Cytotoxic Activity of Piper cubeba Extract in Breast Cancer Cell Lines

This study aimed to evaluate the cytotoxicity of a crude extract of Piper cubeba against normal and breast cancer cell lines. To prepare the extract, P. cubeba seeds were ground, soaked in methanol and dichloromethane and isolated by column chromatography. Fractions were tested for cytotoxicity effects on normal fibroblast (L929), normal breast (MCF-12A) and breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). The most effective fraction was selected for DNA fragmentation assay to detect apoptotic activity. The results showed that the methanolic crude extract had a higher cytotoxic activity against MDA-MB-468 and MCF-7 than a dichloromethane crude extract. Then, the methanolic crude extract was separated into six fractions, designated A to F. Fraction C was highly active against breast cancer cell lines with an IC₅₀ value less than 4 μg/mL. Therefore, Fraction C was further separated into seven fractions, CA to CG. The ¹H-NMR profile showed that Fraction CE was long chain hydrocarbons. Moreover, Fraction CE demonstrated the highest activity against MCF-7 cells with an IC₅₀ value of 2.69 ± 0.09 μg/mL and lower cytotoxicity against normal fibroblast L929 cells with an IC₅₀ value of 4.17 ± 0.77 μg/mL. Finally, DNA fragmentation with a ladder pattern characteristic of apoptosis was observed in MCF-7, MDA-MB-468, MDA-MB-231 and L929 cells, but not in MCF-12A cells.     

内分泌及化疗对乳腺肿瘤干细胞的实验研究

目的探讨研究内分泌及化疗两种治疗方式对ER＋乳腺癌患者中乳腺癌CD44＋CD24low细胞亚群的影响．以期推动ER‘乳腺癌的临床治疗。方法取ER’乳腺癌患者中乳腺癌细胞株MCF-7,进行体外培养,无水乙醇、雌二醇、以及雌二醇联合三苯氧胺的培养观察。随后选取雌二醇组中处于对数生长期的细胞,分别进行紫杉醇、吉西他滨以及阿霉素的培养观察。统计分析各组的分析结果。结果雌二醇培养组与联合培养组,细胞培养7d、10d以及15d后,细胞中CD44＋CD24-aow表现细胞所占的比例相比较或其分别与对照组比较均具有显著性差异（P〈O．05）。而紫杉醇、吉西他滨以及阿霉素培养组中,细胞中CD44＋CD24／Low表现细胞比例在培养1d后与对照组比较有显著性差异（P〈0．05）;在培养15d后与对照组比较无显著性差异（P〉0．05）。结论内分泌以及化疗对Elk＋乳腺癌细胞均有重大影响,对于ER＋乳腺癌根治具有重要意义,值得进一步研究探讨。     

Extract from Aloysia polystachya Induces the Cell Death of Colorectal Cancer Stem Cells

Abstract

Cancer stem cells (CSCs) are an important player in the resistance of cancers to therapy. In this work, we determined the flavonoids composition and biological action of Aloysia polystachya (AP) extracts in colorectal cancer. The chemical characterization of extracts was performed by HPLC. Assays of cytotoxicity, apoptosis, migration and invasion, metalloproteases activity, clonogenic growth, tumorspheres formation, Hoechts efflux, pluripotency marker expression and sensitization to chemotherapeutic drugs were performed in vitro in human HCT116 and murine CT26 colorectal cancer cells. The AP toxicity and effect in tumor growth administered alone or in combination with 5- Fluorouracile was analyzed in vivo, including histopathological studies. We found that AP extracts induced in vitro the apoptosis of colorectal cancer cell lines decreasing the CSC proportion. Moreover, they were capable to kill 5-Fluorouracile resistant side population cells. At not toxic doses in vivo, AP extracts inhibited tumor growth. Regarding the ability to reduce the CSC population, AP extracts deserves to be investigated as a useful therapy for colorectal cancer treatment.     

