre-coated with TP53, TP63, and TP73 antibodies and assayed in triplicate using Human Total p53 DuoSet IC ELISA (R&D systems Inc., Minnesota, USA), Human Tumor Protein P63 ELISA Kit (Abbexa Ltd, Cambridge, UK), and LSBioTM Mouse/Human/Rat TP73/p73 DNA Binding ELISA Kit (LifeSpan BioSciences, Inc., Washington State, USA). After a 30-min incubation at 37ºC, solutions were discarded and the wells were washed five times. Then, horseradish peroxidase-labeled streptavidin was added and incubated for 30 min at 37ºC, followed by five washes, after which the chromogenic reaction was performed. After termination of the reaction, the optical density (OD) value at 450 nm of each well was measured within 15 min by microplate reader (Leidu; Shanghai Science and Technology Co., Beijing, China). The value of the blank well was set to zero. A linear regression equation of the standard curve was generated using standard concentrations and the corresponding OD values. Subsequently, the sample concentrations were calculated from their OD values. The final concentration was calculated by multiplying the measured concentration with the dilution factor. Xenograft model and treatment The inhibition of tumor growth by ACBP-3 was observed in tumor-bearing mice. MGC-803 cells were grafted into the mice. As a result, all of the mice developed single palpable tumors approximately 2 weeks later. Mice with a tumor volume smaller than 80 mm3 or larger than 100 mm 3 or mice with tumor diabrosis were excluded from further experiments. The mice were injected subcutaneously once a day, with ACBP-3 or 5-FU. A similar number of NS was administered to mice as a control. All of the mice were simultaneously sacrificed at the end of the experiment. H&E staining and immunohistochemistry The tissues from the xenograft tumors were dissected after the mice were sacrificed. The tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were then prepared for H&E staining. Immunohistochemistry (IHC) was performed using a kit from MXB Biotechnologies with first antibodies to PUMA (bs-1573R, Bioss Antibodies), Bax (sc-7480, Santa Cruz Biotechnology) and Mcl-1 (4572s, Cell Signaling Technology). Positive staining was in brown (DAB substrate). Negative controls without primary antibody were included for each staining. Hematoxylin was used for counterstaining (in blue). Tissue pathology slides were reviewed and confirmed by two investigators. Positive cells were counted in at least 3 fields using an Olympus CX41 microscope. The overall IHC score was calculated by multiplying intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) by proportion positively stained cells (0, 80%). The IHC score was categorized as negative (IHC score = 0), weak positive (IHC score = 1-4) or strong positive expression (IHC score>4) for further analyses. Statistical analyses SPSS13.0 statistical software was used for all statistical analyses. The results are presented as mean ± standard deviation. The mean difference among the groups was compared by analysis of variance. The results of IHC were analyzed by Kruskal-Wallis test, with P < 0.05 being considered as statistically significant. Results Effect of ACBP on the proliferation of various differentiated human gastric cancer cells As the doses of ACBP increased, the viability of the three differentiated gastric cancer cell lines decreased significantly. ACBP at concentrations exceeding 7.0 µg/ml significantly inhibited the proliferation of NCI-N87 human gastric cells compared with the control (P < 0.01), while at concentrations exceeding 5.25 µg/ml, it inhibited the proliferation of MGC-803 human gastric cancer cells (P < 0.05). ACBP significantly inhibited the proliferation and viability of all three human gastric cancer cell lines as the doses increased (Figure 1A). The effect of four concentrations of 5-Fu, viz., 0.01 mmol/l, 0.1 mmol/l, 1 mmol/l, and 10 mmol/l, were compared with that of the control; 5-Fu at concentrations exceeding 0.1 mmol/l inhibited the proliferation of all three human gastric cancer cell lines (P < 0.01; Figure 1B). The three differentiated gastric cancer cell lines were treated with ACBP at either 5.25 µg/ml or 7.0 µg/ml for 24 h, 48 h, and 72 h, respectively. As the incubation time increased, the growth inhibition of ACBP on the three cell lines increased gradually. 5-Fu was used as positive control. Moreover, the growth inhibition by ACBP increased as the levels of cell differentiation increased, so that the inhibition rate was the lowest in NCI-N87 cells and was the highest in MGC-803 cells. The inhibition rates of ACBP at 48 h already reached more than 30%; therefore, 48 h was used in subsequent experiments (Figure 1C). ACBP induced morphological changes in variously differentiated human gastric cancer cells. Both ACBP and 5-Fu induced different morphological features of apoptosis in the three differentiated gastric cancer cell lines, including ring-shaped nuclei, karyopyknosis, chromatin marginalization, and horseshoe-shaped nuclei (Figure 2A). Changes in cell cycle progression and apoptosis. ACBP promoted cell apoptosis in MGC-803 human gastric cancer cell lines, and the difference between the ACBP group and control group was statistically significant (P < 0.01), while no apoptotic effect of ACBP was found in NCI-N87 cells (Figure 2B, C). After ACBP treatment, the number of NCI-N87 and MGC-803 cells in the G0/G1 phase was statistically significantly higher than that in the control group (P < 0.01). On the other hand, the number of cells in S phase and G2/M phase decreased, indicating that the two types of gastric cancer cells were arrested in G0/G1 phase. The positive control, 5-Fu, did not promote apoptosis in the gastric cancer cell lines, but arrested cells in G0/G1 phase, and the number of such cells were significantly increased compared with that in the control group (P < 0.01; Figures 2D, E). Effect of ACBP on the expression of TP53, TP63, TP73 First, we examine the gene expression of TP53, TP63 and TP73 in three gastric cancer cell lines. Levels of TP53, TP63 and TP73 were increased after ACBP treatment in three cell lines compared to the controls (P < 0.01). After 5-Fu treatment, TP53 expression decreased in MGC-803 cells, while, expression of TP53, TP63 and TP73 were increased in three cell lines compared to the controls (P < 0.01; Figure 3A, B, C). And then, we examined the protein levels of TP53, TP63, and TP73 in three gastric cancer cell lines. Protein levels of TP53, TP63, and TP73 were all increased in NCI-N87 cells after ACBP treatment. Compared with the control, the TP53 level increase was not statistically significant, while those of TP63 and TP73 proteins were statistically significant (both P < 0.01). In MGC-803 cells, the protein levels of TP53, TP63, and TP73 also