Bcap-37 Cells Research Articles

Green synthesis of fluorescent carbon dots from ascorbic acid and their application in sensing and biological imaging

A simple, highly efficient, and environmentally friendly one-step method has been developed to synthesize fluorescent carbon dots (CDs) with the assistance of hydrogen peroxide (H2O2). Here H2O2 acts as a green accelerator to promote the preparation of CDs, while temperature plays a critical role in controlling the entire synthesis process. The prepared CDs demonstrate monodispersion, with an average diameter of ∼6.8 nm, superior stability, and excitation-dependent photoluminescent (PL) behavior. The PL mechanism of the CDs was investigated using time-resolved PL decay, revealing that the PL originates from both the defect state and the intrinsic state of the CDs. Furthermore, the CDs show significant promise as pH sensors and versatile nanothermometry devices, owing to their remarkable sensitivity to both pH and temperature, as demonstrated by the pronounced dependence of their steady-state fluorescence emission spectra. It’s worth noting that temperature and pH sensors present significant reversibility, sensitivity, and linearity. More importantly, the CDs display low cytotoxicity, good biocompatibility, and the ability to enter human breast cancer Bcap-37 cells, making them highly suitable candidates for cellular imaging and labeling.

Pharmacological Mechanism of Mume Fructus in the Treatment of Triple-Negative Breast Cancer Based on Network Pharmacology.

Our study aims to find the relevant mechanism of Mume Fructus in the treatment of triple-negative breast cancer (TNBC) by network pharmacology analysis and experimental validation. The effective compounds of Mume Fructus and TNBC-related target genes were imported into Cytoscape to construct a Mume Fructus-effective compounds-disease target network. The common targets of Mume Fructus and TNBC were determined by drawing Venn diagrams. Then, the intersection targets were transferred to the STRING database to construct a protein-protein interaction (PPI) network. To investigate the mechanism of Mume Fructus in treatment of TNBC, breast cancer cell (MDA-MB-231) was treated with Mume Fructus and/or transfected with small interference RNA-PKM2(siPKM2).CCK-8 assay, cell clonal formation assay, transwell, flow cytometry, qRT-PCR, and western blotting were performed. Eight effective compounds and 145 target genes were obtained, and the Mume Fructus- effective compounds-disease target network was constructed. Then through the analysis of the PPI network, we obtained 10 hub genes including JUN, MAPK1, RELA, AKT1, FOS, ESR1, IL6, MAPK8, RXRA, and MYC. KEGG enrichment analysis showed that JUN, MAPK1, RELA, FOS, ESR1, IL6, MAPK8, and RXRA were enriched in the Th17 cell differentiation signaling pathway. Loss of PKM2 and Mume Fructus both inhibited the malignant phenotype of MDA-MB-231 cells. And siPKM2 further aggravated the Mume Fructus inhibition of malignancy of breast cancer cells. Network pharmacology analysis suggests that Mume Fructus has multiple therapeutic targets for TNBC and may play a therapeutic role by modulating the immune microenvironment of breast cancer.

Baicalin Promotes Apoptosis of Human Medullary Breast Cancer via the ERK/p38 MAPK Pathway

