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OBJECTIVE: To synthesize immunomagnetic nanoparticles with uniform particle size, strong superamagnetism as well as strong immune activity which can be specifically and sensitively combined with circulating tumor cells in peripheral blood of patients with breast cancer.METHODS: Superparamagnetic oxide iron nanoparticles containing active carboxyl groups (SMNP-COOH) were synthesized by polyol methods, thermogravimetric analysis was used to determine the amount of carboxyl groups on the surface of SMNP-COOH, while the content of iron was determined by o-phenanthroline. Mediated by 1-ethyl-3,3-dimethylaminopropyl carbodiimide(EDC) and N-hydroxysuccinimide (NHS), immunomagnetic nanoparticles(IMNP) against human breast carcinoma cell line were constructed by binding the monoclonal antibodies against hMAM with SMNP-COOH. X-Ray diffraction was used to confirm their synthesis,meanwhile,transmission electron microscope (TEM), dynamic light scattering (DLS), and vibrating sample magnetometry (VSM) were applied to characterize their physicochemical properties. The conjugation amount of the antibodies and the activity of IMNPs were evaluated by enzyme linked immunosorbent assay (ELISA).RESULTS: X-Ray diffraction showed that the chracteristic peaks of the crystalline powder of SMNP-COOH and IMNP agreed with the Fe3O4 standard. The concentration of iron in SMMP-COOH and IMNP were 0.205 and 0.164 mol•L-1, respectively.TEM showed that both synthesized SMNP-COOH and IMNP were almost spherical or ellipsoidal. The sizes of SMNP-COOH and IMNP were (13.7±3.6) and (15.4±4.5) nm, respectively. Dynamic light scattering(DLS) demonstrated the intensity particle size and polydispersity index (PDI) of SMNP-COOH and IMNP were 23.4 nm and 0.303, and 71.2 nm and 0.175,respectively. VSM results showed that both SMNP-COOH and IMNP had strong superamagnetism, and the saturation magnetization of SMNP-COOH and IMNP were 71.37 and 67.68 emu•g-1Fe, respectively, which confirmed antibody binding may reduce the magnetism of SMNP-COOH. The ELISA results showed the conjugation amount of antibody was about 93 μg on 1 mg SMNP-COOH by covalent bond. The obtained immunomagnetic nanoparticles (IMNP) which were bound with the hMAM monoclonal antibodies could specifically and sensitively combine with breast cancer cell line MDA-MB-415.CONCLUSION: IMNP with strong superparamagnetic property,excellent stability and perfect antibody activity were successfully synthesized, which demonstrate the potential to magnetically separate circulating tumor cells in peripheral blood from patients with breast cancer, thus providing a favorable weapon to accurately detect CTCs in breast tumor patients.
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PURPOSE: Multidrug resistance (MDR) remains a major obstacle in the treatment of triple-negative breast cancer (TNBC) with conventional chemotherapeutic agents. A previous study demonstrated that hsa-miRNA-143-3p plays a vital role in drug resistance of TNBC. Downregulation of hsa-miRNA-143-3p upregulated the expression of its target protein cytokine-induced apoptosis inhibitor 1 (CIAPIN1) in order to activate MDR, while upregulation of hsa-miRNA-143-3p effectively enhances the sensitivity of drug-resistant TNBC cells to chemotherapeutics. The present study aimed to further verify these findings in vivo. METHODS: We established a hypodermic tumor nude mice model using paclitaxel-resistant TNBC cells. We expressed ectopic hsa-miRNA-143-3p under the control of a breast cancer-specific human mammaglobin promoter that guided the efficient expression of exogenous hsa-miRNA-143-3p only in breast cancer cells. Thereafter, we overexpressed hsa-miRNA-143-3p in xenografts using a recombinant virus system and quantified the expression of hsa-miRNA-143-3p, CIAPIN1 protein, and proteins encoded by related functional genes by western blot. RESULTS: We successfully completed the prospective exploration of the intravenous virus injection pattern from extensive expression to targeted expression. The overexpression of hsa-miRNA-143-3p significantly alleviated chemoresistance of TNBC by inhibiting viability. In addition, we observed that the expression of CIAPIN1 as a hsa-miRNA-143-3p target protein was remarkably decreased. CONCLUSION: We partly illustrated the mechanism underlying the hsa-miRNA-143-3p/CIAPIN1 drug resistance pathway. HsamiRNA-143-3p as a tumor suppressive microRNA may be a novel target to effectively reverse MDR of TNBC in vivo.
