Senescence risk score: a multifaceted prognostic tool predicting outcomes, stemness, and immune responses in colorectal cancer.

Colorectal cancer (CRC) remains a primary cause of cancer mortality globally, necessitating precise prognostic indicators for effective clinical management. Our study introduces the Senescence Risk Score (SRRS), based on several senescence-related genes (SRGs), a potent prognostic tool designed to measure cellular senescence in CRC. The higher SRRS predicts a poorer prognosis, providing a novel and efficient approach to patient stratification. Notably, we found that SRRS correlates with methylation and mutation variations, and increased immune infiltration in the tumor microenvironment, thus revealing potential therapeutic targets. We also discovered an inverse relationship between SRRS and cell stemness, which could have significant implications for cancer treatment strategies. Utilizing bioinformatics resources and machine learning, we identified LIMK1 and WRN as key genes associated with SRRS, further enhancing its prognostic value. Importantly, the modulation of these genes significantly impacts cellular senescence, proliferation, and stemness in CRC cells. In summary, our development of SRRS offers a powerful tool for CRC prognosis and paves the way for novel therapeutic strategies, underscoring its potential in transforming CRC patient management.

Engineered multitargeting exosomes carrying miR-323a-3p for CRC therapy.

Colorectal cancer (CRC) is in the forefront of malignancies for its high incidence and mortality. 5-Fluorouracil (5-FU) is one of the most widely used effective drugs for the treatment of CRC. However, there is an urgent need in reducing its systemic side effects and chemoresistance, in order to make 5-FU-based chemotherapy more effective in the treatment of CRC. In this study, engineered CRC cells were established to overexpress miR-323a-3p, which was a tumor suppressor that targeted both EGFR and TYMS. Then miR-323a-3p-loaded exosomes (miR-Exo) were obtained with suitable methods of collection and purification. We found that miR-Exo significantly inhibited CRC cell proliferation and induced apoptosis by the way of targeting EGFR directly in the cells, which eventually led to desirable tumor regression in the cell derived xenograft (CDX) and patient derived xenograft (PDX) tumor mice models. Moreover, we discovered that miR-323a-3p released from miR-Exo directly inhibited the upregulation of thymidylate synthase (TYMS) induced by 5-FU-resistence in CRC cells, resulting in the revival of tumor cytotoxicity from 5-FU. MiR-Exo could effectively induce the CRC cell apoptosis by targeting EGFR and TYMS, and enhance the therapeutic effects of 5-FU on CRC. Our work demonstrates the potency of miR-Exo for advanced CRC biotherapy.

Individual and combined contamination of oxytetracycline and cadmium inhibited nitrification by inhibiting ammonia oxidizers.

Introduction: The large-scale development of animal husbandry and industrialization lead to more and more serious co-contamination from heavy metals and antibiotics in soils. Ecotoxic effects of residues from antibiotics and heavy metals are of increasing concern. Materials and Methods: In this study, oxytetracycline (OTC) and cadmium (Cd) were selected as target pollutants to evaluate the individual and combined effects on nitrification process using four different soil types sampled from North to South China through a 56-day incubation experiment. Results and Discussion: The results demonstrated that the contaminations of OTC and Cd, especially combined pollution had significant inhibitory effects on net nitrification rates (NNRs) as well as on AOA and AOB abundance. The toxic effects of contaminants were greatly enhanced with increasing OTC concentration. AOB was more sensitive than AOA to exogenous contaminants. And the interaction effects of OTC and Cd on ammonia oxidizers were mainly antagonistic. Furthermore, Cd contaminant (with or without OTC) had indirect effects on nitrification activity via inhibiting mineral N and AOA/AOB, while OTC alone indirectly inhibited nitrification activity by inhibiting ammonia oxidizers. The results could provide theoretical foundation for exploring the eco-environmental risks of antibiotics and heavy metals, as well as their toxic effects on nitrification processes.

MiR-323a regulates ErbB3/EGFR and blocks gefitinib resistance acquisition in colorectal cancer.