Objectives The present study aims to examine the effects of baicalin on the human medullary breast carcinoma (MBC) cell line Bcap-37 and to determine whether baicalin regulates Bcap-37 cell apoptosis through the ERK/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Materials and Methods 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Transwell assay were utilized to measure the effects of baicalin on the proliferation and migration ability of Bcap-37 cells, respectively. Flow cytometer analysis was implied to detect the effects of baicalin on Bcap-37 cell apoptosis. Real-time quantitative polymerase chain reaction (RT-PCR) was conducted to observe the influence of baicalin on mRNA expressions of apoptosis-related genes. Western blot was executed to further explore the action of baicalin on apoptosis-related proteins. PD98059 (a specific inhibitor of ERK) and SB203580 (a specific inhibitor of p38 MAPK) were used to further clarify the intrinsic mechanism of baicalin regulating apoptosis in Bcap-37 cells. Results Compared with the control group, baicalin significantly inhibits the proliferation activity of Bcap-37 cells in a concentration- and time-dependent manner, with a p-value < 0.05. The transwell assay indicated that the migration viability of cells decreased further, followed by the increased concentration of baicalin, and the p-value had a statistical difference. Besides, flow cytometry was conducted to assess the effects of baicalin on the early and late apoptosis rates of Bcap-37 cells, and results showed that baicalin highly promoted the apoptosis level both at the early and late stages with a statistical difference in a concentration-dependent manner ( p < 0.05). Results of RT-PCR presented that, compared with the control group, baicalin significantly activated the mRNA expression of Bax, p38, and p-ERK1 and abolished the mRNA expression of Bcl-2 in every dosing group in a concentration-dependent way ( p < 0.05). Western blot exhibited that, compared with the control group, baicalin promoted the protein expression of caspase-3, caspase-9, Bax, p38, P-ERK, and p53 while playing an opposite function to Bcl-2 in each dosing group ( p values have statistical differences). At last, PD98059 and SB203580 were applied to explore the potential mechanism of baicalin in apoptosis promotion. And results revealed that, compared with the group treated with baicalin alone, protein expression of Bax, p38, p-ERK, caspase-3, and caspase-9 was downregulated obviously in the group treated with both baicalin and PD98059 or SB203580 ( p < 0.05). The p53 expression inhibition showed a difference only when compared to the group adding PD98059. Compared with the blank group, a statistical difference was only observed in Bcl-2 expression in the group treated with baicalin in combination with PD98059 or SB203580 ( p < 0.05). Interestingly, a p53 expression difference was only displayed between the blank group and the group treated with baicalin and SB203580 ( p < 0.05). Conclusion Our study observed that baicalin inhibits the proliferation and migration ability of Bcap-37 cells. In addition, baicalin induces cell apoptosis via the ERK/p38 MAPK signal pathway.

Mitochondrial DNA methylation is a predictor of immunotherapy response and prognosis in breast cancer: scRNA-seq and bulk-seq data insights.

Alterations in Mitochondrial DNA methylation (MTDM) exist in many tumors, but their role in breast cancer (BC) development remains unclear. We analyzed BC patient data by combining scRNA-seq and bulk sequencing. Weighted co-expression network analysis (WGCNA) of TCGA data identified mitochondrial DNA methylation (MTDM)-associated genes in BC. COX regression and LASSO regression were used to build prognostic models. The biological function of MTDM was assessed using various methods, such as signaling pathway enrichment analysis, copynumber karyotyping analysis, and quantitative analysis of the cell proliferation rate. We also evaluated MTDM-mediated alterations in the immune microenvironment using immune microenvironment, microsatellite instability, mutation, unsupervised clustering, malignant cell subtype differentiation, immune cell subtype differentiation, and cell-communication signature analyses. Finally, we performed cellular experiments to validate the role of the MTDM-associated prognostic gene NCAPD3 in BC. In this study, MTDM-associated prognostic models divided BC patients into high/low MTDM groups in TCGA/GEO datasets. The difference in survival time between the two groups was statistically significant (P<0.001). We found that high MTDM status was positively correlated with tumor cell proliferation. We analyzed the immune microenvironment and found that low-MTDM group had higher immune checkpoint gene expression/immune cell infiltration, which could lead to potential benefits from immunotherapy. In contrast, the high MTDM group had higher proliferation rates and levels of CD8+T cell exhaustion, which may be related to the secretion of GDF15 by malignant breast epithelial cells with a high MTDM status. Cellular experiments validated the role of the MTDM-associated prognostic gene NCAPD3 (the gene most positively correlated with epithelial malignant cell proliferation in the model) in BC. Knockdown of NCAPD3 significantly reduced the activity and proliferation of MDA-MB-231 and BCAP-37 cells, and significantly reduced their migration ability of BCAP-37 cell line. This study presented a holistic evaluation of the multifaceted roles of MTDM in BC. The analysis of MTDM levels not only enables the prediction of response to immunotherapy but also serves as an accurate prognostic indicator for patients with BC. These insightful discoveries provide novel perspectives on tumor immunity and have the potentially to revolutionize the diagnosis and treatment of BC.