干细胞标志物ALDH1与大样本乳腺癌患者的预后关系

目的研究干细胞标志物已醛脱氢酶1(ALDH1)在乳腺癌细胞中的表达及其与乳腺癌患者预后的关系。方法采用免疫组化方法检测ALDH1在595例乳腺癌组织芯片中的表达并评分,用ROC方法确定ALDH1表达最佳临界值,检测ALDH1表达情况与患者临床特征之间相关性。结果 ALDH1在乳腺癌细胞中高表达,并与Ki67、CEA和分子分型呈正相关。单因素和多因素分析均表明ALDH1可以作为乳腺癌OS、DFS独立预后因子。结论 ALDH在乳腺癌组织中高表达,且过表达ALDH1可以作为乳腺癌独立预后不良因子。     

Effects of Pomegranate Juice on Hormonal Biomarkers of Breast Cancer Risk

Pomegranate is a rich source of polyphenols. Laboratory studies suggest polyphenols may exert breast cancer preventive effects through modulation of endogenous sex hormone levels. The aim of this study was to determine the effect of pomegranate juice consumption on serum levels of estradiol, estrone, testosterone, androstenedione, and sex hormone binding globulin (SHBG). Sixty-four healthy postmenopausal women were randomly assigned to drink 8 ounces of either 100% commercial pomegranate juice (intervention) or apple juice (control) for 3 weeks. Overall, women in the intervention group did not experience any significant decline in serum sex hormones or SHBG compared to women in the control group. In subgroup analyses restricted to 38 normal weight women, women in the intervention group compared to control group had a significant decline in estrone (pg/mL) and testosterone levels (pg/mL): pomegranate: ?61.6 [95% confidence interval (CI): ?175.8 to 52.6), apple: 1.1 (95% CI: ?5.4 to 7.7), P = 0.05, and pomegranate: ?289.1 (95% CI: ?630.7 to 52.5), apple: 79.6 (95% CI: ?77.8 to 236.9), P = 0.03, respectively. Because of several study limitations, results should be considered preliminary. Additional larger trials would be needed to determine effects in normal versus overweight/obese women.     

Association of Overweight and Obesity with Breast Cancer in India

Background:

In women, cancer of the breast is one of the most common incident cancer and cause of death from cancer. Anthropometric factors of weight, height, and body mass index (BMI) have been associated with breast cancer risk.

Objectives:

To study the association of overweight and obesity with breast cancer in India.

Materials and Methods:

A hospital-based matched case-control study was conducted. Three hundred and twenty newly diagnosed breast cancer patients and three hundred and twenty normal healthy individuals constituted the study population. The subjects in the control group were matched individually with the patients for their age ±2 years and socioeconomic status. Anthropometric measurements of weight and height were recorded utilizing the standard equipments and methodology. The paired ‘t’ test and univariate logistic regression analysis were carried out.

Results:

It was observed that the patients had a statistically higher mean weight, body mass index, and mid upper arm circumference as compared to the controls. It was observed that the risk of breast cancer increased with increasing levels of BMI. Overweight and obese women had Odd''s redio of 1.06 and 2.27, respectively, as compared to women with normal weight.

Conclusions:

The results of the present study revealed a strong association of overweight and obesity with breast cancer in the Indian population.     

Biotransformed Soybean Extract Induces Cell Death of Estrogen-Dependent Breast Cancer Cells by Modulation of Apoptotic Proteins

The process of soybean biotransformation increases the quantity of isoflavones (daidzein and genistein), which besides being considered an alternative to estroprogestive hormone replacement therapy (HRT), are able of hindering the growth and development of tumor cells. We investigated the effects of soybean extract biotransformed by fungus on estrogen-dependent (MCF-7) and nondependent (SK-BR-3) breast cell lines. Cells were treated with different concentrations of biotransformed (BSE) and nonbiotransformed soybean extract (SE), or daidzein (D) and genistein (G) patterns isolated and in combination (D + G). Afterwards, we analyzed cell viability by MTT assay, phosphatidylserine exposure and cell permeability by flow cytometry; expression of apoptotic proteins by Western blotting. BSE promoted reduction in cell viability and increase in DNA degradation in both cell lines. In addition, we verified increase in cell permeability and in the expression of phosphatidylserine, as well as modulation in the expression of apoptotic proteins in MCF-7 cells. The cells did not show any signs of cell death when incubated with the controls (D, G, and D + G). Unknown components found in the BSE induce cell death by apoptosis and necrosis, mainly in MCF-7 cells. These processes depend on the activation of caspase-3 and involve an increase in the expression of proapoptotic molecules.     