The rapid onset of resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) limits its clinical utility in colorectal cancer (CRC) patients, and pan-erb-b2 receptor tyrosine kinase (ErbB) treatment strategy may be the alternative solution. The aim of this study was to develop a possible microRNA multi-ErbB treatment strategy to overcome EGFR-TKI resistance. We detect the receptor tyrosine kinase activity in gefitinib-resistant colorectal cancer cells, ErbB3/EGFR is significantly activated and provides a potential multi-ErbB treatment target. MiR-323a-3p, a tumor suppressor, could target both ErbB3 and EGFR directly. Apoptosis is the miR-323a-3p inducing main biological process by functional enrichment analysis, and The EGFR and ErbB signaling are the miR-323a-3p inducing main pathway by KEGG analysis. MiR-323a-3p promotes CRC cells apoptosis by targeting ErbB3-phosphoinositide 3-kinases (PI3K)/PKB protein kinase (Akt)/glycogen synthase kinase 3 beta (GSK3ß)/EGFR-extracellular regulated MAP kinase (Erk1/2) signaling directly. And miR-323a-3p, as a multi-ErbBs inhibitor, increase gefitinib sensitivity of the primary cell culture from combination miR-323a-3p and gefitinib treated subcutaneous tumors. MiR-323a-3p reverses ErbB3/EGFR signaling activation in gefitinib-resistant CRC cell lines and blocks acquired gefitinib resistance.

Concentrated ambient fine particles exposure affects ovarian follicle development in mice.

BACKGROUND: Ambient fine particles (PM2.5) are known to cause various reproductive and developmental diseases. However, the potential mechanisms of PM2.5 exposure induced female reproductive damage remain unclear. METHODS: Four weeks old female C57BL/6 J mice were exposed to filtered air (FA, n = 10) or concentrated ambient PM2.5 (CAP, n = 10) using a versatile aerosol concentration enrichment system. After 9 weeks of the exposure, mice were sacrificed under sevoflurane anesthesia and tissue samples were collected. Immunohistochemical analysis, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and RNA-sequencing were performed to analyze the effects of PM2.5 exposure on follicle development and elucidate its potential mechanisms. RESULTS: Chronic PM2.5 exposure resulted in follicular dysplasia. Compared to the FA-exposed group, follicular atresia in the CAP-exposed mice were significantly increased. Further studies confirmed that CAP induced apoptosis in granulosa cells, accompanied by a distortion of hormone homeostasis. In addition, RNA-sequencing data demonstrated that CAP exposure induced the alteration of ovarian gene expressions and was associated with inflammatory response. CONCLUSIONS: Chronic exposure to CAP can induce follicular atresia, which was associated with hormone modulation and inflammation.

MiR-103a promotes tumour growth and influences glucose metabolism in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common and high-mortality cancer worldwide. Numerous microRNAs have crucial roles in the progression of different cancers. However, identifying the important microRNAs and the target biological function of the microRNA in HCC progression is difficult. In this study, we selected highly expressed microRNAs with different read counts as candidate microRNAs and then tested whether the microRNAs were differentially expressed in HCC tumour tissues, and we found that their expression was related to the HCC prognosis. Then, we investigated the effects of microRNAs on the cell growth and mobility of HCC using a real-time cell analyser (RTCA), colony formation assay and subcutaneous xenograft models. We further used deep-sequencing technology and bioinformatic analyses to evaluate the main functions of the microRNAs. We found that miR-103a was one of the most highly expressed microRNAs in HCC tissues and that it was upregulated in HCC tissue compared with the controls. In addition, high miR-103a expression was associated with poor patient prognosis, and its overexpression promoted HCC cell growth and mobility. A functional enrichment analysis showed that miR-103a mainly promoted glucose metabolism and inhibited cell death. We validated this analysis, and the data showed that miR-103a promoted glucose metabolism-likely function and directly inhibited cell death via ATP11A and EIF5. Therefore, our study revealed that miR-103a may act as a key mediator in HCC progression.

Synthesis and pharmacological characterization of glucopyranosyl-conjugated benzyl derivatives as novel selective cytotoxic agents against colon cancer.

Glucopyranosyl-conjugated benzyl derivatives containing a [1,2,3]-triazole linker were synthesized. Benzyl served as an important pharmacophore in anti-cancer compounds. Compound 8d inhibited the proliferation of colorectal cancer cells with the potency comparable to 5-fluorouracil (5-FU) with improved selectivity towards cancer cells. The antiproliferative activity of 8d is achieved through triggering apoptotic cell death.

COL4A1 promotes the growth and metastasis of hepatocellular carcinoma cells by activating FAK-Src signaling.