Sauchinone inhibits breast cancer cell proliferation through regulating microRNA‐148a‐3p/HER‐2 axis

Sauchinone is extracted from the root of Saururus chinensis and exhibits potent antitumor effects in various human cancers. However, how sauchinone is involved in breast cancer has not been well studied. Cells apoptosis, cell proliferation, and cycle distribution were evaluated. Xenograft tumor mouse model was constructed to investigate the roles of sauchinone. The relevant protein expression was detected by western blot. We found that sauchinone significantly reduced proliferation and survival, also induced apoptosis of MCF-7 and Bcap-37 cells in vitro. Sauchinone significantly increased miR-148a-3p expression, and human epidermal growth factor receptor (HER)-2 targeted on miR-148a-3p. Sauchinone exposure downregulated HER-2 expression whose overexpression partly eliminated the inhibitory effect of sauchinone. Further, sauchinone efficiently inhibited breast cancer progression through downregulating HER-2 expression in vivo. Our results indicate that sauchinone efficiently inhibits breast cancer progression through regulating miR-148a-3p/HER-2 axis, suggesting that sauchinone could be an effective anticancer agent for breast cancer.

Expression and clinical significance of undifferentiated embryonic cell transcription factor 1 in breast cancer.

At present, due to the heterogeneity of breast cancer, common tumor markers have certain limitations in clinical prognostic evaluation. This suggests an unmet need for markers to predict clinical outcomes and potentially guide targeted therapies. The present study sought to explore the expression level and clinical significance of undifferentiated embryonic cell transcription factor 1 (UTF1) in breast cancer. Immunohistochemistry (IHC) was used to detect the expression of UTF1 in 221 breast cancer samples. The clinical significance of UTF1 protein expression in breast cancer tissues was evaluated by combining clinicopathological parameters and UTF1 expression profile. We performed 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and clone formation assays to evaluate the effect of UTF1 on Bcap37 cell proliferation. Wound healing assay and transwell migration assay were used to evaluate the changes of cell invasion and migration ability, respectively. All experiments were performed with 3 biological replicates. Genomic differences after UTF1 overexpression were evaluated by RNA sequencing technology and the possible functions and regulatory mechanisms were elucidated. The findings showed that UTF1 expression level was significantly correlated with tumor size (P=0.004), but not with patient age, tumor histological stage, lymph node metastasis, as well as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2), and Ki67 expression levels. Kaplan-Meier survival analysis and Cox proportional hazard model indicated that UTF1 expression was significantly associated with overall survival (OS) time of breast cancer patients. The median survival time of patients with high expression level of UTF1 was shorter compared with that of patients with low UTF1 expression level. The results of cell experiments showed that UTF1 overexpression could significantly promote the growth, proliferation, migration, and invasion of breast cancer cells. The RNA sequencing results showed that UTF1 was not only closely related to apoptosis genes, but also closely related to the nuclear factor (NF)-kappa B pathway. The findings of the current study indicate that UTF1 is involved in occurrence and tumor progression and is significantly associated with prognosis of breast cancer patients.

New anti-proliferative triterpenes from hydrolyzate of total Gynostemma pentaphyllum saponins induces cell cycle arrest and apoptosis in human breast cancer cells