increased after ACBP treatment; however, again, only the increase in TP73 was significant (P < 0.05) compared to the level for the control group. The positive control 5-Fu induced a statistically significant increase in TP53, TP63, and TP73 protein levels in cell lines compared with the control (P < 0.01; Figure 3D, E). Effects of ACBP on xenograft tumor ACBP and 5-FU inhibited MGC-803 growth in xenograft model. The tumor weight was decreased after ACBP or 5-FU treatment (Figure 4A). The inhibition rate of tumor was 31.76% for ACBP, and 35.24% for 5-FU (Figure 4B). Compare to the control, the number of tumor cells in the G0/G1 phase decreased, and those in the G2/M phase increased after ACBP treatment (Figure 4C). ACBP induced apoptosis in xenograft tumor, while 5-FU did not (Figure 4D). Expression of apoptotic related proteins in xenograft tumor We also examined the expression of PUMA, Bax and Mcl-1 in tumor tissue of xenograft model. After H&E staining, the sections showed focal tumor and tumor cells. Immunohistochemical results showed that the positive staining of PUMA and Bax was mainly in cancer cell cytoplasm and cell membrane, while, Mcl-1 positive expression was found in cancer cell nucleus and cytoplasm. Compared with control group, PUMA expression was more in 5-Fu, ACBP (P < 0.01) and combined treatment. The positive staining of Bax was also found more in 5-Fu (P < 0.05), ACBP (P < 0.05) and combined treatment. Mcl-1 expression in 5-Fu and ACBP treatment did not change, but in combined treatment its expression disappeared (P < 0.05) (Figure 5). Discussion ACBP is a biologically active, low molecular weight polypeptide separated and purified from the spleens of goats immunized with gastric cancer lysates (4,9). Over the past decade, acute toxicity and chronic toxicity tests completed in mice and rats have indicated that ACBP does not interfere with normal physiological processes and enzyme metabolism, and has no toxic side effects. In vitro studies have shown that ACBP strongly inhibits the proliferation of multiple tumor cell lines (10-12). Previous in vivo animal experiments indicated that the total tumor inhibition rates of ACBP could reach 30-50%, and confirmed that ACBP exerts its anti-cancer effects through the inhibition of DNA synthesis and induction of apoptosis and cell differentiation, leading to inhibition of cell proliferation (5,13). In this study, we have used flow cytometry, ELISA, and RT-PCR to examine the expression of TP53, TP63, and TP73 after ACBP treatment in two different differentiated human gastric cancer cell lines, NCI-N87 (well-differentiated) and MGC-803 (poorly differentiated). Additionally, we analyzed the changes in the two cell lines and the effects of ACBP on cell cycle progression and apoptosis, and explored the possible mechanisms for the inhibition of ACBP on gastric cancer cell growth in vitro and in vivo. The data presented here indicated that the inhibitory effects of ACBP on cancer cell growth in the two differentiated gastric cancer cell lines increased gradually as the concentration and incubation time increased. The effect of ACBP correlated positively with the degree of malignancy of cancer cells, i.e., the higher the malignancy degree, the stronger the inhibitory effect. For instance, 7.0 µg/ml of ACBP significantly inhibited NCI-N87 cell growth, while for MGC-803 cells, a concentration of only 5.25 µg/ml ACBP was sufficient to inhibit tumor cell growth significantly. Morphological observation and flow cytometry showed that ACBP induced apoptosis in all three tumor cell lines. Furthermore, ACBP also induced cell cycle arrest. To confirm whether these series of ACBP-induced changes were associated with the induced changes in expression of members of the TP53 tumor suppressor gene family, we examined the expression of TP53, TP63, and TP73 at gene and protein levels. TP53 is an important tumor suppressor gene. Mutations in this gene have been found in 50% of human tumor tissues and are the most common genetic alterations in cancer (12,14). The biological functions of TP53 can be summarized as tumor inhibition (including the cell cycle arrest in G1 and the inhibition of G2/M transition) and induction of apoptosis. It plays an important role in the formation of various tumors and tissue injury. TP63 and TP73 are newly discovered members of the TP53 family, with a high degree of homology to TP53, and are important factors in tumorigenesis (3). TP63, identified in 1998 by Okada et al. and is located on chromosome 3q27-28, is a candidate tumor suppressor gene and can induce apoptosis in TP53-deficient tumor cells. Together with TP53, TP63 can also activate p21, Bax, and other relevant molecular (8). It has been found that TP63 not only regulates the growth and differentiation of normal squamous cells but is also important for the initiation and progression of squamous cell carcinoma (15,16) TP73 was identified in 1997 by Kaghad et al. and is located on chromosome 1p36 (17). When overexpressed, TP73 can activate transcription from TP53-responsive promoters and inhibit apoptosis, leading to cell proliferation (18, 19). Moreover, elevated expression of TP53, TP63, and TP73 proteins is involved in the initiation and development of osteosarcoma and is closely related to the progression of osteosarcoma. TP53 mutations may be associated with the abnormal expression of TP63 and TP73. Our data indicated that ACBP induces expression of the tumor suppressor genes TP53, TP63, and TP73, which may ultimately lead to cell cycle arrest and apoptosis, therefore shedding more light on the relationship between ACBP and TP53 family members. B cell leukemia-2 (Bcl-2) protein family plays an important role in regulation of apoptosis. These proteins can be divided into two subfamilies according to different structure and function, the family of antiapoptotic proteins (such as the Bcl-2, the Bcl-xl, myeloid leukemia cells-1 (Mcl-1) and A1) and promoting apoptosis protein family. The latter can be divided into two subtribes including the multiple structure domain proteins (Bak, Bax) and BH3-only protein (Bim, the Bid, Noxa, Puma) (22). Bax and PUMA were apoptosis promoting genes. Bax proteins play a pivotal role in the process of apoptosis. When the apoptosis occurred under the regulation of TP53, Bcl-2 protein decreased, while, Bax protein increased, the Bcl-2/Bax ratio and the Bcl-2/Bax heterologous dimer decreased, Bax homologous dimer increased. Bax bound to mitochondrial membrane, forming permeability membrane pore complex and mediating mitochondrial to release factors, including: cytochrome C, apoptosis inducing factor (AIF). Bax gene mutations are often found in human digestive tract tumors and leukemia. About 50% of human colonic cancer (25) and 60% of the gastric cancers (26) with microsatellite instability had reading frame shift mutation of Bax gene. PUMA can be rapidly induced by TP53 and has strong activity in promoting apoptosis; therefore, it was named PUMA as p53 up-regulated modulator of apoptosis (PUMA). PUMA has not only attenuated the inhibition of and sensitized Bax, but also directly activate Bax. The alpha 1 spiral carboxyl terminal area of Bax can directly interact with the BH-3 structure domain of PUMA (2). PUMA directly activate Bax/Bak polymerization, form the apoptosis process was known as "direct model" to promote apoptosis. Recent study also found that phosphorylation of PUMA protein was involved in mitochondrial autophagy (23). In tumor cells, PUMA is inhibited and cannot effectively establish apoptosis. Therefore, elevated PUMA expression in tumor cell can increase the effect of tumor treatment (24). Our study showed that after ACBP treatment, Puma and Bax protein increased in tumor tissue of xenograft model. ACBP treatment induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP 63 and TP73 and its downstream genes Puma and Bax. Mcl-1, an antiapoptotic protein, is involved in cell apoptosis, differentiation and cell cycle regulation, is critical to the survival and growth of cells. The lack of Mcl-1 protein expression can cause neurodegenerative diseases; while excessive expression can lead to the occurrence of malignant tumor, and is closely related to the drug resistance of tumor (27). Our study showed that after ACBP treatment, Mcl-1 expression is significantly reduced in tumor tissue of xenograft model, indicating that ACBP induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP 63 and TP73 and down-regulating antiapoptotic gene Mcl-1. Conclusions In summary, our study found that ACBP inhibited growth of gastric cancers in vitro and in vivo by inducing cell cycle arrest and apoptosis. ACBP induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP 63 and TP73 and its downstream apoptosis promoting genes Puma and Bax, down-regulating antiapoptotic gene Mcl-1. Declarations Ethical Approval and Consent to Participate The study on animals were approved by the Ethics Committee for Animal Experiments of Inner Mongolia Medical College, China (number: YKD2016152), all procedures reporting in this study on the animals were carried in accordance with the ARRIVE guidelines, and all methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not Applicable. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Authors Contributions XLS and XL conceived and designed research. YLS conducted experiments and contributed new reagents or analytical tools and analyzed data. XL wrote the manuscript. All authors read and approved the manuscript and all data were generated in-house and that no paper mill was used. Funding We are grateful to all participants in this study. This work was supported financially by the National Natural Science Foundation of China (81450047, 81660468 and 22168028), and Inner Mongolia Natural Science Fund surface project (2021MS02005), and the Inner Mongolia Autonomous Region Health Science and Technology Plan Project (202201274), and Inner Mongolia Autonomous Region "Grassland Talent" plan project. Our manuscript was translated and edited by the Editage. Acknowledgement Not Applicable. References Furukawa K, Hatakeyama K, Terashima M, Nagashima T, Urakami K, Ohshima K,et al. Molecular classification of gastric cancer predicts survival in patients undergoing radical gastrectomy based on project HOPE. Gastric Cancer. 25(1):138-148, 2022. Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D. Global cancer statistics. CA Cancer J Clin. 61: 69-90, 2012. Cui R, He J, Mei R, de Fromentel CC, Martel-Planche G, Taniere P, et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4286067","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":293533506,"identity":"d615df4c-3f89-466c-8e5c-31b0350288e4","order_by":0,"name":"Qimuge Suyila","email":"","orcid":"","institution":"Inner Mongolia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qimuge","middleName":"","lastName":"Suyila","suffix":""},{"id":293533507,"identity":"2f2faed6-a37f-4e3a-bfb7-59f5dae1dbc8","order_by":1,"name":"Xian Li","email":"","orcid":"","institution":"Inner Mongolia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Li","suffix":""},{"id":293533508,"identity":"0b6ad0be-ef59-4b24-a48d-d39d66b7627c","order_by":2,"name":"Xiulan Su","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYDACdiD+2ABiMTYeIE4LM1DtzAYGCSDVQLwWZl6wFgYG4rQYHGZ/Jm27w6ZOt/0w0JYam2iCWiSbecykc8+kSZidSQRqOZaW20BICz8zD5t0btthCbMDQC2MDYcJa2FjBjrMEqTl/EMitfAzM5hJM4K03CDWFqBfjC1729Ikt90A2pJAjF8Mjrc/vPGzzYbf7Hz6wwcfamwIa0EFCaQpHwWjYBSMglGACwAAuag+6YvRyQkAAAAASUVORK5CYII=","orcid":"","institution":"Inner Mongolia Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiulan","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2024-04-18 07:59:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4286067/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4286067/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55228135,"identity":"bb5a7e56-4be1-4411-8a6f-a78e6207c3b4","added_by":"auto","created_at":"2024-04-24 11:38:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":190596,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effect of ACBP at different concentrations on three differentiated human gastric cancer cell lines. NCI-N87 and MGC-803 were treated with ACBP at 5.25, 7, 8.75, and 10.5 µg/ml. ACBP significantly inhibited the proliferation and viability of all three human gastric cancer cell lines as the doses increased (Compared with the control, *P \u0026lt; 0.05, **P \u0026lt; 0.01). (B)\u003cstrong\u003e \u003c/strong\u003eEffect of different concentrations of 5-Fu on three differentiated human gastric cancer cell lines. NCI-N87 and MGC-803 was treated with 5-Fu at 0.01, 0.1, 1, and 10 mmol/l. 5-Fu at 0.1 mmol/l inhibited the proliferation of all three human gastric cancer cell lines (**P \u0026lt; 0.01). (C)\u003cstrong\u003e \u003c/strong\u003eThe inhibition rate of proliferation in gastric cancer cell lines with ACBP and 5-Fu. NCI-N87 was treated with 7 µg/ml ACBP, MGC-803 were treated with 5.25 µg/ml ACBP, and all three cell lines were treated with 0.1 mmol/l 5-Fufor 24, 48, and 72 h. ACBP and 5-Fu inhibited cell growth time-dependently in all cell lines.\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4286067/v1/5045d4479ad5d00858e807d2.png"},{"id":55228136,"identity":"9ed35a7f-3a55-4a4f-9229-23c5fb4afc76","added_by":"auto","created_at":"2024-04-24 11:38:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":369285,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Hematoxylin and eosin staining of three human gastric cancer cell lines after ACBP treatment (×100). Morphological analysis showed apoptotic features a higher number of apoptotic cells were found in the ACBP than in the control group.