BACKGROUND: Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood. METHODS: In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression. RESULTS: COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor. CONCLUSION: COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC.

Zinc pyrithione induces immobilization of human spermatozoa and suppresses the response of the cAMP/PKA signaling pathway.

Zinc pyrithione (ZPT), a zinc coordination complex, is used as an antimicrobial agent. This study investigated the molecular mechanisms underlying ZPT-induced spermatozoa immobilization by examining plasma membrane integrity, mitochondrial dysfunction, and the cAMP/PKA signaling pathway response. ZPT inhibited spermatozoa motility and movement patterns in a concentration-dependent manner. The 100% effective concentration (EC100) and median effective concentration (EC50) at which ZPT-induced spermatozoa immobilization at 20 s were 40 µmol/L and 16.19 µmol/L, respectively. ZPT did not significantly disrupt spermatozoa plasma membranes, but it exerted a strong and significant effect on the depolarization of mitochondria. In addition, ZPT exposure induced intracellular H+ accumulation and Ca2+ dissipation in spermatozoa, accompanied by suppression of the cAMP/PKA signaling pathway. Thus, ZPT induces spermatozoa immobilization without significant plasma membrane injury and so could be a candidate microbicidal spermicide.

Expression of PD-L1 in mononuclear cells, multinucleated cells, and foam cells in tenosynovial giant cell tumors.

Tenosynovial giant cell tumor (TGCT) is a rare, proliferative and inflammatory disease with activation of colony stimulating factor 1 (CSF1) expression, and exhibits abnormal proliferation of mononuclear cells, multinucleated cells and foam cells. PD-L1 inhibitors represent a promising strategy in a variety of tumors. However, PD-L1 expression has never been studied in CSF1 activated TGCT. In this study, we determined the expression of programmed cell death ligand 1 (PD-L1) in 40 TGCT cases by immunohistochemistry and evaluated its clinical significance. We found that PD-L1 was positively expressed in 52.5% of all patients, and among them, the mononuclear cells, multinucleated cells, and foam cells with positive PD-L1 expression were observed in 21 (52.5%), 10 (25.0%), and 7 (17.5%) patients, respectively. The mononuclear cells and foam cells exhibited PD-L1 expression on the membrane or in the cytoplasm, and the multinucleated cells showed membranous PD-L1 expression. In addition, the PD-L1-positive mononuclear cells, multinucleated cells, and foam cells co-expressed CD68. Moreover, the patients with positive PD-L1 expression had a larger tumor size than those with negative PD-L1 expression. We further found that the foam cells of human coronary atherosclerosis also exhibited the expression of PD-L1 in two of three patients. These findings provide valuable evidence that PD-L1 is highly positive in CSF1-activated TGCT, and treatment with anti-PD-L1 agents may be a valuable therapeutic option for those diseases with PD-L1 expression on mononuclear cells, multinucleated cells, or foam cells.

Carbohydrate-conjugated 4-(1,3,2-dithiarsolan-2-yl)aniline as a cytotoxic agent against colorectal cancer.

Arsenic trioxide (As2O3) has been approved for the treatment of acute promyelocytic leukemia (APL); however, its use in the treatment of solid tumors is limited due to its pharmacokinetic properties. Organic arsenic compounds provide better options for pharmaceutical optimization. p-Aminophenyl arsenoxide (p-APAO), an organic arsenic compound, was found to interact with the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) fusion protein in a similar manner to arsenic trioxide. Analogs of p-APAO such as 4-(1,3,2-dithiarsolan-2-yl)aniline (p-APDTAs) were recently found to show improved cytotoxicity toward several solid tumor cell lines with lower toxicity to normal cells. Here, we synthesized a carbohydrate-conjugated 4-(1,3,2-dithiarsolan-2-yl)aniline (p-APDTAs) and showed that it exhibited reduced cytotoxicity to normal cells, suggesting a feasible approach to improve the therapeutic index of arsenic-containing compounds as chemotherapeutic agents.

U0126 inhibits pancreatic cancer progression via the KRAS signaling pathway in a zebrafish xenotransplantation model.