Phellinus Linteus Extract Induces Autophagy and Synergizes With 5-Fluorouracil to Inhibit Breast Cancer Cell Growth

Phellinus linteus (PL) is a medicinal mushroom due to its several biological properties, including anticancer activity. However, the mechanisms of its anticancer effect remain to be elucidated. We evaluated the inhibitory effects of the ethanolic extract from the PL combined with 5-FU on MDA-MB-231 breast cancer cell line and to determine the mechanism of cell death. Individually, PL extract and 5-FU significantly inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner. PL extract (30 mg/mL) in combination with 5-FU (10 μg/mL) synergistically inhibited MDA-MB-231 cells by 1.8-fold. PL did not induce apoptosis, as demonstrated by the DNA fragmentation assay, the sub-G1 population, and staining with annexin V-FITC and propidium iodide. The exposure of MDA-MB-231 cells to PL extracts resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine staining, the formation of acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, and ultrastructural observation of autophagic vacuoles by transmission electron microscopy. We concluded that PL extracts synergized with low doses of 5-FU to inhibit triple-negative breast cancer cell growth and demonstrated that PL extract can induce autophagy-related cell death.     

Effects of Thymus serpyllum Extract on Cell Proliferation,Apoptosis and Epigenetic Events in Human Breast Cancer Cells

Thymus (T.) serpyllum (wild thyme) is an aromatic medicinal plant due to its several biological properties, including anticancer activity. Breast cancer is one of the most common malignancies and increasing evidence supports that it is not only a genetic but also an epigenetic disease. Epigenetics investigates changes in gene expression caused by mechanisms that do not involve alterations in DNA sequence. DNA methylation and histone acetylation are the most widely studied epigenetic changes in cancer cells. This study evaluated the effects of T. serpyllum on apoptosis and epigenetic events in breast cancer cells. XTT cell viability assay was used to determine cytotoxicity. DNA fragmentation and caspase 3/7 activity assays were used in the assesment of apoptosis. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities were evaluated by ELISA and verified by qRT-PCR. T. serpyllum extract induced significant cytotoxicity in breast cancer cells (MCF-7 and MDA-MB-231) but not in normal cells. It also induced apoptosis and inhibited the DNMT and HDAC activities in MDA-MB-231 cells. In the present study, the first preliminary data on the effects of the methanolic extract of T. serpyllum in normal and breast cancer cells were obtained and suggest that T. serpyllum may be a promising candidate in the development of novel therapeutic drugs for breast cancer treatment.     

Trans-Resveratrol Alters Mammary Promoter Hypermethylation in Women at Increased Risk for Breast Cancer

Trans-resveratrol, present in high concentration in the skin of red grapes and red wine, has a dose-dependent antiproliferative effect in vitro, prevents the formation of mammary tumors, and has been touted as a chemopreventive agent. Based upon in vitro studies demonstrating that trans-resveratrol downregulates the expression of 1) DNA methyltransferases and 2) the cancer promoting prostaglandin (PG)E₂, we determined if trans-resveratrol had a dose-related effect on DNA methylation and prostaglandin expression in humans. Thirty-nine adult women at increased breast cancer risk were randomized in double-blind fashion to placebo, 5 or 50 mg trans-resveratrol twice daily for 12 wk. Methylation assessment of 4 cancer-related genes (p16, RASSF-1α, APC, CCND2) was performed on mammary ductoscopy specimens. The predominant resveratrol species in serum was the glucuronide metabolite. Total trans-resveratrol and glucuronide metabolite serum levels increased after consuming both trans-resveratrol doses (P < .001 for both). RASSF-1α methylation decreased with increasing levels of serum trans-resveratrol (P = .047). The change in RASSF-1α methylation was directly related to the change in PGE₂ (P = .045). This work provides novel insights into the effects of trans-resveratrol on the breast of women at increased breast cancer risk, including a decrease in methylation of the tumor suppressor gene RASSF-1α. Because of the limited sample size, our findings should be validated in a larger study.     

乳腺癌干细胞的研究现况

乳腺癌是当今严重危害女性健康及生命的头号杀手,目前有多项研究认为乳腺癌起源于乳腺癌干细胞。乳腺癌干细胞理论的提出较为合理地解释了乳腺癌的根源、复发及转移等生物学行为,为根治乳腺癌提供了一个新的方向。     