\u003cstrong\u003e \u003c/strong\u003e(B) Apoptotic effect of ACBP in various differentiated human gastric cancer cell lines. (C)\u003cstrong\u003e \u003c/strong\u003eACBP promoted significant cell apoptosis in MGC-803 human gastric cancer cell lines.\u003cstrong\u003e \u003c/strong\u003e(D, E)\u003cstrong\u003e \u003c/strong\u003eEffect of ACBP on cell cycle progression in various differentiated human gastric cancer cell lines. Compared with the control (*, P \u0026lt; 0.05, **, P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4286067/v1/1c92ec09011047d653780679.png"},{"id":55228137,"identity":"5b7969bc-274a-49cf-afb6-cfebd73769d1","added_by":"auto","created_at":"2024-04-24 11:38:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90802,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of TP53 family members during ACBP-induced apoptosis in various differentiated human gastric cancer cells. (A) Agarose gel (2%) showed the products of semi-quantitative reverse transcription-polymerase chain reaction analysis of the expression of TP53, TP63, and TP73 mRNA in the three gastric cancer cell lines after treatment with ACBP, in comparison to control and 5-Fu. Graphical representation of the changes in mRNA expression of TP53, TP63, and TP73 in the three gastric cancer cell lines after treatment with ACBP, in comparison to control and 5-Fu in NCI-N87 (B) and MGC-803(C). Changes in the protein levels of TP53 family members during ACBP-induced apoptosis in various differentiated human gastric cancer cells in NCI-N87 (D) and MGC-803(E). Compared with the control (*, P \u0026lt; 0.05, **, P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4286067/v1/e158fd46985c51b12b19a08e.png"},{"id":55228606,"identity":"998b8ab4-c6eb-4a70-ac4e-e69a5395b7c0","added_by":"auto","created_at":"2024-04-24 11:46:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":860169,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ACPB on MGC-803 in xenograft mouse model. (A) ACBP and 5-Fu inhibited growth of MGC-803 in xenograft mouse model. (B) The inhibition rate of tumor growth in xenograft mouse model treated with ACBP and 5-Fu. (C) Effect of ACBP on cell cycle progression of MGC-803 in xenograft mouse model. Compare to the control, the number of tumorcells in the G2/M phase increased after ACBP and 5-Fu treatment. (D) Apoptotic effect of ACBP on MGC-803 in xenograft mouse model. ACBP induced apoptosis in xenograft tumor.\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4286067/v1/0406934ff5cfdd90a21361dc.png"},{"id":55228138,"identity":"49456bac-d7e9-454a-91fe-a18f28bc1eb2","added_by":"auto","created_at":"2024-04-24 11:38:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1813239,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effect of ACBP on the expression of Puma, Bax and Mcl-1 in xenograft mouse. Immunohistochemical staining showed expression of Puma, Bax and Mcl-1 in gastric cancer from xenograft mouse. Immunostaining showed a clear nuclear or cytoplasm staining. Original magnifications: ×400. (B) The expression differences between Ctrl, ACBP and 5-Fu. Compared with the control (*, P \u0026lt; 0.05, **, P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4286067/v1/c30a64e3cc20b82d29d3f3a6.png"},{"id":56715951,"identity":"8f753005-3373-46ee-9aa4-69397fff4d3c","added_by":"auto","created_at":"2024-05-18 19:08:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4724651,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4286067/v1/53a01c98-3917-4bd7-a296-8a5cd4a05678.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-cancer bioactive peptide induces apoptosis in gastric cancer cells through TP53 signaling cascade","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite the continuous decline in the worldwide incidence of gastric cancer over the past decade, it remains one of the leading causes of cancer-related death (1,2). Currently, in China, the incidence and mortality rates of gastric cancer are higher than the global average, ranking in the top 20, suggesting that the prevention and treatment of gastric cancer in China are important for its prevention and control globally (3). To date, most studies on gastric cancer have focused on drugs that target different risk factors. 5-Fluorouracil (5-Fu) is the first antimetabolite drug synthesized from design and is the most widely used anti-gastric cancer drug in clinical practice. Clinical application has proven the prominent efficacy of 5-Fu in gastrointestinal cancer and other solid tumors, and it is often used as a positive control in studies on new anti-cancer drugs (4-6). However, 5-Fu has significant toxic side effects, which affects its clinical use. Therefore, finding drugs that target the proliferation of gastric cancer cells, promote apoptosis of gastric cancer cells, but also exert less harmful effects on human bodies has become the focus of the research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnti-cancer bioactive peptide (ACBP) was identified from goat spleen extract after immunization with gastric cancer lysates in our laboratory. ACBP is a polypeptide with low molecular weight (ca. 8,000 Da), which was enriched and purified by centrifugation, and microcolumn high-performance liquid chromatography (MHPLC). Our previous in vivo and in vitro studies have demonstrated that ACBP can effectively inhibit the initiation and progression of gastric cancer by promoting apoptosis and inducing cell cycle arrest (7, 8). However, the relationships between the effects of ACBP and the levels of cell differentiation as well as the mechanisms of function of ACBP have yet to be investigated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, three different differentiated human gastric cancer cell lines, viz., NCI-N87 (well-differentiated tubular adenocarcinoma) and MGC-803 (poorly differentiated adenocarcinoma) were used. The effects of ACBP on apoptosis and cell cycle progression of these gastric cancer cells and xenograft tumor were observed, and the possible mechanisms of ACBP-induced apoptosis, with a focus on the expression of the tumor suppressor genes TP53 signaling cascade.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell lines and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human gastric cancer cell lines NCI-N87 and MGC-803 were kindly provided by Professor Ke Yang (Medical Healthy Center, Peking University, China). The human gastric cancer cell line MKN45 was purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College (Beijing, China). Cells were cultured in RPMI 1640 medium (HyClone, Thermo Scientific, Waltham, MA, USA) containing 10% FBS (HyClone, Thermo Scientific, Waltham, MA, USA) in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator (Heal Force Inc., Hong Kong, China) at 37\u0026ordm;C. The medium was changed every other day, and cells were passaged every 3-4 days.