Luteolin suppresses androgen receptor-positive triple-negative breast cancer cell proliferation and metastasis by epigenetic regulation of MMP9 expression via the AKT/mTOR signaling pathway.

BACKGROUND: Triple-negative breast cancer (TNBC) represents up to 20% of all breast cancers. This cancer lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The current therapeutic strategy for patients with this subtype is the use of cytotoxic chemotherapy and surgery. Luteolin is a natural herbal flavonoid and a potential therapeutic candidate for multiple diseases. The use of a treatment that combines Chinese herbal medicine and western medicine is rising in Asia. PURPOSE: The present study evaluates the effects and molecular mechanisms involved with luteolin treatment and evaluates whether this herb affects androgen receptor-positive breast cancer cell proliferation or metastasis. STUDY DESIGN: In vitro evaluation of the effect of luteolin on androgen receptor-positive TNBC cell proliferation and metastasis METHODS: Cell viability analysis was used for the cytotoxicity test. Colony formation and Bromodeoxyuridine (BrdU) staining-based proliferation experiments were used for cell proliferation. Wound healing and transwell assays were used for in vitro migration/invasion. The RT-qPCR analysis was used for gene expression. Furthermore, ChIP-qPCR analysis was used for epigenetic modification of gene promoters. RESULTS: Luteolin significantly inhibited the proliferation and metastasis of androgen receptor-positive TNBC. Furthermore, luteolin inactivated the AKT/mTOR signaling pathway and reversed the epithelial-mesenchymal transition (EMT). The combination of luteolin and inhibitors of AKT/mTOR synergistically repressed an androgen receptor-positive TNBC cell proliferation and metastasis. Luteolin also downregulated MMP9 expression by decreasing the levels of the AKT/mTOR promoting H3K27Ac and H3K56A on the MMP9 promoter region. CONCLUSION: Our findings indicate that luteolin inhibited the proliferation and metastasis of androgen receptor-positive TNBC by regulating MMP9 expression through a reduction in the levels of AKT/mTOR-inducing H3K27Ac and H3K56Ac.

Imbalances in the disposition of estrogen and naphthalene in breast cancer patients: a potential biomarker of breast cancer risk.

Elevation of naphthoquinones and estrogen quinones, which are reactive metabolites of naphthalene and estrogen, is thought to be an important indicator of naphthalene- and estrogen-induced carcinogenesis. We compared background levels of naphthalene and estrogen quinone-derived adducts in serum albumin (Alb) from 143 women with breast cancer and 119 healthy controls. Cysteinyl adducts of naphthoquinones, including 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ), and estrogen quinones, including estrogen-2,3-quinones (E2-2,3-Q) and estrogen-3,4-quinones (E2-3,4-Q), were characterized after adduct cleavage. Levels of estrogen quinones and naphthoquinones were positively correlated in healthy controls, but not in breast cancer patients (p < 0.05). Compared with controls, levels of 1,2-NPQ and E2-3,4-Q were elevated by two- to ten-fold in cancer patients (p < 0.001). To explore the correlation between estrogen- and naphthalene-derived quinone adducts and disease status, we performed linear discriminant analysis of the ratio of 1,2-NPQ-Alb to (1,2-NPQ-Alb plus 1,4-NPQ-Alb) versus the ratio of E2-3,4-Q-2-S-Alb to (E2-2,3-Q-4-S-Alb plus E2-3,4-Q-2-S-Alb) in patients and controls. These two groups were separable using albumin adducts of estrogen quinones and naphthoquinones, with 99.6% overall correct classification rate (overall accuracy). The findings of this study suggest that differences in the disposition of estrogen and naphthalene, and the subsequent elevation of cumulative E2-3,4-Q and 1,2-NPQ may serve as biomarkers of breast cancer risk.

Association of surgical margins with local recurrence in patients undergoing breast-conserving surgery after neoadjuvant chemotherapy.