Pancreatic cancer is one of the most aggressive human cancers, and the pharmaceutical outcomes for its treatment remain disappointing. Proper animal models will provide an efficient platform for investigating novel drugs, and the zebrafish has become one of the most promising and comprehensive model animal in cancer research. In the present study, we used a novel xenograft model in zebrafish by transplanting human pancreatic cancer cells to study the progression and metastasis of pancreatic cancer cells and to assay the pharmacological effects of new drug U0126 in vivo. We first established a primary xenograft model of pancreatic cancer by injecting human pancreatic cancer cells into both live larval and adult zebrafish, and then investigated the behaviors of CM-DiI­labeled human pancreatic cancer cells. Subsequently, we tested the potential of this model for drug screening by evaluating a known small-molecule inhibitor, U0126, which targets the KRAS signaling pathway. Cells with KRAS mutations exhibited significant proliferative and migratory behaviors and invaded the zebrafish vasculature system. In contrast, the proliferation and migration of Mia PaCa-2 cells in zebrafish larvae were substantially repressed following U0126 treatment. These results suggest that zebrafish xenotransplantation can be used as a simple and efficient tool to screen and identify new anti-pancreatic cancer compounds.

Use of antagonists and morpholinos in loss-of-function analyses: estrogen receptor ESR2a mediates the effects of 17alpha-ethinylestradiol on primordial germ cell distribution in zebrafish.

BACKGROUND: Various chemicals released into the aquatic environment adversely affect the reproductive system of fish, particularly by changing gonad structure and function. 17alpha-ethinylestradiol (EE2) is a potent environmental estrogen that disrupts sexual differentiation and normal reproduction in fish. Previous studies have shown that exposure to endocrine-disrupting chemicals (EDCs) disrupts the migration of primordial germ cells (PGCs) in zebrafish. METHODS: To investigate the effects of EE2 exposure on PGC migration, zebrafish embryos were injected with gfp-nanos mRNA to label PGCs and subsequently exposed to different concentrations of EE2. Typical estrogen receptor antagonist treatment and morpholino knockdown experiments were used to identify functional estrogen receptors that mediate the effects of EE2. RESULTS: The migration of PGCs was disrupted after exposure to high concentrations of EE2 (1 mirog/L). Loss-of-function analyses were performed for estrogen receptor ESR1, ESR2a, and ESR2b, and only loss of ESR2a resulted in a decreased number of ectopic PGCs following exposure to 1 mirog/L EE2. CONCLUSIONS: EE2 exposure disrupts PGC migration and distribution, and this effect is mediated through the estrogen receptor ESR2a.

MicroRNA-200a promotes anoikis resistance and metastasis by targeting YAP1 in human breast cancer.

PURPOSE: The process of metastases involves the dissociation of cells from the primary tumor, penetration into the basement membrane, invasion, and exiting from the vasculature to seed and colonize distant tissues. miR-200a is involved in this multistep metastatic cascade. This study aimed to test the hypothesis that miR-200a promotes metastasis through increased anoikis resistance in breast cancer. EXPERIMENTAL DESIGN: Breast cancer cells transfected with mimic or inhibitor for miR-200a were assayed for anoikis in vitro. miR-200a expression was assessed by quantitative real-time PCR (qRT-PCR). Luciferase assays, colony formation assays, and animal studies were conducted to identify the targets of miR-200a and the mechanism by which it promotes anoikis resistance. RESULTS: We found that overexpression of miR-200a promotes whereas inhibition of miR-200a suppresses anoikis resistance in breast cancer cells. We identified Yes-associated protein 1 (YAP1) as a novel target of miR-200a. Our data showed that targeting of YAP1 by miR-200a resulted in decreased expression of proapoptotic proteins, which leads to anoikis resistance. Overexpression of miR-200a protected tumor cells from anoikis and promoted metastases in vivo. Furthermore, knockdown of YAP1 phenocopied the effects of miR-200a overexpression, whereas restoration of YAP1 in miR-200a overexpressed breast cancer cells reversed the effects of miR-200a on anoikis and metastasis. Remarkably, we found that YAP1 expression was inversely correlated with miR-200a expression in breast cancer clinical specimens, and miR-200a expression was associated with distant metastasis in patients with breast cancer. CONCLUSIONS: Our data suggest that miR-200a functions as anoikis suppressor and contributes to metastasis in breast cancer.

Generation of a fluorescent transgenic zebrafish for detection of environmental estrogens.

