中药复方BBYNG对乳腺癌骨转移小鼠的作用观察

目的透过对中药复方BBYNG与西药双磷酸盐OSTAC的动物实验研究,观察中药治疗骨转移的疗效。方法参照文献建立高发骨转移的小鼠乳腺癌动物模型后,分别对不同组别的荷瘤小鼠灌喂相当于临床病人服用剂量的BBYNG或OSTAC,对比观察荷瘤小鼠的肿瘤生长、活动状态、生存时间、骨转移及骨破坏的程度等。结果BBYNG可减慢荷瘤小鼠的肿瘤生长,但不能显著缩小肿瘤的体积;可减轻肿瘤骨转移引起的活动障碍和骨破坏,延长生存时间。此结果与作者已进行的临床观察结果相似。结论BBYNG有减轻动物模型肿瘤引起的骨转移和骨破坏、延长荷瘤小鼠生存时间的作用;值得推广应用及开展用于预防肿瘤骨转移的探讨,并深化对其作用机理的研究。     

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and genetic polymorphisms in breast cancer patients

Reactive oxygen species (ROS) causes damage to DNA, but the role of ROS in breast carcinoma is still not clear. The objective of this study was to measure the urinary 8-OHdG levels of breast cancer patients at each stage of carcinogenesis and assess its association with the development of breast cancer. Sixty patients with malignant breast tumors were matched with 60 control subjects of the same ages in this case control study. Urinary 8-OHdG levels were significantly higher among breast cancer patients than among the control subjects, after making adjustments for confounders such as smoking, coffee consumption and use of oral contraceptives. The breast cancer patients were divided into three groups based on the stages of their cancer; urinary 8-OHdG levels decreased with each stage of breast carcinoma. Using multiple regression and logistic models adjusted for other covariates, urinary 8-OHdG levels significantly correlated with the development of breast cancer. However, it was found that breast cancer was not significantly influenced by CYP1A1, CYP1M1 or NAT2 polymorphisms. In conclusion, it was found that oxygen radical generation occurred within carcinoma cells, but the role of polymorphism of specific genes in the development of breast cancer should be evaluated.     

Hair Iron and Other Minerals’ Level in Breast Cancer Patients

Little is known about hair minerals in cancer patients, and serum iron level has been shown to be elevated in breast cancer patients. Therefore, the aim of this study was to evaluate hair iron and hair minerals’ level related to hair iron in breast cancer patients compared to controls. We compared hair mineral analysis data of 40 breast cancer subjects with age and body mass index-matched normal control data (n?=?144) by cross-sectional analysis. All breast cancer patients were newly diagnosed at one Breast Cancer Center in Ajou University and had their hair cut before anti-cancer chemotherapy, and the normal controls (without breast cancer) also had their hair cut for various reasons in out-patient clinics of the Department of Family Practice and Community Health. Breast cancer patients had low calcium, magnesium, iron, copper, manganese, and zinc, whereas they had high arsenic, sodium, and potassium compared with the normal control. The hair iron level was positively correlated with hair calcium (r?=?0.761, P?<?0.001), magnesium (r?=?0.643, P?<?0.001), and manganese (r?=?0.550, P?<?0.001) and negatively correlated with arsenic (r?=??0.537, P?<?0.001). The hair iron level was significantly associated with the hair calcium (β?=?0.778, P?<?0.001) and manganese (β?=?0.240, P?<?0.001) by using multiple linear regression analysis. We observed different hair mineral patterns in breast cancer patients compared to normal controls. Especially, hair iron level was significantly reduced and associated with hair calcium and manganese levels.     