BACKGROUND: The aim of the current study was to report a single-institution experience using breast-conserving surgery after neoadjuvant chemotherapy (NACT), focusing on the association between microscopic resection margin status and locoregional recurrence (LRR). METHODS: Our institutional prospectively maintained database was reviewed to identify patients who were treated with NACT between January 2008 and April 2018. RESULTS: Among the main partial mastectomy specimens available for analysis (n = 161), 28 had margins < 1 mm, 21 had margin width of 1-2 mm and the remaining 112 had margins > 2 mm. LRR occurred in 16 patients (9.9%) and distant metastases were detected in 27 (16.8%) patients. There was no significant difference in the LRR between the > 2 mm margin group with a 60-month cumulative survival of 85.2% compared with 76.2% for the ≤2 mm group (P = 0.335) in the Kaplan-Meier analysis. When we stratified patients by margin widths of ≥1 mm or <  1 mm, there was no LRR-free survival benefit observed for the ≥1 mm pathologic excision margin group in the univariate analysis (hazard ratio = 0.443; 95% confidence interval = 0.142-1.383; P = 0.161) with a 60-month cumulative LRR-free survival of 84.9% compared with 69.5% for the < 1 mm margin cohort (P = 0.150). CONCLUSIONS: In the absence of multiple scattered microscopic tumour foci, a negative margin of no ink on tumour maybe sufficient for stage I-III invasive breast cancer treated with NACT and breast-conserving surgery.

Indocyanine green fluorescence method for sentinel lymph node biopsy in breast cancer.

BACKGROUND/OBJECTIVE: Breast biopsy and analysis of sentinel lymph nodes (SLNs) accurately predict tumor status in the affected basin and help in avoiding unnecessary axillary lymph node dissection, which is associated with remarkable morbidity risk. Blue dye and radioisotope are the most widely used mapping agents, but non-radioactive tracers of comparable accuracy warrant further investigation. This study aimed to investigate utilization of indocyanine green (ICG) fluorescence in sentinel node localization compared with blue dye and to assess the incremental value of ICG. METHODS: A total of 39 consecutive patients underwent sentinel lymph node biopsy (SLNB) (40 cases: 38 unilateral and 1 bilateral) with combined blue dye and ICG for localization. The obtained fluorescence images of the lymphatic system were investigated. RESULTS: All 84 lymph nodes removed in 40 procedures were identified by ICG, but only 37 were identified by blue dye. The ICG method identified an average of 2.1 SLNs in 39 of 40 cases with a detection rate of 97.5%, but only 0.93 SLN per case with blue dye. Subcutaneous lymphatic channel patterns were also detected by fluorescent imaging in 37 procedures, which all revealed lymphatic drainage toward the axilla except in one case with internal mammary pathway. CONCLUSION: This study demonstrated the accuracy and safety of ICG for SLNB and its superiority to blue dye method in SLN localization. Therefore, ICG fluorescence method is safe and effective addition in breast clinical settings, wherein blue dye alone is used.

Albumin and hemoglobin adducts of estrogen quinone as biomarkers for early detection of breast cancer.

Cumulative estrogen concentration is an important determinant of the risk of developing breast cancer. Estrogen carcinogenesis is attributed to the combination of receptor-driven mitogenesis and DNA damage induced by quinonoid metabolites of estrogen. The present study was focused on developing an improved breast cancer prediction model using estrogen quinone-protein adduct concentrations. Blood samples from 152 breast cancer patients and 71 healthy women were collected, and albumin (Alb) and hemoglobin (Hb) adducts of estrogen-3,4-quinone and estrogen-2,3-quinone were extracted and evaluated as potential biomarkers of breast cancer. A multilayer perceptron (MLP) was used as the predictor model and the resultant prediction of breast cancer was more accurate than other existing detection methods. A MLP using the logarithm of the concentrations of the estrogen quinone-derived adducts (four input nodes, 10 hidden nodes, and one output node) was used to predict breast cancer risk with accuracy close to 100% and area under curve (AUC) close to one. The AUC value of one showed that both data sets were separable. We conclude that Alb and Hb adducts of estrogen quinones are promising biomarkers for the early detection of breast cancer.

Changing blood lead levels and DNA damage (comet assay) among immigrant women in Taiwan.