Subject(s)
Animals , Humans , Mice , Apoptosis , Blotting, Western , Breast , Breast Neoplasms , Down-Regulation , Drug Resistance , Drug Resistance, Multiple , Heterografts , Mice, Nude , MicroRNAs , Prospective Studies , Triple Negative Breast Neoplasms , Up-RegulationABSTRACT
Objective: To explore the serum levels and the positive expression rates of human mammaglobin (hMAM) and extracellular matrix metalloproteinase inducer (CD147) of the patients with breast cancer, and to clarify their clinical significnaces in the diagnosis of breast cancer. Methods: A total of 122 patients with breast cancer (breast cancer group), 21 patients with breast fibroadenoma (breast fibroadenoma group) and 16 healthy controls (healthy control group) were selected as the subjects. The serum samples of subjects in various groups were collected. The serum levels and the positive expression rates of hMAM and CD147 of the subjects in various groups were measured by ELISA method. The serum levels and the positive expression rates of hMAM and CD147 of the breast cancer patients with different clinicopathological features were analyzed. The cut-off values of serum levels of hMAM and CD147 of the patients with breast cancer and their sensitivities and specificities in the dignosis of breast cancer were comfirmed by receiver operating characteristic (ROC) curve. Results: The serum levels of hMAM and CD147 of the patients in breast cancer group were higher than those in healthy control group and breast fibroadenoma group (P<0. 05). The positive expression rates of hMAM and CD147 of the patients in breast cancer group were higher than those in healthy control group and breast fibroadenoma group (P<0. 01); while the positive expression rates of serum hMAM combined with CD147 of the patients in breast cancer group were higher than those in healthy control group and breast fibroadenoma group (P<0. 01). There were significant differences in the positive expression rates of serum hMAM and CD147 of breast cancer patients with lymph node metastasis or not (χ2=10. 375, P<0. 01; χ2=15. 556, P<0. 01). There were significant differences in the positive expression rates of serum CD147 of the breast cancer patients with different TNM stages, ER and Her-2 (χ2= 8157, P<0.05; χ2= 6. 035, P<0. 05;χ2 = 5. 385, P<0. 05). With the increasing of TNM stages, the positive expression rates of serum hMAM and CD147 were increased. The area under ROC curve (AUC) of serum level of hMAM was 0. 809, 95%CI; 0.703-0.916; the AUC of serum level of CD147 was 0. 721, 95% Cl: 0.582-0.861. When the cut-off value of hMAM was 6. 51 μg · L-1, its sensitivity and specificity were 61. 1% and 87. 5%, respectively. The sensitivity and specificity were 73.6% and 68.7% respectively when the cut-off value of CD147 was 144. 92 ng · L-1. The AUC of detection of hMAM combined with CD147 was 0. 880, 95% Cl: 0. 798-0. 962; the sensitivity and specificity were 80. 6% and 81. 2%, respectively. Conclusion; The serum level of hMAM has the high sensitivity and specificity in the diagnosis of breast cancer. Combined detection of hMAM and CD147 can improve the positive rate of diagnosis.