Claudin-16 reduces the aggressive behavior of human breast cancer cells

Claudin-16 (Paracellin-1) is a transmembrane tight junction (TJ) protein originally described as having a critical role in the re-absorption of magnesium and calcium in the kidney. This study examined expression of Claudin-16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between Claudin-16 levels and patient prognosis. Insertion of the Claudin-16 gene into MDA-MB-231 human breast cancer cells resulted in cells that were significantly less motile and invasive in behavior, with increased adhesion to matrix. These cells also exhibited significantly increased TJ functionality and "tighter" colony morphology. Moreover, growth rates were reduced in both in vitro and in vivo assays (P < 0.002). Frozen sections from breast cancer primary tumors (matched tumor 124 and background 33) were immuno-stained. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, normalized with cytokeratin-19 levels). Levels of expression of Claudin-16 were significantly decreased in node positive tumors compared to negative (P = 0.016). Expression was significantly lower in patients with node positive tumors (P = 0.016) and in those who had died from breast cancer or had general poor prognosis (P < 0.015). Immunohistochemical staining showed decreased expression of Claudin-16 in tumor sections (P < 0.00001). In conclusion, forced expression of Claudin-16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumor volume. Claudin-16 expression was reduced in human breast cancer, particularly in patients with aggressive tumors and high mortality. This suggests that Claudin-16 plays a role beyond that of an initial metastasis repressor in this cancer type.     

Urinary pS2/TFF1 levels in the management of hormonodependent breast carcinomas

pS2/TFF1 overexpression in breast carcinomas correlates with response to hormonotherapy. We evaluated the clinical relevance of urinary pS2/TFF1 in breast cancer patients. In healthy controls (100 cases), it represents an individual and relatively stable parameter. Although 24 out 83 pre-operative breast cancer patients showed elevated levels, both the sensitivity and specificity of the test were too low for breast cancer screening. However, neoadjuvant hormonotherapy decreased pS2/TFF1 levels in nine out of 20 patients. Furthermore, among 22 patients receiving long-term adjuvant hormonotherapy, four exhibited elevated levels, two of them at the time of relapse. Thus, urinary pS2/TFF1 quantification might be suitable as an in vivo diagnosis for tumor hormonodependency, and disease follow-up during hormonotherapy.     

Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Introduction

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method

We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results

Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions

Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.     

T型钙通道与乳腺癌

T型钙通道是激活电位低、失活速度快、单通道电导小的电压依赖性钙通道,具有高组织特异性、突出的生理功能及药理学选择性等特点。近年来的研究表明,T型钙通道通过独特的激活失活效应参与细胞内外钙流的振荡,影响肿瘤细胞的增殖过程。值得关注的是正常人乳腺上皮细胞中没有T型钙通道,而在不同分化阶段的乳腺癌细胞中该通道却有表达。实验证实,T型钙通道的表达影响乳腺癌细胞的增殖,通道拮抗剂能够显著地抑制乳腺癌细胞增殖。这一发现为乳腺癌的诊断及靶向治疗药物的研发提供了新的思路。本文概要介绍了近年来T型钙通道与乳腺癌关系的研究进展。     

Abnormal Excretion of Corticosteroid Sulphates in Patients with Breast Cancer

In a preliminary study, the 24-hour urinary excretion of corticosteroid sulphates and free cortisol have been measured in a group of patients with breast cancer and compared with the excretion of the same compounds in a group of normal women of similar age. Excretion of corticosteroid sulphates in the breast cancer group was found to be markedly raised. In a small number of patients with localized cancer of sites other than the breast the level of corticosteroid sulphate was not raised. If proved metastases were present a noticeable rise was observed.     

Feasibility of Breast Cancer Screening by PIXE Analysis of Hair

To reveal the role of key elements present in the hair of breast cancer patients on cancer development, the levels of a number of elements in scalp hair samples of 82 people including healthy individuals, people suffering from benign breast disease, and breast cancer patients were measured by PIXE analysis. Pellets of hair samples were prepared and bombarded by 2.2 MeV proton beam of a 3-MV Van de Graaff accelerator. The number of incident ions hitting the sample was indirectly measured using the RBS spectrum of a thin Ag film placed in the beam path. The concentrations of S, Cl, K, Ca, Fe, and Cu in the hair of healthy individuals were in agreement with those observed in the hair of hyperplasia and cancer patients within standard deviations. However, a lower average level of zinc was found in samples from hyperplasia and breast cancer patients. Strong positive correlations were found between iron and potassium as well as between calcium and potassium in the cancer patients. These results could be of significance in the screening for breast cancer.     