BACKGROUND: International marriage has had a rapid growth in recent years in Taiwan. However, little is known about the blood lead levels and DNA damage levels among immigrant women from resource-limited countries. OBJECTIVE: This study (a) explored differences between immigrant women and native women in demographic characteristics, blood lead levels, and DNA damage levels, and (b) identified risk factors that are associated with blood lead concentrations and DNA damage levels after immigration. METHODS: We used a structured questionnaire to collect data on socio-demographic status from (a) 71 immigrant women who had resettled in 2006 in Taichung, Taiwan and (b) 83 native women who live in the same area. Each study participant provided blood samples for lead and metal measurements, complete blood count examination, and the comet assay to measure degree of DNA damage. RESULTS: Immigrant women had higher mean blood lead concentration (2.23+/-1.63 vs. 1.63+/-1.00 microg/dl; p=0.04) and lower mean blood zinc level (6.22+/-2.22 vs. 6.89+/-2.44 mg/l; p=0.07) than native women. Resettlement time was a determinant to decrease blood lead and DNA damage levels among immigrants in Taiwan. Multiple linear regression analysis confirmed a statistically significant association between blood lead level and DNA damage, while zinc had a protective effect. CONCLUSIONS: Public health agencies should focus on primary prevention and providing screening programs for this vulnerable population. An immigrant women's cohort should been established to follow-up and improve for elevated lead exposure families.

Possible effect of probiotics and breast milk in short bowel syndrome: report of one case.

Primary volvulus means idiopathic volvulus without predisposing factor and is rare in children. The etiology is unknown. The incidence is relatively higher in neonates. The most common symptoms are abdominal distension and bilious vomiting. Our patient was a preterm baby at age of 89 days. Acute onset of abdominal distension and sepsis-like symptoms were noted. After operation, no anatomical anomaly was noted. Probable primary midgut volvulus was diagnosed. Early diagnosis of primary volvulus of the small intestine is difficult. Operation should be performed as soon as possible in a neonate with quick progression toward unstable hemodynamics and acidosis with ileus. Postoperative short bowel syndrome was noted. There are often sepsis, enterocolitis, and poor body weight gain noted among short bowel patients. With breast milk feeding and probiotics usage, there were few complications of short bowel syndrome noted in our patient. The duration for establishing intestinal adaptation was shorter than for other patients. The patient's body weight, body length and development caught up gradually within 18 months.

Ulinastatin alone does not reduce caspase 3-mediated apoptosis in protease-positive Aeromonas hydrophilia-induced sepsis.

BACKGROUND/PURPOSE: To evaluate the effect of ulinastatin, a protease inhibitor, on survival and apoptosis in protease-positive Aeromonas hydrophilia (PPAH)-induced sepsis. METHODS: Thirty mice were randomly allocated to receive intraperitoneal injection of either phosphate buffered saline (PBS) (control mice, n = 10) or PPAH (PPAH mice, n = 20). After 30 minutes, control mice received an additional intraperitoneal PBS injection, 10 PPAH mice received intraperitoneal PBS injection (non-treated PPAH mice), and the remaining 10 PPAH mice received an intraperitoneal injection of ulinastatin (ulinastatin-treated PPAH mice). RESULTS: Survival at 24 hours was 100% in control mice, and 35% (p < 0.05) in PPAH mice; the survival rate in non-treated and ulinastatin-treated PPAH mice were 30% and 40% (p > 0.05), respectively. The thymus weight (mg) decreased significantly in PPAH mice (51.1 +/- 14.9) compared to control mice (69.7 +/- 14.4; p < 0.001); there was no difference between ulinastatin-treated (52 +/- 13.9; p > 0.05) and non-treated PPAH mice (50.4 +/- 16). The thymus gland cell count reduced significantly in PPAH mice (8.1 +/- 4.7 x 10(7)) compared to control mice (12.8 +/- 6.6 x 10(7); p < 0.01), and immunofluorescence analysis demonstrated that the reduced cells were mostly CD4+ CD8+, in contrast to the increase in CD4+ CD8- cells. There was no difference in cell count between ulinastatin-treated (8.7 +/- 4.9 x 10(7)) and non-treated PPAH mice (7.4 +/- 4.6 x 10(7); p > 0.05). Caspase 3-mediated apoptosis was not detectable in control mice in contrast to the pronounced manifestation in PPAH mice. CONCLUSION: PPAH-induced sepsis has a high mortality that is related to lymphocyte apoptosis. Ulinastatin alone does not significantly reduce caspase 3-mediated lymphocyte apoptosis.