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Objective:To explore the positive expressions of biological markers human mammaglobin (hMAM) combined with matrix metallopeptidase 9 (MMP-9) and human epidermal growth factor receptor 2 (C-erbB2) mRNA in peripheral blood of the breast cancer patients with micrometastases,and to clarify its clinical application value in diagnosis of the micrometastases in peripheral blood of the breast cancer patients.Methods:A total of 74 patients with breast cancer,21 patients with breast fibroadenoma and 10 healthy controls were selected as the subjects.All the patients received surgical treatment and the peripheral blood was collected.The mRNA expression levels of hMAM,MMP-9 and C-erbB2 in peripheral blood were measured by the real-time fluorescent quantitative PCR.The positive expression rates of detection of hMAM,MMP-9 and C-erbB2 were compared,and the differences in detection of hMAM combined with MMP-9 and C-erbB2 between the patients with different clinicopathologic features were analyzed.Results:In the breast cancer patients with lymph node metastasis,the differences of positive expression rates of MMP-9 and C-erbB2 mRNA were significant (x2=6.450,P＜0.05;x2=5.636,P＜0.05),and the difference of positive expression rate of hMAM mRNA was sigificant between HER-2 positive and negative patients (x2=5.804,P＜0.05).The positive expression rates of individual hMAM and combined with MMP-9 and C-erbB2 were 37.84％ (28/74),59.46％ (44/74) and 48.65％ (36/74) in the breast cancer patients,the combined postive expression rate of these three kinds of markers was 64.86 ％ (48/74),which were higher than those in healthy controls group (x2=5.676,P＜0.05;x2=3.102,P＞0.05;x2=5.339,P＜0.05;x2 =2.310,P＞0.05),fibroadenoma of breast group (x2 =8.438,P＜0.01;x2 =4.491,P＜ 0.05;x2 =7.982,P＜0.01;x2 =4.844,P＜0.05) and non-breast cancer group (healthy controls group+ breast fibroadenoma group) (x2 =13.093,P＜0.01;xx2 =6.471,P＜0.05;x2 =11.837,P＜0.01;x2 =6.103,P＜ 0.05).The positive expression rates of individual hMAM and the joint detection in the breast cancer patients at stage Ⅲ + Ⅳ were higher than those in the patients at stage Ⅰ + Ⅱ;the positive expression rates of individual hMAM and combined with C-erbB2 were statistically significant (x2 =5.157,P＜0.05;x2 =4.912,P＜0.05).Conclusion:hMAM has a low positive rate in the diagnosis of micrometastases in the breast cancer patients,while hMAM combined with MMP-9 and C-erbB2 detection could improve the positive rates.which presents some clinical application value for the early diagnosis of breast cancer micrometastases.
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Objective:To explore the positive expressions of biological markers human mammaglobin (hMAM) combined with matrix metallopeptidase 9 (MMP-9) and human epidermal growth factor receptor 2 (C-erbB2) mRNA in peripheral blood of the breast cancer patients with micrometastases,and to clarify its clinical application value in diagnosis of the micrometastases in peripheral blood of the breast cancer patients.Methods:A total of 74 patients with breast cancer,21 patients with breast fibroadenoma and 10 healthy controls were selected as the subjects.All the patients received surgical treatment and the peripheral blood was collected.The mRNA expression levels of hMAM,MMP-9 and C-erbB2 in peripheral blood were measured by the real-time fluorescent quantitative PCR.The positive expression rates of detection of hMAM,MMP-9 and C-erbB2 were compared,and the differences in detection of hMAM combined with MMP-9 and C-erbB2 between the patients with different clinicopathologic features were analyzed.Results:In the breast cancer patients with lymph node metastasis,the differences of positive expression rates of MMP-9 and C-erbB2 mRNA were significant (x2=6.450,P＜0.05;x2=5.636,P＜0.05),and the difference of positive expression rate of hMAM mRNA was sigificant between HER-2 positive and negative patients (x2=5.804,P＜0.05).The positive expression rates of individual hMAM and combined with MMP-9 and C-erbB2 were 37.84％ (28/74),59.46％ (44/74) and 48.65％ (36/74) in the breast cancer patients,the combined postive expression rate of these three kinds of markers was 64.86 ％ (48/74),which were higher than those in healthy controls group (x2=5.676,P＜0.05;x2=3.102,P＞0.05;x2=5.339,P＜0.05;x2 =2.310,P＞0.05),fibroadenoma of breast group (x2 =8.438,P＜0.01;x2 =4.491,P＜ 0.05;x2 =7.982,P＜0.01;x2 =4.844,P＜0.05) and non-breast cancer group (healthy controls group+ breast fibroadenoma group) (x2 =13.093,P＜0.01;xx2 =6.471,P＜0.05;x2 =11.837,P＜0.01;x2 =6.103,P＜ 0.05).The positive expression rates of individual hMAM and the joint detection in the breast cancer patients at stage Ⅲ + Ⅳ were higher than those in the patients at stage Ⅰ + Ⅱ;the positive expression rates of individual hMAM and combined with C-erbB2 were statistically significant (x2 =5.157,P＜0.05;x2 =4.912,P＜0.05).Conclusion:hMAM has a low positive rate in the diagnosis of micrometastases in the breast cancer patients,while hMAM combined with MMP-9 and C-erbB2 detection could improve the positive rates.which presents some clinical application value for the early diagnosis of breast cancer micrometastases.