• Three undescribed triterpenes were obtained from hydrolyzate of G. pentaphyllum . • All the isolated compounds exhibited significant cytotoxic activity. • Gypensapogenin II possessed good anti-proliferative selectivity against Bcap37 cells. • It significantly inhibited proliferation and migration led to G1 cells cycle arrest. • It also induced apoptosis in Bcap37 cells through mitochondrial pathway. Phytochemical investigation of the hydrolysate in total Gynostemma pentaphyllum saponins led to the isolation of three previously undescribed triterpenes, Gypensapogenin II ( 1 ), Gypensapogenin III ( 2 ), and Gypensapogenin IV ( 3 ), together with five known compounds. Their structures were mainly identified by NMR and HR-ESI-MS data. All isolated compounds were evaluated in Bcap37, A549, Hela, and HepG-2 cells and showed significant anti-cancer activities, while having a much weaker effect on the growth of normal human breast cells (MCF-10a) and lung fibroblast cells (MRC5). Among the compounds, compound 1 possessed good anti-proliferative selectivity against Bcap37 cells (IC 50 value of 6.13 ± 0.86 μM). Furthermore, basic mechanism studies revealed that compound 1 significantly decreased survival and inhibited proliferation and migration leading to G1 cell cycle arrest and apoptosis in Bcap37 cells through mitochondrial pathway. Taken together, these results indicated compound 1 exerted promising anti-proliferative activity, and inhibited the migration and promoted the apoptosis of Bcap37 cells.

CACNA1B facilitates breast cancer cell growth and migration by regulating cyclin D1 and EMT: the implication of CACNA1B in breast cancer

Purpose This study mainly aimed to explore the influences of Calcium Voltage-Gated Channel Subunit Alpha1 B (CACNA1B) on the development of breast cancer and the related mechanism. Materials and methods The information of patients with breast cancer from TCGA database was used for analyses of CACNA1B expression and its prognostic value. Loss- and gain- of functions of CACNA1B were conducted in MCF7 and Bcap-37 cells, respectively. CCK-8, colony formation and transwell assays were applied for evaluating the cell viability and motility. Western blot was used for protein expression detection. Results We revealed that highly expressed CACNA1B in breast cancer tissues was related to poor prognosis according to the data gained from TCGA database. The outcomes of functional assays showed that depletion of CACNA1B restrained MCF7 cell growth, invasion and migration and high-expression of CACNA1B fortified the growth, invasion and migration in Bcap-37 cells. Finally, we manifested that silencing CACNA1B obviously raised the protein expression level of E-cadherin and reduced the protein levels of Cyclin D1, N-cadherin and Snail in MCF7 cells, whilst, over-expression of CACNA1B reduced the level of E-cadherin and increased the expression of Cyclin D1, N-cadherin and Snail in Bcap-37 cells. Conclusions These results identified CACNA1B as a forwarder of the growth, invasion and migration in breast cancer cells.

MiR-301b and NR3C2 co-regulate cells malignant properties and have the potential to be independent prognostic factors in breast cancer.

This study intends to address the function of miR-301b/nuclear receptor subfamily 3 group C member 2 (NR3C2) in breast cancer. The Cancer Genome Atlas database was processed to investigate the expression of miR-301b/NR3C2 in breast cancer samples, as well as the relationship between their expression and the prognosis of the patients. Cox regression analysis was performed to determine whether miR-301b/NR3C2 was an independent predictor of the patient's prognosis. Associations between miR-301b and NR3C2 were analyzed by prediction website, dual-luciferase assay, and Pearson correlation coefficient. Quantitative polymerase chain reaction and Western blot analyses were implemented to detect gene expression. The relevant biological characteristics of MCF7 and BCAP-37 cells were tested by cell counting kit-8, colony formation, and transwell assays. Lower expression of NR3C2, which was closely related to the bad prognosis of breast cancer patients, was presented in breast cancer samples and can be used as an independent predictor. miR-301b, as an upstream regulator of NR3C2, was highly expressed in breast cancer samples and can be used as an independent predictor as well. Notably, a higher level of miR-301b and lower level of NR3C2 were related to the reduced overall survival in patients with breast cancer. The proliferative and migratory behaviors of cells were elevated or blocked after overexpression of miR-301b or NR3C2, respectively. However, the above situation was attenuated after together upregulation of miR-301b and NR3C2. The present data afforded evidence that miR-301b may be a tumor-promoting miRNA in breast cancer, and that miR-301b/NR3C2 axis mediated tumor development from cell proliferation and migration.