To establish a novel in vivo test system for rapid detection of environmental estrogens, an ere-zvtg1: gfp transgenic zebrafish line has been generated. In this transgenic line, under control conditions, GFP was exclusively expressed in the liver of mature adult female fish. Male and larval transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17-alpha-ethynylestradiol (EE(2)). Concurrent accumulation of zvtg1 and gfp mRNAs in embryos and larvae after EE(2) exposure was observed, which indicated that the expression of gfp transgene was driven by the zvtg1 promoter. Green fluorescence was first observed in the liver at 53, 74, 100 or 131h post-fertilization (hpf) after exposure to 100, 10, 1 or 0.1ng/L EE(2) from 1 to 2 cell stage, respectively. As for mature male transgenic zebrafish, green fluorescence was observed after exposure to 100, 10, 1 or 0.1ng/L EE(2) for 2, 3, 4 or 7 days, respectively; as for mature female, fluorescence was increased after exposure to relatively high concentrations of EE(2) (10 and 100ng/L). Green fluorescence in the liver was increased with prolonging of exposure time and was repeatedly induced after removal and re-addition of EE(2). We also demonstrated that GFP expression could be induced by other estrogenic compounds, including beta-estradiol (E(2), 0.1microg/L), cadmium chloride (CdCl(2), 10microg/L), zearalenone (50microg/L), estriol (E(3), 1microg/L), diethylstilbestrol (DES, 50ng/L) bisphenol A (BPA, 1mg/L) but not by weakly estrogenic compounds such as nonylphenol (NP, up to 10mg/L), or non-estrogenic steroid hormones such as progesterone (up to 100mg/L) and 17-hydroxysteroid (up to 50mg/L). These data suggest the transgenic zebrafish is sensitive and specific for detection of estrogenic compounds. Because the observed-effect concentrations are as low as those of environment and the observed-effect exposure times are very short, this transgenic fish is a promising candidate system for monitoring environmental estrogens directly, rapidly and easily.

Critical role of connexin43 in zebrafish late primitive and definitive hematopoiesis.

In vitro studies have suggested that connexin43 (cx43) expression is of particular importance during establishment and regeneration of the mammalian hematopoietic system. However, little is known about its in vivo functions during hematopoiesis due to the embryonic lethality of mammalian knockout models. In this study, we observed that zebrafish cx43 is not only expressed in the eyes, cerebellum, heart, and vasculature, but also expressed, albeit at low levels, in intermediate cell mass (ICM, the primitive hematopoietic site). Knockdown of cx43 leads to vacuolization in the wedge of the ICM and an apparent reduction in the number of circulating blood cells, but does not affect their cellular morphology. Whole-mount in situ hybridization analysis revealed that the hemangioblastic marker flk-1 and the primitive hematopoietic markers lmo2 and scl are basically maintained at normal levels in cx43 morphant embryos at 12-13 h postfertilization (hpf) compared with the con-MO injected embryos. However, subsequent expression of the definitive hematopoietic stem cell (HSC) marker c-myb was severely downregulated in the ventral wall of the dorsal aorta of cx43-depleted embryos at 36 hpf. Furthermore, we confirmed this phenotype by injection of cx43-MO into Tg(gata1:EGFP) embryos. Together, our results show that cx43 contributes to late primitive and definitive hematopoiesis in zebrafish embryos.

Potent anti-hepatoma efficacy of HCCS1 via dual tumor-targeting gene-virotherapy strategy.

We previously demonstrated that hepatocellular carcinoma suppressor 1 (HCCS1) exerts potent anti-tumor activity. In this study, we constructed a new dual tumor-targeting oncolytic adenovirus vector, PD55-HCCS1, in which E1A was driven by the promoter of progression elevated gene-3, which is hepatoma-specific, and a CMV-HCCS1 expression cassette replaced E1B55. The PD55-HCCS1-mediated selective expression of E1A and HCCS1 in hepatoma cells and tumor-selective cytotoxicity in vitro and in vivo demonstrated the strongest inhibition of BEL-7404 cell xenografts in nude mice among a number of control Ad vectors. These data indicated the efficacy and safety of the PD55-HCCS1 system for HCC treatment.

[Tumor-targeting expression of a new tumor suppressor gene HCCS1 and its tumor-selective inhibitory effects on hepatocellular carcinoma].