检测PSCA和Oct-4蛋白在乳腺癌早期诊断的临床意义

目的研究前列腺干细胞抗原（PSCA）和Oct-4蛋白在乳腺良性增生性疾病和乳腺癌组织中的表达及临床意义。方法利用免疫组织化学技术检测乳腺浸润性导管癌、纤维囊性乳腺病和导管内乳头状瘤组织中PSCA和Oct-4蛋白的表达。结果PSCA和Oct-4蛋白阳性信号主要位于细胞质和细胞膜；乳腺浸润性导管癌和纤维囊性乳腺病组织中PSCA和Oct-4蛋白的阳性率和阳性表达强度明显高于导管内乳头状瘤；2种蛋白在乳腺浸润性导管癌和纤维囊性乳腺病组织中的阳性率和阳性表达强度未见明显差异；PSCA和Oct-4蛋白的阳性表达强度与乳腺浸润性导管癌的组织学分级无关。结论高表达PSCA和Oct-4蛋白的细胞可能是“癌干细胞”，可能主要参与乳腺癌的发生，检测乳腺良性增生性病变中PSCA和／或Oct-4蛋白的表达水平对预防和早期发现乳腺癌可能有较重要临床价值。     

Inhibition of Cancer Cell Replication by Inorganic Nitrite

Nitrite has been implicated in carcinogenesis, especially under acidic conditions such as in the stomach or in urine, where it forms nitrosating species that can react with secondary amines to form nitrosamines. Recent studies have shown that nitrite and acid form a variety of other nitrogen oxides in vivo including nitric oxide—a compound with documented antitumor activity. Here we tested the effects of nitrite on bladder tumor cells incubated in mildly acidified urine. Nitrite (50 μM) inhibited thymidine incorporation in human T24 bladder cancer cells. This inhibition required slight acidification (pH 5.5–6), and no effect of nitrite could be observed at pH 7. Nitrite effects were further augmented in the presence of ascorbic acid, whereas ascorbic acid alone had no effect. The effects were paralleled by formation of nitric oxide gas. We here demonstrate an inhibitory effect of nitrite on cancer cell replication at concentrations and acidity commonly found in urine and gastric juice. The inhibitory effect is likely caused by nitric oxide and possibly other reactive nitrogen oxides formed from acidified nitrite.     

Antiproliferative Effect of Trifolium Pratens L. Extract in Human Breast Cancer Cells

Breast cancer is currently the most common cancer in women worldwide. Tamoxifen is used for the treatment of both early and advanced estrogen receptor positive breast cancer. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. The effect of T. pratense extract on breast cell lines (MCF-7 and MDA-MB-231) was investigated here. The cells were treated with T. pratense extract and cell viability was evaluated using trypan blue staining, MTT assay and lactate dehydrogenase activity measurement. Apoptosis, autophagy cell death was detected using fluorescent dye staining. Griess assay was performed to nitric oxide (No) measurement. Change in expression level of some apoptotic and autophagic- related genes was detected using real-time PCR. The combination effect of exteract and tamoxifen was evaluated by calculating the combination index and dose reduction index (DRI) values. T. pratense extract reduced the cell viability in dose and time- dependent manner. Apoptosis and autophagy was increased after treatment with T. pratense extract. Nitric oxide production significantly reduced in cells and combination of tamoxifen and T. pratense extract had a synergistic cytotoxic effect on breast cancer cell lines. T. pratense as a dietary supplement can be beneficial for some patients with breast cancer who receive conventional chemotherapy.     

Pepper Seed Extract Suppresses Invasion and Migration of Human Breast Cancer Cells

This study was performed to determine the antimetastatic activities of chili pepper seed on human breast cancer cells. The water extract of chili pepper seeds was prepared and it contained a substantial amount of phenols (131.12 mg%) and no capsaicinoids. Pepper seed extract (PSE) suppressed the proliferation of MDA-MB-231 and MCF-7 cells at the concentration of 10, 25, and 50 μg/ml (MDA-MB-231: IC₅₀ = 20.1 μg/ml, MCF-7: IC₅₀ = 14.7 μg/ml). PSE increased the expression level of E-cadherin up to 1.2-fold of the control in MCF-7 cells. PSE also decreased the secretion of matrix metalloproteinase (MMP)-2 and MMP-9 in MDA-MB-231 and MCF-7 cells at the concentration of 25 and 50 μg/ml. PSE treatment significantly suppressed the invasion of MDA-MB-231 and MCF-7 cells in a dose-dependent manner. The motility of cancer cells was apparently retarded in the wound healing assay by the PSE treatment. Although our data collectively demonstrate that PSE inhibits invasion and migration of breast cancer cells, further study is needed to identify specific mechanisms and bioactive components contributing to antimetastatic effects of chili pepper seed.     