Synthesis and antitumor activity of novel pyridazinone derivatives containing 1,3,4-thiadiazole moiety

A series of novel pyridazinone derivatives containing the 1,3,4-thiadiazole moiety were synthesized and characterized by 1H NMR, 13C NMR, spectroscopies HRMS and IR. Among them, the structure of compound 5c (2-(Tert-butyl)−4-chloro-5-((5-((2-ethylphenyl)amino)−1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One) was unambiguously confirmed via single crystal X-ray diffraction analysis. The inhibitory activity of all the target compounds against MGC-803 and Bcap-37 was determined by MTT assay, with doxorubicin (the inhibition rates were 95.5 ± 0.4% and 95.7 ± 1.0% respectively) as a control. The preliminary results showed that the inhibitory activity of compound 5n (2-(Tert-butyl)−4-chloro-5-((5-((3-fluorophenyl)amino)−1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One) was superior to the others. The inhibition rates of MGC-803 and Bcap-37 cells were 86.3 ± 2.2% and 92.3 ± 0.6% at a concentration of 10 μmol/L, respectively. The preliminary structure-activity relationship showed that when the 2-position of the benzene ring was substituted by a methyl group, such as compound 5j (2-(Tert-butyl)−4-chloro-5-((5-((2,3-dimethylphenyl)amino)−1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One), it exhibited good anticancer activity on MGC-803 cells. Besides, introducing fluorine, chlorine, or trifluoromethyl group onto the benzene ring, such as compound 5 m (2-(Tert-butyl)−4-chloro-5-((5-((4-(trifluoromethoxy)phenyl)amino)−1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One), displayed good anticancer activity on MGC-803 and Bcap-37 cells.

Peptide specifically binding to human medullary breast carcinoma Bcap-37 cells selected by phage display in vivo and its property as well as combination effect

Objective To investigate the property and the combination effect of the peptide specifically binding to human medullary breast cancer Bcap-37 cells by using phage display in vivo and to provide molecular targeting probe for early diagnosis of breast cancer. Methods The human medullary breast carcinoma Bcap-37 cells tumor-bearing nude mice model was prepared and three rounds in vivo were performed by using Ph.D.-C7CTM phage display peptide library. The distribution of screened phages in tumors and normal tissues was detected by using immunohistochemistry. The affinity of monoclonal phage to Bcap-37 cells was identified by using enzyme linked immunosorbent assay (ELISA). The positive monoclonal phage DNA was taken and sequenced, and the sequence with high repetition rate was selected to synthesize peptide by using chemical methods. Optical molecular probe was prepared and fluorescence molecular imaging was used to test its specificity and targeting ability for breast transplantation tumor of tumor-bearing nude mice in vivo. Results The recovery rate of phage in the third round screening in vivo was 107.2 times than that in the first round. Immunohistochemical results showed that the phages binding to the tumor tissues were increased successively with the increasing screening rounds in vivo. The number of phages binding to tumor tissues was more than that binding to normal tissues (lung, skeletal muscle, liver and kidney). The absorbancy (A) value of section scanning image in the tumor tissues was higher than that in the normal tissues, and the difference was statistically significant different (P < 0.05). ELISA results showed that 22 phages (affinity≥2) among the 50 randomly selected monoclonal phage were positive. After DNA sequencing analysis of the positive monoclonal phage, 4 repeat amino acid sequences were obtained. The fluorescein isothiocyanate-labelled (FITC)-CSPLNTRFC with the highest repetition rate was synthesized for FITC-CSPLNTRFC peptide. Bcap-37 cells tumor-bearing nude mice model assay in vivo showed that FITC-CSPLNTRFC peptide could significantly enriched in the breast xenograft tissues. Conclusion CSPLNTRFC peptide specifically binding to human medullary breast cancer Bcap-37 cells can be screened out successfully by using phage display in vivo, which will be helpful to vitro studies on the early diagnosis of breast cancer. Key words: Breast neoplasms; Peptide library; Phage display; Specifically binding peptide

Synthesis and evaluation of antitumor activity of dibenzodiazepine derivatives.