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Objective: To explore the clinical value of cytokeratin 19 (CK19) and human mammaglobin (hMAM) in the detection of circulating tumor cells (CTCs) in peripheral blood of patients with breast cancer. Methods: Expressions of CK19 and hMAM in blood were identified by real-time fluorescent quantitative PCR, and the correlations between CK19, hMAM and the clinicopathologic features were analyzed. Results: In breast cancer patients, the rates of diagnostic positive detection of individual CK19 or hMAM and the combination of these two biomarkers were 45.5% (20/44), 38.6% (17/44) and 56.8% (25/44), respectively. The expression of combined biomarkers was correlated with lymph node metastasis (P = 0.031). The rates of diagnostic positive detection of CK19, hMAM and the combination biomarkers were significantly higher in breast cancer patients prior to treatment than those in the healthy control group (P = 0.000 3, P = 0.000 1 and P = 0.000 1, respectively). After two-cycle chemotherapy, CTCs which were positive at baseline presented as negative in 5 stage III patients and 4 stage IV patients (P = 0.025 3 and P = 0.045 5). However, the number of CTCs-positive patients at stagesI-II decreased no matter using CK19, hMAM or CK19 plus hMAM as biomarker, leaving more CTCs-positive patients in combined biomarker group than that in single biomarker group, but there was no statistical significance. Conclusion: The combined panel of both hMAM and CK19 may serve as representative biomarkers for CTCs, thus it presents potentially significant value for monitoring early metastasis, therapeutic efficacy and prognosis for the patients with breast cancer. Copyright © 2014 by TUMOR.
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Objective To study the expression of human-mammaglobin (hMAM) and vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR-3) in breast cancer tissues, and to analyze its relationship with biological characteristics and prognosis of breast cancer. Methods The expression of hMAM, VEGF-C andVEGFR-3 was determined by immunohistochemical technique in 116 breast cancer tissues and 44 adjacent normal tissues (from cancer tissue≥ 5 cm) using tissue microarray technology (TMA). Results (1) The positive rates of hMAM, VEGF-C and VEGFR-3 in primary breast carcinoma tissues were significantly higher than those in the adjacent normal tissues (59. 48% [69/116] vs 0. 00%, 50. 86% [59/116] vs 9. 09% and 61. 20% [71/116] vs 18. 18%, respectively, all P<0. 01). (2)The positive rates of hMAM, VEGF-C and VEGFR-3 were significantly correlated with axillary lymph node metastasis, and the positive rates of hMAM, VEGFR-3 were significantly correlated with histological gradings (all P<0. 05). (3) Spearman rank correlation analysis showed that the positive rates of hMAM, VEGF-C and VEGFR-3 in breast cancer tissues were all significantly correlated with each other, with correlation coefficients being 0. 278, 0. 280, and 0. 244, respectively, all P<0. 05). (4)Kaplan-Meier survival analysis showed that the disease-free survival and 5-year overall survival of patients with negative expression of HMAM, VEGF-Cand VEGFR-3 were significantly prolonger compared to those with positive expression (Log-rank test, P< 0. 05). Conclusion hMAM, VEGF-C and VEGFR-3 are highly expressed in breast cancer patients, which might be associated with the invasion and metastasis of breast cancer, and hMAM expression might be related to the lymphangiogenesis of breast cancer.