Osteoblasts suppress high bone turnover caused by osteolytic breast cancer in-vitro

The skeleton is the most common site of breast cancer metastasis, which can occur in up to 85% of patients during their lifetime. The morbidity associated with bone metastases in patients with breast cancer includes pathological fractures, bone pain, hypercalcaemia, and spinal cord compression. When breast cancer metastasizes to bone, the balance of bone resorption (mediated by osteoclasts) and bone formation (mediated by osteoblasts) favors bone resorption, which leads to net bone destruction (i.e., osteolysis). Anti-resorptive agents such as bisphosphonates are commonly used to treat bone resorption in osteoporosis or osteolytic cancer patients. However, bisphosphonates by themselves are unable to rebuild lost bone tissue, and can cause severe side effects. In this study, we developed a bovine bone explant culture system and have observed that murine osteoblasts can modulate the activity of osteotropic human breast cancer cells on this substrate. Using markers of bone metabolism, we observe diminished bone turnover in organ culture following the addition of exogenous osteoblasts. The data presented in this study supports further investigation into the use of cytotherapies to limit breast cancer mediated osteolysis.     

Small molecule inhibition of arylamine N-acetyltransferase Type I inhibits proliferation and invasiveness of MDA-MB-231 breast cancer cells

Arylamine N-acetyltransferase 1 is a phase II metabolizing enzyme that has been associated with certain breast cancer subtypes. While it has been linked to breast cancer risk because of its role in the metabolic activation and detoxification of carcinogens, recent studies have suggested it may be important in cell growth and survival. To address the possible importance of NAT1 in breast cancer, we have used a novel small molecule inhibitor (Rhod-o-hp) of the enzyme to examine growth and invasion of the breast adenocarcinoma line MDA-MB-231. The inhibitor significantly reduced cell growth by increasing the percent of cells in G2/M phase of the cell cycle. Rhod-o-hp also reduced the ability of the MDA-MB-231 cells to grow in soft agar. Using an in vitro invasion assay, the inhibitor significantly reduced the invasiveness of the cells. To test whether this effect was due to inhibition of NAT1, the enzyme was knocked down using a lentivirus-based shRNA approach and invasion potential was significantly reduced. Taken together, the results of this study demonstrate that NAT1 activity may be important in breast cancer growth and metastasis. The study suggests that NAT1 is a novel target for breast cancer treatment.     

miR-188-5p suppresses cellular proliferation and migration via IL6ST: A potential noninvasive diagnostic biomarker for breast cancer

Previously, serum miR-188-5p is differentially expressed in breast cancer, but the diagnostic potential of circulating miR-188-5p as well as its regulatory mechanism in breast cancer remain uncertain. Herein, serum miR-188-5p was detected by real-time polymerase chain reaction in patients with breast cancer, breast fibroadenoma, and healthy subjects. Circulating miR-188-5p was abnormally elevated in patients with breast cancer as compared with these other two groups, and was reduced in patients with breast cancer following surgical treatment. Increased serum miR-188-5p corresponded to lymph node metastasis status and TNM stages of breast cancer. A receiver operating characteristic curve analysis of the ability to circulate miR-188-5p to distinguish between patients with breast cancer and either noncancerous patients or patients with breast fibroadenoma yielded corresponding areas under the curve of 0.894 and 8.814. miR-188-5p was downregulated in the highly malignant cancer line MDA-MB-231 relative to the less malignant MCF-7 cells. In vitro, functional analyses conducted via transfecting cells with mimics and inhibitors revealed miR-188-5p to suppress breast cancer cell proliferation and migration, which was mediated by its downstream target IL6ST. Comparison of intracellular and exosomal miR-188-5p levels indicated that miR-188-5p was selectively sorted into exosomes derived from MDA-MB-231 cells rather than those from MCF-7 cells. However, exosomal miR-188-5p levels in the serum of patients with breast cancer were reduced compared to healthy controls and did not differ relative to patients with breast fibroadenoma. In summary, miR-188-5p acts in a tumor-suppressive manner in breast cancer progression and may serve as a noninvasive early diagnostic biomarker and therapeutic target in breast cancer.     