A series of dibenzodiazepine 2-position derivatives, bearing N-methylpiperazine at the C-11 position, were prepared by using a concise approach. Their inhibitory activities of tumor cell proliferation in vitro were tested in five cell lines, including breast cancer cell BCAP37, gastric cancer cell SGC7901, liver cancer cell HepG2, cervical cancer cell HeLa and acute promyelocytic leukemia cell HL-60. Several compounds showed efficient tumor activity with IC50 values down to 0.30μM. These compounds are expected to be a new class of potential anticancer lead compounds.

Long-chain non-coding RNA GACAT1 promotes development and progression of breast cancer by targeting microRNA-875-3p.

Role of long-chain non-coding ribonucleic acid (lncRNA) GACAT1 in the development of breast cancer and its possible mechanism were investigated. The levels of GACAT1, microRNA-875-3p and Stonin2 (STON2) in breast cancer tissues and adjacent normal tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The level of GACAT1 in breast cancer cell lines was further explored. The effects of GACAT1 and microRNA-875-3p on cell proliferation and cycle were detected by cell counting kit-8 (CCK-8) and flow cytometry. The binding relationship between microRNA-875-3p and STON2, microRNA-875-3p and GACAT1 was examined by a dual luciferase reporting assay. It was confirmed by rescue experiments whether GACAT1 canregulate the level of STON2 by binding to microRNA-875-3p. GACAT1 level was clearly enhanced in breast cancer tissues compared to that of the adjacent tissues. Similar result was observed in breast cancer cell lines. Upregulation of GACAT1 promoted the proliferation and cycle of breast cancer cells including MCF-7 and BCap-37. The dual luciferase reporting assay results indicated that GACAT1 had a binding relationship with microRNA-875-3p. Further experiments confirmed that microRNA-875-3p was conspicuously downregulated in breast cancer tissues, and upregulation of microRNA-875-3p could inhibit the proliferation ability of MCF-7 and BCap-37 cells, and partially reversed the promoting effect of GACAT1 on cell cycle. Through bioinformatics prediction and dual luciferase reporter gene experiments, we found that STON2 might be a target gene of microRNA-875-3p. Overexpression of STON2 could partially abolish the effect of microRNA-875-3p on cell proliferation and cycle of MCF-7 and BCap-37 cells. GACAT1 can participate in the progression of breast cancer by promoting the proliferation and cycle of breast cancer cells. The mechanism may be through the regulation of the level of STON2 by adsorbing microRNA-875-3p.

High dose vitamin C inhibits proliferation of breast cancer cells through reducing glycolysis and protein synthesis

To investigate the effects of high dose vitamin C (VC) on proliferation of breast cancer cells and to explore its mechanisms. Human breast cancer cells Bcap37 and MDA-MB-453 were treated with VC at low dose (0.01 mmol/L), medium dose (0.10 mmol/L) and high dose (2.00 mmol/L). Cell proliferation was determined with CCK-8 assay, protein expression was evaluated by Western blot, and the secretion of lactic acid in tumor cells was detected by colorimetric method. Bcap37 cells were inoculated in nude mice, and tumor baring nude mice were intraperitoneally injected with high VC(4 g/kg, VC group, n=5)or normal saline (control group, n=5) for 24 d. Tumor weight and body weight were calculated. In vitro experiments demonstrated that high dose VC significantly inhibited cell proliferation in Bcap37 and MDA-MB-453 cells (all P<0.01); the expressions of Glut1 and mTOR signaling pathway-related proteins were decreased (all P<0.05); and the secretion of lactic acid was also markedly reduced (all P<0.05). In vivo experiment showed that the tumor weight was decreased in mice treated with high-dose VC as compared with control group (P<0.05), but no difference in body weights between two groups was observed. High dose VC may inhibit proliferation of breast cancer cells both in vitro and in vivo through reducing glycolysis and protein synthesis.

Nanoformulation of a Novel Pyrano[2,3-c] Pyrazole Heterocyclic Compound AMDPC Exhibits Anti-Cancer Activity via Blocking the Cell Cycle through a P53-Independent Pathway.