OBJECTIVE: To construct a tumor-targeting recombinant adenovirus vector containing hepatocellular carcinoma suppressor gene HCCS1 to enhance the safety of tumor treatment. METHODS: CCK-8 assay was used to observe different inhibitory effects on normal and malignant liver cells with high expressions of HCCS1 protein. The relative transcriptional activity of PEG-3p was quantified by luciferase assay. Recombinant adenovirus Ad-PEG-3p-HCCS1 was packaged with AdEasy system and confirmed by PCR. The tumor-targeted expression of HCCS1 protein in cells infected with Ad-PEG-3p-HCCS1 was determined by Western blot. Crystal violet assay and MTT assay were applied to observe the selective anti-tumor effects of the newly constructed virus in vitro. RESULTS: A higher inhibitory rate of about 60% was found in BEL-7404 and SW-620 than that in L02 and NHLF 96 h after the high expression of HCCS1. Luciferase assay showed 3.9-, 4.7-, and 1.5-fold transcriptional activity in BEL-7404, BEL-7405 and QGY-7703 respectively, in comparison with that in L02. Ad-PEG-3p-HCCS1 was constructed successfully and was verified by PCR. Western blot indicated that high expression of HCCS1 could be induced in BEL-7404 and QGY-7703 but not in L02. Crystal violet assay and MTT assay showed that it remarkably reduced the toxicity to L02 but still had enough antitumoral effect on Ad-CMV-HCCS1. CONCLUSIONS: With high expression of HCCS1 the tumor cells we used are being inhibited more. PEG-3p has the tumor-selective driving function in malignant liver cells. Our recombinant adenovirus Ad-PEG-3p-HCCS1 can tumor-targeting induce HCCS1 expression in tumor cells, which can improve the safety of gene therapy with HCCS1.

[The anti-tumour effect of Wuxing soup and its mechanism in inducing apoptosis of tumour cells mediated by calcium].

OBJECTIVE: To confirm the anti-cancer effect and mechanism of Wuxing soup. METHODS: Inhibition of cellular growth under Wuxing soup treatment was observed by MTT; Apoptosis was detected by gel electrophoresis, transmission electron microscopy and FACS; The concentration of calcium was measured by fluorescence probe. RESULTS: After SGC-7901 cell being treated by Wuxing soup, it showed that: 1) Wuxing soup could specifically inhibit cancer cells proliferation in a time and dose dependent manner; 2) Typical apoptotic morphological changes and DNA ladder of SGC-7901 cells were observed; 3) calcium inhibitor Bapta AM could reduce the apoptotic rate and protect SGC-7901 cells in a dose dependent manner. CONCLUSION: Wuxing soup has an effective inhibition on cancer cells, and can induce SGC-7901 cells to apoptosis by calcium.

[Preparation of anti-human integrin beta 3 monoclonal antibody and study on its antitumor activity].

AIM: To prepare the monoclonal antibody (mAb) against human integrin beta 3, and explore its role in tumor therapy. METHODS: Total RNA was isolated from human lymphocytes, and the DNA fragment encoding extra-cellular domain of human integrin beta 3 was amplified by RT-PCR. The integrin beta 3 gene was cloned into the prokaryotic expression vector pQE30, and the expression plasmid pQE30-beta 3 was transformed into E.coli M15. The expression of beta 3 protein was induced by IPTG, and the expressed beta 3 protein was purified by Ni-affinity agarose. BALB/c mice were immunized with the purified beta 3 protein, and mAb was prepared by hybridoma technique. The specificity of the mAbs was identified by Western blot. The mAbs were screened by their inhibitory effect on the tumor growth in vivo. The inhibition of the mAb to HUVEC cell growth was detected by MTT assay. The role of the mAb in inducing HUVEC apoptosis was analyzed by flow cytometry (FCM). RESULTS: The extra-cellular gene fragment of human integrin beta 3 was amplified by RT-PCR, and the expression plasmid pQE30-beta 3 was constructed. Human integrin beta 3 protein was expressed and purified, and used for immunization. Eight clones of mAb against human integrin beta 3 were successfully prepared. The mAb 4F12 was found to inhibit tumor growth in vivo and reduce blood vessels in tumor. Furthermore, the mAb 4F12 could inhibit HUVEC growth and induce apoptosis in vitro. CONCLUSION: The human integrin beta 3 protein was obtained and the mAbs against it have been prepared successfully. The mAb 4F12 can inhibit tumor growth in vivo and reduce blood vessels in tumor. One of the mechanisms of the antitumor effect is inhibition of growth and induction of apoptosis of endothelial cells of blood vessels in tumor.