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Background: Human mammaglobin (hMAG) is a secreted protein which has been detected in breast epithelial cells of mammary glands and has been used as a specific marker for breast cancer. Objectives: This study aims at studying the hMAG expression and identifying the significant predictors of hMAG expression in breast cancer tissues. Materials and Methods: The tissue samples were obtained from two major teaching hospitals in the country. They were examined by immunohistochemistry (IHC) and the hMAG expression was evaluated using an established scoring system. Results: Out of 84 breast cancer tissue samples, hMAG was expressed in 50 samples (59.6%). The expression of hMAG was found to be increased with cancer grade. The output of logistic regression model showed that hMAG was overexpressed in breast cancer samples from the first hospital (P = 0.014), but not with those from the second hospital. Conclusions: It can be concluded that hMAG may serve in the diagnosis and the assessment of progression with the increased cancer grade. The dominance in hMAG expression in samples from HUSM may correlate with ethnic, environmental or genetic factors.
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Adult , Aged , Aged, 80 and over , Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Disease Progression , Female , Gene Expression , Hospitals, Teaching , Humans , Immunohistochemistry , Malaysia , Mammaglobin A , Middle Aged , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Uteroglobin/biosynthesis , Uteroglobin/geneticsABSTRACT
Objective To investigate the relationship between bone marrow micrometastasis of patients with breast cancer and clinical pathological parameters, some molecular markers as well as prognosis.Methods The expression of hMAM mRNA in BM of patients with breast cancer was detected by RT-PCR.The expressions of ER, PR in cancer tissues were detected by immunohistochemical SP method. Results About 38.2 % positive expression rate of hMAM mRNA in 102 patients with stage Ⅰ -Ⅳ breast cancer was found.The expression of hMAM increased more in patients with T2-3 (＞2 cm) tumors than T1 (≤2 cm) (P =0.001)and with stage Ⅱ ,Ⅲ than stage Ⅰ (P =0.001). The expression of hMAM in the BM of breast cancer with grade I was lower than that of grade Ⅱ or Ⅲ (P =0.014). The expression of hMAM in the BM was related to the pathological type (P =0.032) and the axillary lymph node metastasis (P =0.001). The expressions of hMAM in BM were much higher with ER negative in breast cancer tissues (P ＜0.05). There was a correlation between patients with positive expression of hMAM in BM and distant metastasis (P =0.009). Conclusion The micrometastasis in BM is correlated with some clinical pathological parameters and some tumor markers. The patients with positive expression of hMAM in BM have more chances with distant metastasis and poor prognosis. The detection of micrometastasis may be as one of targets to predict the prognosis of breast cancer.
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Objective:To clone the cDNA in full-length of human mammaglobin,do prokaryotic expression and purify hMAM protein,as a basis for early diagnosis of breast cancer.Methods :hMAM cDNA was amplified through RT-PCR from breast cancer tissue and breast cancer cell line MD-MB453,and the recombination pQE40-hMAM vector was constructed and expressed in E.Coli.M15 after induction by IPTG.The fusion protein was purified with Ni-NTA-His affinity chromatography. Results: Two subtypes of hMAM cDNA and the hMAM(Isoform) cDNA were found,in which consisted of 270 bp,different from the wildtype hMAM cDNA of 279 bp on nine continuous base pair missing.The fusion protein formed inclusion body in prokaryotic expression system and the renatured protein was purified which purity was about 97%.Conclusion: The recombinant hMAM was successfully purified.