Pyrano[2,3-c]pyrazole derivatives have been reported as exerting various biological activities. One compound with potential anti-tumor activity was screened out by MTT assay from series of dihydropyrazopyrazole derivatives we had synthesized before using a one-pot, four-component reaction, and was named as 6-amino-4-(2-hydroxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (hereinafter abbreviated as AMDPC). The IC50 of AMDPC against Bcap-37 breast cancer cells was 46.52 μg/mL. Then the hydrophobic AMDPC was encapsulated in PEG-PLGA block copolymers, and then self-assembled as polymeric micelle (mPEG-PLGA/AMDPC) to improve both physiochemical and release profiles. The effect of mPEG-PLGA/AMDPC on BCAP-37 cancer cells showed similar anti-tumor effects as AMDPC. Furthermore, the anti-tumor mechanism of mPEG-PLGA/AMDPC was investigated, which can probably be attributed to stimulating the expression of P21 gene and therefore protein production on BCAP-37 cells, and then blocked the cell cycle through the P53-independent pathway both in S phase and G2 phase. Thus, mPEG-PLGA/AMDPC is a promising therapeutic agent for cancer treatment, and further in vivo studies will be developed.

Inhibition of Suv39H1 enhances transgenic IFNα-2b gene expression in Bcap-37 cells

The low expression of exogenous transferred gene limited the application of transgenic animal technology. Suppressor of variegation 3 ∼ 9 homolog 1(SUV39H1) gene plays a prominent role on repressive heterochromatin and transcription. To understand if exogenous transgenic gene expression was affected by SUV39H1 epigenetic modification, in this paper, the effective shRNA fragments targeting SUV39H1 gene were first screened, their roles on expression of exogenous transgenic genes were determined by using Bcap-37 cell line with stable expressing IFNα-2b gene as a model, the preliminary regulation mechanism of SUV39H1 gene was investigated. The results showed that the designed shRNA1/2 fragments of SUV39H1 gene had an obvious inhibition effect on the expression of SUV39H1 gene, reached 53.07 and 31.28%, respectively by qRT-PCR analysis. Compared with the control group, the expression of IFNα-2b gene in transgenic Bcap-37 cells infected with shRNA1 and 2 viruses significantly increased by 96.25 and 121.08%, respectively (p < 0.05). In addition, the expression of DNMT1, HDAC1 and G9a gene in the shRNA infected cells reduced significantly, and the expression of the HAT1 gene increased significantly (p < 0.05). The above results indicated that the expression of exogenous transgenic gene could be promoted by suppressing SUV39H1 gene at the cell level.

In vitro expansion of γδT cells and their killing effect on different tumor cells

Objective To study the killing effect of γδT cells on different tumor cells by inducing and expanding γδT cells in vitro. Methods Collected the peripheral venous blood of 20 healthy volunteers recruited at Xinqiao Hospital of Third Military Medical University from January to March 2018. Peripheral blood mononuclear cells (PBMCs) were induced and expanded using zoledronic acid combined with interleukin-2 (IL-2) to obtain γδT cells. The cell purity was detected on day 0, 7, 10, 14 and 16 using flow cytometry. The killing activity was detected by CCK-8 kit. Cells cultured on the 14th day were used as effector cells, and breast cancer cell BCap-37 and liver cancer cell Bel-7402 were used as target cells, and the cell killing activity was detected at the effective target ratio (E∶T) of 5∶1, 10∶1 and 20∶1 respectively. Results The purity of γδT cells were respectively (3.35±1.32)%, (50.76±5.76)%, (80.43±4.53)%, (90.56±3.34)%, (89.54±4.42)% on day 0, 7, 10, 14 , 16, with significant difference (F=18.431, P=0.012). The efficiency of γδT cells killing BCap-37 tumor cells were (31.3±2.0)% (E∶T=5∶1), (48.7±1.6)% (E∶T=10∶1), (71.3±2.4)% (E∶T=20∶1) respectively, with significant difference (F=16.724, P=0.016), further comparison between each two groups: 10∶1 vs. 5∶1 (P=0.013), 20∶1 vs. 5∶1 (P=0.017), 20∶1 vs. 10∶1 (P=0.011). The killing rate increased with the increase of target ratio. The efficiency of γδT cells killing Bel-7402 tumor cells were (34.5±2.0)% (E∶T=5∶1), (52.4±1.9)% (E∶T=10∶1), (74.5±1.6)% (E∶T=20∶1) respectively, with significant difference (F=18.253, P=0.013), further comparison between each two groups: 10∶1 vs. 5∶1 (P=0.015), 20∶1 vs. 5∶1 (P=0.012), 20∶1 vs. 10∶1 (P=0.015). The killing rate increased with the increase of target ratio. Conclusion We can obtain high purity γδT cells using zoledronic acid combined with IL-2 induced PBMCs, and the amplified γδT cells have significant killing effect on BCap-37 and Bel-7402 tumor cells. Key words: γδT cells; Zoledronic acid; Killing function