\u003c/p\u003e\n\u003cp\u003eTumor cell survival determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells in logarithmic phase were digested with 0.25% trypsin (HyClone, Thermo Scientific, Waltham, MA, USA), and then seeded in a 96-well plate at a concentration of 4 \u0026times; 103 cells/well. ACBP, at a concentration of 5.25 \u0026micro;g/ml, 7 \u0026micro;g/ml, 8.75 \u0026micro;g/ml, or 10.5 \u0026micro;g/ml, was added to the cells. 5-Fu at a concentration of 0.01 \u0026micro;mol/l, 0.1 \u0026micro;mol/l, 1 \u0026micro;mol/l, or 10 \u0026micro;mol/l was used as the positive control and the same volume of saline was used as the negative control. After 24, 48, or 72-h incubation, 20 \u0026micro;l of MTT (Sigma-Aldrich, St Louis, MO, USA), at a concentration of 5 mg/ml, was added to each well and the cells were cultured for another 4 h. Then, the supernatant was discarded, 150 \u0026micro;l dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA) was added, and the plate was shaken for 10 min to fully dissolve the formazan before using a microplate reader to measure the absorbance at 570 nm. Samples exposed to each drug concentration were processed in triplicate and the average was calculated. Samples of each drug concentration were run in triplicate and the average cell inhibition rate (IR) was calculated using the following formula: IR = (absorbance of control group\u0026nbsp;-\u0026nbsp;absorbance of the experimental group)/absorbance of control group \u0026times; 100%, and an average value was calculated for each well.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and eosin (HE) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree cell lines were seeded on coverslips. Cells were treated with saline (Ctrl), active peptide (ACBP), or positive control group (5-Fu) were stained with hematoxylin and eosin. Cellular morphology was observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis and cell cycle analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were routinely digested with 0.25% trypsin and washed thoroughly with phosphate-buffered saline (PBS) The cells for apoptosis assay were used the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) and proceeded according to manufacturer\u0026rsquo;s instruction. Briefly, 5 \u0026times; 105 cells were collected and resuspended in 500 ml Binding Buffer. Then, 5 \u0026micro;l Annexin V-FITC and 20 \u0026micro;l propidium iodide (PI) was added and the mixture was mixed well. The reaction proceeded in the dark at room temperature for 15 min and cells were analyzed by flow cytometry (Becton Dickinson Co., Franklin Lakes, CA, USA) within 1 h to detect cell apoptosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cells for cell cycle analysis were used Cycletest Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA) and proceeded according to manufacturer\u0026rsquo;s instruction. Briefly, cells were collected and washed three times in cold PBS, and then incubated with 15 \u0026micro;l RNAse (100 \u0026micro;g/ml) at 4\u0026ordm;C in the dark for 15 min. Subsequently, after addition of 500 \u0026micro;l PBS containing 50 \u0026micro;g/ml PI, cells were incubated in the dark at 4\u0026ordm;C in the dark for 30 min and then analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003eA part of xenograft tumor was minced into small pieces by scissors and digested in collagenase IV solution for 1-2 hr at 37 \u0026deg;C to get single cell suspension. Apoptosis and cell cycle analysis was performed as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSemi-quantitative RT-PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from control cells or cells treated with ACBP or 5-Fu by using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The amount of total RNA was measured by a UV spectrophotometer. At 37\u0026ordm;C, 2 \u0026micro;g of total RNA was digested with DNase, and cDNA was synthesized using PrimeScript\u0026trade;\u0026nbsp;1st Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan). RT-PCR was conducted in a 20-\u0026mu;L reaction volume containing 4 \u0026mu;L PrimeScript buffer (5 \u0026times;), 20 units RNase Inhibitor, 200 units PrimeScript RTase, 1 \u0026mu;L Oligo dT primer (50 \u0026mu;M), 1 \u0026mu;L dNTP (10 mM), and 2 \u0026mu;g RNA. The RT-PCR mix was incubated at 30\u0026deg;C for 10 min, followed by incubation at 42\u0026deg;C for 60 min, and 95\u0026deg;C for 5 min. The primer sequence for TP53 was F: 5\u0026prime;-ATGAAGCTCCCAGAATGCCA-3\u0026prime;, R: 5\u0026prime;-AATCAACCCACA GCTGCACA-3\u0026prime;, and the amplified product was 232 bp. The primer sequence of TP63 was F: 5-\u0026prime;CTGCCCTGACCCTTACATC-3\u0026prime;, tR: 5\u0026prime;-CAGTCCATGCTAACTCAATC-3\u0026prime;, and the amplified product was 267 bp. The primer sequence of TP73 was F: 5\u0026prime;-GGAACCAGACAGCACCTAC-3\u0026prime;, R: 5\u0026prime;-GTTGTGCGTAGGG CGAGTG-3\u0026prime;, and the amplified product was 167 bp. The primer sequences of GAPDH were F: 5\u0026prime;-AGAAGGCTGGGGCTCATTTG-3\u0026prime;, R: 5\u0026prime;-AGGGGCCATCCACAGTCTTC-3\u0026prime;, and the amplified fragment was 258 bp. PCR was conducted in a 50-\u0026mu;L reaction volume containing 5 \u0026mu;L Ex Taq buffer (10 \u0026times;; TaKaRa, Otsu, Japan), 4 \u0026mu;L dNTP (2.5 mM; TaKaRa, Otsu, Japan), 0.2 \u0026mu;M of each forward and reverse primer, 0.25 \u0026mu;L (5 U/\u0026mu;L) TaKaRa Ex Taq (TaKaRa, Otsu, Japan), and 1 \u0026mu;L cDNA. PCR reaction conditions were as follows: initial denaturation at 94\u0026ordm;C for 4 min; 28-32 cycles of denaturation for 30 s at 94\u0026ordm;C, annealing for 30 s at 60-62\u0026ordm;C (temperature according to the gene of interest), and extension for 30 s at 72\u0026ordm;C; and final extension at 10 min at 72\u0026ordm;C. PCR products were separated by 2% agarose gel electrophoresis (containing 0.15 g/l ethidium bromide), and the images obtained were analyzed with a gel imaging and analysis system (Heal Force Inc., Hong Kong, China). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA assay for protein levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell suspension samples were collected from the ACBP, 5-Fu, and control groups. The samples or standards were added to the 96-well plates pre-coated with TP53, TP63, and TP73 antibodies and assayed in triplicate using Human Total p53 DuoSet IC ELISA (R\u0026amp;D systems Inc., Minnesota, USA), Human Tumor Protein P63 ELISA Kit (Abbexa Ltd, Cambridge, UK), and LSBioTM Mouse/Human/Rat TP73/p73 DNA Binding ELISA Kit (LifeSpan BioSciences, Inc., Washington State, USA). After a 30-min incubation at 37\u0026ordm;C, solutions were discarded and the wells were washed five times. Then, horseradish peroxidase-labeled streptavidin was added and incubated for 30 min at 37\u0026ordm;C, followed by five washes, after which the chromogenic reaction was performed. After termination of the reaction, the optical density (OD) value at 450 nm of each well was measured within 15 min by microplate reader (Leidu; Shanghai Science and Technology Co., Beijing, China). The value of the blank well was set to zero. A linear regression equation of the standard curve was generated using standard concentrations and the corresponding OD values. Subsequently, the sample concentrations were calculated from their OD values. The final concentration was calculated by multiplying the measured concentration with the dilution factor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXenograft model and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inhibition of tumor growth by ACBP-3 was observed in tumor-bearing mice. MGC-803 cells were grafted into the mice. As a result, all of the mice developed single palpable tumors approximately 2 weeks later. Mice with a tumor volume smaller than 80 mm3 or larger than 100 mm\u003csup\u003e3\u003c/sup\u003e or mice with tumor diabrosis were excluded from further experiments. The mice were injected subcutaneously once a day, with ACBP-3 or 5-FU. A similar number of NS was administered to mice as a control. All of the mice were simultaneously sacrificed at the end of the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E staining and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The tissues from the xenograft tumors were dissected after the mice were sacrificed. The tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were then prepared for H\u0026amp;E staining. Immunohistochemistry (IHC) was performed using a kit from MXB Biotechnologies with first antibodies to PUMA (bs-1573R, Bioss Antibodies), Bax (sc-7480, Santa Cruz Biotechnology) and Mcl-1 (4572s, Cell Signaling Technology). Positive staining was in brown (DAB substrate). Negative controls without primary antibody were included for each staining. Hematoxylin was used for counterstaining (in blue). Tissue pathology slides were reviewed and confirmed by two investigators. Positive cells were counted in at least 3 fields using an Olympus CX41 microscope. The overall IHC score was calculated by multiplying intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) by proportion positively stained cells (0, \u0026lt;10%; 1, 11-50%; 2, 51%-80%; 3, \u0026gt;80%). The IHC score was categorized as negative (IHC score = 0), weak positive (IHC score = 1-4) or strong positive expression (IHC score\u0026gt;4) for further analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;SPSS13.0 statistical software was used for all statistical analyses. The results are presented as mean \u0026plusmn; standard deviation. The mean difference among the groups was compared by analysis of variance. The results of IHC were analyzed by Kruskal-Wallis test, with P \u0026lt; 0.05 being considered as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of ACBP on the proliferation of various differentiated human gastric cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs the doses of ACBP increased, the viability of the three differentiated gastric cancer cell lines decreased significantly. ACBP at concentrations exceeding 7.0 \u0026micro;g/ml significantly inhibited the proliferation of NCI-N87 human gastric cells compared with the control (P \u0026lt; 0.01), while at concentrations exceeding 5.25 \u0026micro;g/ml, it inhibited the proliferation of MGC-803 human gastric cancer cells (P \u0026lt; 0.05). ACBP significantly inhibited the proliferation and viability of all three human gastric cancer cell lines as the doses increased (Figure 1A). The effect of four concentrations of 5-Fu, viz., 0.01 mmol/l, 0.1 mmol/l, 1 mmol/l, and 10 mmol/l, were compared with that of the control; 5-Fu at concentrations exceeding 0.1 mmol/l inhibited the proliferation of all three human gastric cancer cell lines (P \u0026lt; 0.01; Figure 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe three differentiated gastric cancer cell lines were treated with ACBP at either 5.25 \u0026micro;g/ml or 7.0 \u0026micro;g/ml for 24 h, 48 h, and 72 h, respectively. As the incubation time increased, the growth inhibition of ACBP on the three cell lines increased gradually. 5-Fu was used as positive control. Moreover, the growth inhibition by ACBP increased as the levels of cell differentiation increased, so that the inhibition rate was the lowest in NCI-N87 cells and was the highest in MGC-803 cells. The inhibition rates of ACBP at 48 h already reached more than 30%; therefore, 48 h was used in subsequent experiments (Figure 1C).\u003c/p\u003e\n\u003cp\u003eACBP induced morphological changes in variously differentiated human gastric cancer cells. Both ACBP and 5-Fu induced different morphological features of apoptosis in the three differentiated gastric cancer cell lines, including ring-shaped nuclei, karyopyknosis, chromatin marginalization, and horseshoe-shaped nuclei (Figure 2A).\u003c/p\u003e\n\u003cp\u003eChanges in cell cycle progression and apoptosis. ACBP promoted cell apoptosis in MGC-803 human gastric cancer cell lines, and the difference between the ACBP group and control group was statistically significant (P \u0026lt; 0.01), while no apoptotic effect of ACBP was found in NCI-N87 cells (Figure 2B, C). After ACBP treatment, the number of NCI-N87 and MGC-803 cells in the G0/G1 phase was statistically significantly higher than that in the control group (P \u0026lt; 0.01). On the other hand, the number of cells in S phase and G2/M phase decreased, indicating that the two types of gastric cancer cells were arrested in G0/G1 phase. The positive control, 5-Fu, did not promote apoptosis in the gastric cancer cell lines, but arrested cells in G0/G1 phase, and the number of such cells were significantly increased compared with that in the control group (P \u0026lt; 0.01; Figures 2D, E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ACBP on the expression of TP53, TP63, TP73\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, we examine the gene expression of TP53, TP63 and TP73 in three gastric cancer cell lines. Levels of TP53, TP63 and TP73 were increased after ACBP treatment in three cell lines compared to the controls (P \u0026lt; 0.01). After 5-Fu treatment, TP53 expression decreased in MGC-803 cells, while, expression of TP53, TP63 and TP73 were increased in three cell lines compared to the controls (P \u0026lt; 0.01; Figure 3A, B, C). And then, we examined the protein levels of TP53, TP63, and TP73 in three gastric cancer cell lines. Protein