Measurement of estrone-3-glucuronide and pregnanediol-3α-glucuronide in early morning urine samples to monitor ovarian function

The determination of the concentration of estrone-3-glucuronide and pregnanediol-3α-glucuronide has been performed by a chemiluminescent immunoassay in early morning urine samples of 14 normal menstruating women and 11 women affected by luteal phase defect. The early morning urine samples were daily collected for an entire menstrual cycle. We have employed a timed and measured volume collection procedure as correction factor. The integrated values of the hormonal data in definite time intervals were used to create a nomogram. By means of this method, it was possible to completely separate normal from luteal insufficiency subjects and to distinguish two different types of luteal phase defects. Moreover, the same approach was applied to the study of the role and the frequency of luteal phase defect in 15 patients affected by habitual abortion and in 17 premenopausal women who had undergone quadrantectomy for T1a No Mo breast cancer. A luteal phase defect was detected in nine of the aborting patients (60%) and in eight women affected by breast cancer (47%). Finally estrone-3-glucuronide was measured in early morning urine samples of 96 prepubertal and pubertal girls in different pubertal stages and in one patient affected by precocious puberty, before and during an agonist GnRH treatment. The urinary test of ovarian function seems to be suitable for diagnostic purposes and for clinical studies.     

Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells

Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8(+)CXCR6(+) activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into "inflamed" peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.     

ADAM 12 cleaves extracellular matrix proteins and correlates with cancer status and stage

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers.     

Nucleoside excretion in breast cancer: comparison with other biochemical tumour-index-substances

[We have measured four urinary nucleosides (dimethylguanosine, 1-methylinosine, pseudouridine and beta-aminoisobutyric acid)in patients with benign breast disease and patients with early and advanced breast cancer in order to assess their value as tumour-index-substances. We compared the results with other biochemical indices of breast cancer and sought and correlations between these indices. The results indicate that few abnormalities occurred in patients without overt metastases and these did not predict early relapse. In those with metastatic disease, dimethylguanosine excretion was most frequently elevated. Correlations were observed between some of the nucleosides and lysozyme and alpha1-antirypsin.     

Urinary Pyrophosphate in Normal Subjects and in Stone Formers

The urinary excretion of inorganic pyrophosphate was determined in nine normal subjects and also in eight patients with recurrent calcium-containing renal stones during varied levels of phosphate intake. The excretion of pyrophosphate and orthophosphate is virtually the same in the two groups at all levels of phosphate intake. It appears unlikely that a consistently reduced urinary excretion of pyrophosphate is a factor in the formation of urinary calculi. Pyrophosphate excretion rose and calcium excretion fell with increasing phosphate intake; this might be expected to have a beneficial effect in patients with recurrent calcium stones.     

Aromatase inhibitors and their application in breast cancer treatment*

Estrogens are known to be important in the growth of breast cancers in both pre- and postmenopausal women. The number of breast cancer patients with hormone-dependent disease increases with age, as does the incidence of breast cancer. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. Because aromatase catalyzes the rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme have been developed in the last few years as a logical treatment strategy. Two classes of aromatase inhibitors, steroidal and nonsteroidal compounds, are now in use. Among the steroid substrate analogs, formestane and examestane have been shown to be effective in breast cancer patients with advanced disease. Highly potent and selective nonsteroidal inhibitors have recently been found to suppress plasma and urinary estrogens by more than 95% in breast cancer patients. Two of these compounds recently were approved in the United States and have been shown to be more effective than other second-line agents in terms of overall response rates and treatment failure, as well as better tolerated. Although studies of the efficacy of these agents in earlier stage disease are awaited, it is evident that aromatase inhibitors can extend the duration of treatment in breast cancer patients.