Abstract A46: KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway in breast cancer

Abstract Krüppel-like factor 4 (KLF4) has critical roles in breast cancer development and progression and several solid tumors. Tamoxifen (TAM) resistance represents a daunting challenge to the successful treatment of breast cancer. KLF4 expression, function, and regulation in the efficacy of TAM therapy in breast cancer have yet to be demonstrated. Here, we investigated the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 was correlated with increased TAM sensitivity in breast cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERa and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness of MCF-7/TAM and T47D cells to TAM. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion, and migration ability. Besides, KLF4 expression was downregulated in breast cancer tumor tissues and high expression of KLF4 linked to favorable outcome. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and P38 were relatively activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors can significantly suppress cell viability. Knockdown of KLF4 activated ERK and P38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and P38 signaling and resulted in enhanced sensitivity to TAM. Therefore, our findings suggest that KLF4 contributes to TAM efficiency in breast cancer via phosphorylation modification of ERK and P38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting the KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for TAM-resistant patients. Note: This abstract was not presented at the conference. Citation Format: Yunlu Jia, Wang Linbo. KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A46.

Aporphine Alkaloids from Illigera aromatica from Guangxi Province, China

Three undescribed aporphine alkaloids laurodionine B (1), illigerine A (2), and N-formyl-laurolitsine (3) were isolated from the methanolic extracts of the Chinese medicinal plant, Illigera aromatica, together with three known analogues (4–6). The chemical structures of 1–6 were identified by spectroscopic methods including 1D and 2D NMR (1H, 13C, COSY, HSQC, and HMBC) and high resolution mass spectrometry (HRESIMS). Compounds 1–3 showed moderate inhibitory activities in vitro against two cultured tumor cell lines, Hela and SMMC7721, with IC50 values of 32.42–62.90 μM. Only compound 1 had in vitro cytotoxic activity against Bcap37 cells, with the IC50 value of 90.61 μM.

Bcl-2 promotes metastasis through the epithelial-to-mesenchymal transition in the BCap37 medullary breast cancer cell line.

Metastatic breast cancer is one of the major types of cancer in women. However, despite being the focus of considerable research efforts, its molecular mechanism remains to be fully elucidated. The B-cell lymphoma/leukemia gene-2 (Bcl-2) protein is well known for its role in inhibiting programmed cell death/apoptosis. However, little is known concerning its function in cell invasion and migration. In the present study, cell migration and invasion assays revealed that anti-apoptotic Bcl-2 protein induced migration and invasion without affecting cell proliferation in the BCap37 breast cancer cell line. In addition, it was found that the overexpression of Bcl-2 in BCap37 cells increased metastasis to the lung in a mouse model. Using western blotting and RT q-PCR analysis, it was demonstrated that the overexpression of Bcl-2 inhibited the expression of E-cadherin, an epithelial marker, whereas it increased the levels of mesenchymal markers N-cadherin and vimentin. Therefore, the results suggested that Bcl-2 may induce cellular metastasis in breast cancer via the epithelial-to-mesenchymal transition.