levels of TP53, TP63, and TP73 were all increased in NCI-N87 cells after ACBP treatment. Compared with the control, the TP53 level increase was not statistically significant, while those of TP63 and TP73 proteins were statistically significant (both P \u0026lt; 0.01). In MGC-803 cells, the protein levels of TP53, TP63, and TP73 also increased after ACBP treatment; however, again, only the increase in TP73 was significant (P \u0026lt; 0.05) compared to the level for the control group. The positive control 5-Fu induced a statistically significant increase in TP53, TP63, and TP73 protein levels in cell lines compared with the control (P \u0026lt; 0.01; Figure 3D, E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of ACBP on xenograft tumor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eACBP and 5-FU inhibited MGC-803 growth in xenograft model. The tumor weight was decreased after ACBP or 5-FU treatment (Figure 4A). The inhibition rate of tumor was 31.76% for ACBP, and 35.24% for 5-FU (Figure 4B). Compare to the control, the number of tumor cells in the G0/G1 phase decreased, and those in the G2/M phase increased after ACBP treatment (Figure 4C). ACBP induced apoptosis in xenograft tumor, while 5-FU did not (Figure 4D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of apoptotic related proteins in xenograft tumor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also examined the expression of PUMA, Bax and Mcl-1 in tumor tissue of xenograft model. After H\u0026amp;E staining, the sections showed focal tumor and tumor cells. Immunohistochemical results showed that the positive staining of PUMA and Bax was mainly in cancer cell cytoplasm and cell membrane, while, Mcl-1 positive expression was found in cancer cell nucleus and cytoplasm. Compared with control group, PUMA expression was more in 5-Fu, ACBP (P \u0026lt; 0.01) and combined treatment. The positive staining of Bax was also found more in 5-Fu (P \u0026lt; 0.05), ACBP (P \u0026lt; 0.05) and combined treatment. Mcl-1 expression in 5-Fu and ACBP treatment did not change, but in combined treatment its expression disappeared (P \u0026lt; 0.05) (Figure 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eACBP is a biologically active, low molecular weight polypeptide separated and purified from the spleens of goats immunized with gastric cancer lysates (4,9). Over the past decade, acute toxicity and chronic toxicity tests completed in mice and rats have indicated that ACBP does not interfere with normal physiological processes and enzyme metabolism, and has no toxic side effects. In vitro studies have shown that ACBP strongly inhibits the proliferation of multiple tumor cell lines (10-12). Previous in vivo animal experiments indicated that the total tumor inhibition rates of ACBP could reach 30-50%, and confirmed that ACBP exerts its anti-cancer effects through the inhibition of DNA synthesis and induction of apoptosis and cell differentiation, leading to inhibition of cell proliferation (5,13). In this study, we have used flow cytometry, ELISA, and RT-PCR to examine the expression of TP53, TP63, and TP73 after ACBP treatment in two different differentiated human gastric cancer cell lines, NCI-N87 (well-differentiated) and MGC-803 (poorly differentiated). Additionally, we analyzed the changes in the two cell lines and the effects of ACBP on cell cycle progression and apoptosis, and explored the possible mechanisms for the inhibition of ACBP on gastric cancer cell growth in vitro and in vivo.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data presented here indicated that the inhibitory effects of ACBP on cancer cell growth in the two differentiated gastric cancer cell lines increased gradually as the concentration and incubation time increased. The effect of ACBP correlated positively with the degree of malignancy of cancer cells, i.e., the higher the malignancy degree, the stronger the inhibitory effect. For instance, 7.0 \u0026micro;g/ml of ACBP significantly inhibited NCI-N87 cell growth, while for MGC-803 cells, a concentration of only 5.25 \u0026micro;g/ml ACBP was sufficient to inhibit tumor cell growth significantly. Morphological observation and flow cytometry showed that ACBP induced apoptosis in all three tumor cell lines. Furthermore, ACBP also induced cell cycle arrest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm whether these series of ACBP-induced changes were associated with the induced changes in expression of members of the TP53 tumor suppressor gene family, we examined the expression of TP53, TP63, and TP73 at gene and protein levels. TP53 is an important tumor suppressor gene. Mutations in this gene have been found in 50% of human tumor tissues and are the most common genetic alterations in cancer (12,14). The biological functions of TP53 can be summarized as tumor inhibition (including the cell cycle arrest in G1 and the inhibition of G2/M transition) and induction of apoptosis. It plays an important role in the formation of various tumors and tissue injury. TP63 and TP73 are newly discovered members of the TP53 family, with a high degree of homology to TP53, and are important factors in tumorigenesis (3). TP63, identified in 1998 by Okada et al. and is located on chromosome 3q27-28, is a candidate tumor suppressor gene and can induce apoptosis in TP53-deficient tumor cells. Together with TP53, TP63 can also activate p21, Bax, and other relevant molecular (8). It has been found that TP63 not only regulates the growth and differentiation of normal squamous cells but is also important for the initiation and progression of squamous cell carcinoma (15,16) TP73 was identified in 1997 by Kaghad et al. and is located on chromosome 1p36 (17). When overexpressed, TP73 can activate transcription from TP53-responsive promoters and inhibit apoptosis, leading to cell proliferation (18, 19). Moreover, elevated expression of TP53, TP63, and TP73 proteins is involved in the initiation and development of osteosarcoma and is closely related to the progression of osteosarcoma. TP53 mutations may be associated with the abnormal expression of TP63 and TP73. Our data indicated that ACBP induces expression of the tumor suppressor genes TP53, TP63, and TP73, which may ultimately lead to cell cycle arrest and apoptosis, therefore shedding more light on the relationship between ACBP and TP53 family members. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB cell leukemia-2 (Bcl-2) protein family plays an important role in regulation of apoptosis. These proteins can be divided into two subfamilies according to different structure and function, the family of antiapoptotic proteins (such as the Bcl-2, the Bcl-xl, myeloid leukemia cells-1 (Mcl-1) and A1) and promoting apoptosis protein family. The latter can be divided into two subtribes including the multiple structure domain proteins (Bak, Bax) and BH3-only protein (Bim, the Bid, Noxa, Puma) (22).