Serine protease inhibitors nafamostat mesilate and gabexate mesilate attenuate allergen-induced airway inflammation and eosinophilia in a murine model of asthma.

BACKGROUND: Serine proteases such as mast cell tryptase and certain allergens are important in the pathogenesis of allergic inflammation of asthma. OBJECTIVE: We sought to investigate the effects of serine protease inhibitors nafamostat mesilate (FUT), gabexate mesilate (FOY), and ulinastatin (UTI) on airway inflammation in a mouse model of allergic asthma. METHODS: BALB/c mice were sensitized to Dermatophagoides pteronyssinus (Der p) and intratracheally challenged with Der p (0.5 mg/mL). Therapeutic doses of FUT (0.0625 mg/kg), FOY (20 mg/kg), and UTI (10,000 U/kg) were intra-peritoneally injected into 3 corresponding sensitized mice during the sensitization phase (protocol 1) or 24 hours after allergen challenge (protocol 2). RESULTS: Both FUT-treated and FOY-treated sensitized mice had reduced mast cells activation, airway hyperresponsiveness, attenuated eosinophils infiltrations, and decreased Der p-induced IL-4 and TNF-alpha, but increased IL-12 cytokine production in bronchoalveolar lavage fluid compared with nontreated mice. Furthermore, FUT treatment downregulated the expression of IL-1beta, TNF-alpha, IL-6, eotaxin, inducible NO synthase, CD86, and nuclear factor-kappaB activation, but enhanced the expression of IL-12 and IL-10 in Der p-stimulated alveolar macrophages. UTI-treated mice have no significant change of the aforementioned measurements compared with nontreated sensitized mice. CONCLUSION: Nafamostat mesilate and FOY exerting the therapeutic effect in allergen-induced airway inflammation was a result not only of their inhibitory action in the early phase of mast cells activation but also of immunoregulatory function in the late phase of allergic inflammation. Such properties of FUT and FOY might be a potential therapeutic approach for asthma. CLINICAL IMPLICATIONS: The clinical used of serine protease inhibitors FUT and FOY may also have implications for treating airway inflammation of asthma.

Enhanced insulin sensitivity using electroacupuncture on bilateral Zusanli acupoints (ST 36) in rats.

In this study, intravenous glucose tolerance test (ivGTT) and insulin challenge test (ICT) were applied to evaluate the influence of electroacupuncture (EA) on insulin sensitivity in rats. Firstly, hypoglycemic activity was confirmed on normal Wistar rats (36+/-12%) and streptozotocin (STZ)-induced diabetic rats (13+/-8%) after 60 min of 15 Hz EA on bilateral Zusanli acupoints. The rats were divided into the experiment group (EG) and control group (CG) randomly. After fasting, plasma glucose and insulin levels were assayed in the normal Wistar rats undergoing ivGTT. Plasma glucose levels and hypoglycemic activity were also evaluated in the normal Wistar rats and STZ diabetic rats during ICT. As the data showed, EA improved the glucose tolerance from 15 to 90 min (p<0.005 compared with the plasma glucose levels of the CG) during ivGTT. In addition, significant improvement in the Homeostasis Model Assessment (HOMA) index was found in the EG from 15 to 90 min (p<0.005 compared with the CG). More hypoglycemic activity was achieved in normal Wistar and STZ diabetic rats in the EG than in the CG (from 30 to 60 min) during ICT. In conclusion, the results suggest that 15 Hz EA at bilateral Zusanli acupoints improved glucose tolerance. Thus, EA should be considered as an alternative method for improving insulin sensitivity and/or increase insulin-hypoglycemic activity in rats.