Selective Growth Inhibition of Human Breast Cancer Cells by Graviola Fruit Extract In Vitro and In Vivo Involving Downregulation of EGFR Expression

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC₅₀ = 4.8 μg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.     

Association of Selected Medical Conditions With Breast Cancer Risk in Korea

Objectives

To estimate the effect of medical conditions in the population of Korea on breast cancer risk in a case-control study.

Methods

The cases were 3242 women with incident, histologically confirmed breast cancer in two major hospitals interviewed between 2001 and 2007. The controls were 1818 women each admitted to either of those two hospitals for a variety of non-neoplastic conditions. Information on each disease was obtained from a standardized questionnaire by trained personnel. Odds ratios (ORs) for each disease were derived from multiple logistic regression adjusted for age, age of menarche, pregnancy, age of first pregnancy, and family history of breast cancer.

Results

Among all of the incident breast cancer patients, pre-existing diabetes (OR, 1.33; 95% confidence interval [CI], 0.99 to 1.78), hypertension (OR, 1.46; 95% CI, 1.18 to 1.83), thyroid diseases (OR, 1.26; 95% CI, 1.00 to 1.58), and ovarian diseases (OR, 1.70; 95% CI, 1.23 to 2.35) were associated with an increased risk of breast cancer when other factors were adjusted for. In a stratified analysis by menopausal status, pre-existing hypertension (pre-menopause OR, 0.80; 95% CI, 0.48 to 1.34 vs. post-menopause OR, 1.87; 95% CI, 1.44 to 2.43; p-heterogeneity <0.01) and ovarian disease (pre-menopause OR, 4.20; 95% CI, 1.91 to 9.24 vs. post-menopause OR, 1.39; 95% CI, 1.02 to 1.91; p-heterogeneity 0.01) showed significantly different risks of breast cancer.

Conclusions

Our results suggest the possibility that medical conditions such as hypertension affect breast cancer development, and that this can differ by menopausal status. Our study also indicates a possible correlation between ovarian diseases and breast cancer risk.     

Association between Dietary Intake of Flavonoids and Cancer Recurrence among Breast Cancer Survivors

Intake of flavonoids is associated with the incidence of breast cancer, but the association between the intake of flavonoids and cancer recurrence is unclear. This study aimed to investigate the hypothesis that intake of flavonoids and flavonoid-rich foods is negatively associated with cancer recurrence. Among 572 women who underwent breast cancer surgery, 66 patients had a cancer recurrence. Dietary data were collected using a structured 24-h dietary recall, and intake of flavonoids was calculated based on the Korea Rural Development Administration flavonoid database. Among overweight and obese patients, disease-free survival was associated with intake of flavonoids (p = 0.004) and flavonoid-rich foods (p = 0.003). Intake of flavonoids (hazard ratio (HR) = 0.249, 95% confidence interval (CI): 0.09–0.64) and flavonoid-rich foods (HR = 0.244, 95% CI: 0.09–0.66) was negatively associated with cancer recurrence after adjusting for confounding factors in overweight and obese patients. Consumption of flavonoids and flavonoid-rich foods was lower in overweight and obese patients with cancer recurrence than those without recurrence and in normal-weight patients. This study suggests that intake of flavonoids and flavonoid-rich foods could have beneficial effects on cancer recurrence in overweight and obese breast cancer survivors.     

乳腺癌细胞核差异蛋白筛选

目的 比较体外培养的正常乳腺细胞和乳腺癌细胞核蛋白指纹图谱,筛选差异蛋白,为乳腺癌亚细胞蛋白标志物的研究奠定基础.方法 提取正常乳腺细胞株HBL-100和乳腺癌细胞株MCF-7、MDA-MB-231的核蛋白,表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)检测蛋白表达,重复试验30次,Biomaker Wizard Software分析软件统计结果并筛选差异蛋白.结果 分子量(Mr) 2～100 kD范围内MCF-7和MDA-MB-231与HBL-100相比差异蛋白点数目分别为78个和69个(P＜0.05),其中5.8 kD、8.3 kD和18.2 kD的蛋白在两株癌细胞中表达均降低,9.9 kD和15.7 kD的蛋白在两株癌细胞中表达均增高.结论 SELDI-TOF-MS能够快速灵敏地检测分析乳腺癌细胞核差异表达蛋白,为乳腺癌亚细胞蛋白标志物的研究提供了新思路.