\u003c/p\u003e\n\u003cp\u003eBax and PUMA were apoptosis promoting genes. Bax proteins play a pivotal role in the process of apoptosis. When the apoptosis occurred under the regulation of TP53, Bcl-2 protein decreased, while, Bax protein increased, the Bcl-2/Bax ratio and the Bcl-2/Bax heterologous dimer decreased, Bax homologous dimer increased. Bax bound to mitochondrial membrane, forming permeability membrane pore complex and mediating mitochondrial to release factors, including: cytochrome C, apoptosis inducing factor (AIF). Bax gene mutations are often found in human digestive tract tumors and leukemia. About 50% of human colonic cancer (25) and 60% of the gastric cancers (26) with microsatellite instability had reading frame shift mutation of Bax gene. PUMA can be rapidly induced by TP53 and has strong activity in promoting apoptosis; therefore, it was named PUMA as p53 up-regulated modulator of apoptosis (PUMA). PUMA has not only attenuated the inhibition of and sensitized Bax, but also directly activate Bax. The alpha 1 spiral carboxyl terminal area of Bax can directly interact with the BH-3 structure domain of PUMA (2). PUMA directly activate Bax/Bak polymerization, form the apoptosis process was known as \u0026quot;direct model\u0026quot; to promote apoptosis. Recent study also found that phosphorylation of PUMA protein was involved in mitochondrial autophagy (23). In tumor cells, PUMA is inhibited and cannot effectively establish apoptosis. Therefore, elevated PUMA expression in tumor cell can increase the effect of tumor treatment (24). Our study showed that after ACBP treatment, Puma and Bax protein increased in tumor tissue of xenograft model. ACBP treatment induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP 63 and TP73 and its downstream genes Puma and Bax.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMcl-1, an antiapoptotic protein, is involved in cell apoptosis, differentiation and cell cycle regulation, is critical to the survival and growth of cells. The lack of Mcl-1 protein expression can cause neurodegenerative diseases; while excessive expression can lead to the occurrence of malignant tumor, and is closely related to the drug resistance of tumor (27). Our study showed that after ACBP treatment, Mcl-1 expression is significantly reduced in tumor tissue of xenograft model, indicating that ACBP induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP 63 and TP73 and down-regulating antiapoptotic gene Mcl-1.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our study found that ACBP inhibited growth of gastric cancers in vitro and in vivo by inducing cell cycle arrest and apoptosis. ACBP induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP 63 and TP73 and its downstream apoptosis promoting genes Puma and Bax, down-regulating antiapoptotic gene Mcl-1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study on animals were approved by the Ethics Committee for Animal Experiments of Inner Mongolia Medical College, China (number: YKD2016152), all procedures reporting in this study on the animals were carried in accordance with the ARRIVE guidelines, and all methods were carried out in accordance with relevant guidelines and regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXLS and XL conceived and designed research. YLS conducted experiments and contributed new reagents or analytical tools and analyzed data. XL wrote the manuscript. All authors read and approved the manuscript and all data were generated in-house and that no paper mill was used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all participants in this study.\u0026nbsp;This work was supported financially by the National Natural Science Foundation of China (81450047, 81660468 and\u0026nbsp;22168028), and Inner Mongolia Natural Science Fund surface project (2021MS02005), and the Inner Mongolia Autonomous Region Health Science and Technology Plan Project (202201274), and Inner Mongolia Autonomous Region \u0026quot;Grassland Talent\u0026quot; plan project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur manuscript was translated and edited by the Editage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFurukawa K, Hatakeyama K, Terashima M, Nagashima T, Urakami K, Ohshima K,et al. 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Cells\u0026nbsp;3(2): 418-437,\u0026nbsp;2014.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chemotherapy, anti-cancer bioactive peptide, differentiation, human gastric cancer cells, tumor suppressor gene","lastPublishedDoi":"10.21203/rs.3.rs-4286067/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4286067/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Our previous studies show that anti-cancer bioactive peptide (ACBP) inhibits the initiation and progression of gastric cancer via apoptosis and cell cycle arrest, however, the mechanisms is not clear. Here, we studied the effects of ACBP in three human gastric cancer cell lines, NCI-N87 and MGC-803, and investigated effect of ACBP on the survival and morphology of the cancer cell lines, examined apoptosis and cell cycle progression, detected the expression of TP53, TP63, and TP73 in cancer cells and the expression of Bax, PUMA and Mcl-1 in xenograft mouse model. ACBP inhibited the proliferation of three cancer cells dose-dependently as did the positive control and 5-fluorouracil (5-Fu). The effect of ACBP correlated with the degree of differentiation of cancer cells, the lower the differentiation degree, the stronger the inhibitory effect. After ACBP treatment, TP53, TP63 and TP73 expression was increased in all cell lines. In xenogragft mouse model, ACBP inhibited growth of MGC-803 cell in vivo. Apoptotic related genes Bax and PUMA were up-regulated, and Mcl-1 was down-regulated. ACBP inhibited tumor cell growth by induced apoptosis through TP53 signaling cascade by up-regulating TP53, TP63 and TP73 and its downstream apoptosis promoting genes Bax and PUMA, down-regulating antiapoptotic gene Mcl-1.","manuscriptTitle":"Anti-cancer bioactive peptide induces apoptosis in gastric cancer cells through TP53 signaling cascade","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-24 11:38:13","doi":"10.21203/rs.3.rs-4286067/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bce0d832-708c-4386-ba7c-751e79a20569","owner":[],"postedDate":"April 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-12T02:38:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-24 11:38:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4286067","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4286067","identity":"rs-4286067","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}